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[ "<title>1. INTRODUCTION</title>", "<p>Peroxisome proliferator-activated receptors (PPARs) are ligand-activated\nnuclear receptors that function as transcription\nfactors regulating the expression of genes involved in lipid biosynthesis, glucose\nmetabolism, as well as cell proliferation, differentiation, and survival [##REF##16476485##1##–##REF##11729302##4##]. Their\ndiscovery was driven by search of a molecular target for peroxisome\nproliferators, a group of agents named after their property to increase\nperoxisomes in rodent liver [##REF##2129546##5##, ##UREF##0##6##]. Later on, activity studies helped elucidate\nthe versatile role of these molecules in modulating diverse biological\nfunctions such as metabolism, tissue remodeling, inflammation, angiogenesis,\nand carcinogenesis [##REF##10839530##7##–##UREF##2##11##]. Three\nPPAR gene types have been identified: <italic>α</italic>, <italic>β</italic>/<italic>δ</italic>, and <italic>γ</italic> [##REF##10022237##12##, ##REF##12059785##13##]. Between them,\nPPAR<italic>γ</italic> is the most intensively investigated [##UREF##3##14##, ##REF##16360030##15##].</p>" ]
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[ "<title>5. CONCLUSION</title>", "<p>Existing\ndata suggest that peroxisome proliferator-activated receptor-gamma (PPAR<italic>γ</italic>) is a\npotential target ally to cancer chemopreventive agents. Although PPAR<italic>γ</italic> was first understood as a key\nregulator of adipocyte differentiation and glucose homeostasis, it is now\nrecognized that it\nis also involved in cell proliferation, differentiation, apoptosis, and\nangiogenesis. Meticulous research for PPAR<italic>γ</italic> agonists with potency to function as cancer\nchemopreventive agents is highly warranted.</p>" ]
[ "<p>Recommended by Dipak Panigrahy</p>", "<p>Peroxisome proliferator-activated receptor-gamma (PPAR<italic>γ</italic>), one of three ligand-activated transcription factors named PPAR, has been identified as a molecular target for cancer chemopreventive agents. PPAR<italic>γ</italic> was initially understood as a regulator of adipocyte differentiation and glucose homeostasis while later on, it became evident that it is also involved in cell differentiation, apoptosis, and angiogenesis, biological processes which are deregulated in cancer. It is now established that PPAR<italic>γ</italic> ligands can induce cell differentiation and yield early antineoplastic effects in several tumor types. Moreover, several bioactive natural products with cancer protecting potential are shown to operate through activation of PPAR<italic>γ</italic>. Overall, PPAR<italic>γ</italic> appears to be a prevalent target ally to cancer chemopreventive agents and therefore pursuing research in this area is of great relevance.</p>" ]
[ "<title>2. THE HUMAN PPAR<italic>γ</italic> GENE</title>", "<p>The human PPAR<italic>γ</italic> gene consists of six coding exons located at chromosome 3p25.2 and\nextends approximately over 100 kb of genomic DNA [##REF##9168928##16##]. \nThree major transcriptional\nstart sites have identified where three mature mRNAs originate from, differing\nin their 5′ untranslated regions \n[##REF##9228052##17##, ##REF##9821958##18##]. Notably PPAR<italic>γ</italic>1 and\nPPAR<italic>γ</italic>3 mRNAs code for the same protein of 475 amino\nacids, while PPAR<italic>γ</italic>2 transcript codes for a different protein\nwhich contains an additional 28 N-terminal amino acids [##REF##7926726##19##].</p>", "<title>2.1. Tissue\ndistribution of different PPAR<italic>γ</italic> isoforms</title>", "<p>The PPAR<italic>γ</italic>1 is found in virtually all tissues, such as liver, skeletal\nmuscle, prostate, kidney, breast, intestine, and the gonads. The PPAR<italic>γ</italic>2 is the\nmajor PPAR<italic>γ</italic> isoform expressed mainly in adipose tissue where it normally operates as\nan adipocyte-specific\ntranscription factor in preadipocytes and regulates\nadipose tissue differentiation, and the PPAR<italic>γ</italic>3 isoform is restricted to adipose\ntissue and large intestine [##REF##9821958##18##, ##REF##9065481##20##],</p>", "<title>2.2. PPAR<italic>γ</italic> protein structure and function</title>", "<p>Similar to other members of the nuclear hormone receptors superfamily,\nPPAR<italic>γ</italic> protein has three functional domains: the N-terminal domain, the DNA-binding\ndomain, and a carboxy-terminal ligand-binding pocket \n(##FIG##0##Figure 1##).</p>", "<p>PPAR<italic>γ</italic> protein receptor is activated by a number of endogenous and exogenous\nligands of various potencies. Among pharmaceutical compounds, thiazolidinedione\n(TZD) class of insulin-sensitizing drugs (also called glitazones) are best\nknown to operate as ligands to PPAR<italic>γ</italic> [##REF##9744270##21##, ##REF##9813012##22##] while long-chain\npolyunsaturated fatty acids are the most well-characterized endogenous ligands [##REF##8240670##23##].</p>", "<p>The activated PPAR<italic>γ</italic>\nprotein becomes operational following its heterodimerization with retinoid X\nreceptors (RXR) [##REF##1324435##24##]. The PPAR<italic>γ</italic>/RXR complex translocates to the nucleus where it binds to target genes\nwhich contain a peroxisome proliferator response element (PPRE). A PPRE consist of a direct repetition of the\nconsensus sequence AGGTCA separated by a single nucleotide (Direct repetition; DR1) [##REF##9228052##17##]. To initiate transcriptional regulation of PPRE-bearing\ngenes, the PPAR<italic>γ</italic>/RXR complex requires accessory\nproteins to bind on. These proteins can\neither trigger (coactivators) or represses gene transcription (corepressors) \n(##FIG##0##Figure 1##). It must be noted though that besides their PPAR<italic>γ</italic>-dependent genomic effects, PPAR<italic>γ</italic> ligands can also influence\ncellular biology via nongenomic, PPAR<italic>γ</italic>-independent events [##REF##17611413##25##] \n(##FIG##0##Figure 1##).</p>", "<p>As a rule, the\ntranscriptional activity of PPAR<italic>γ</italic> is negatively\nmodulated through phosphorylation by MAPK [##REF##8953045##26##–##REF##17560826##28##]. Phosphorylation of human PPAR<italic>γ</italic>1 protein at Ser-84 site\nrestrains its function [##REF##9030579##27##], and\nphosphorylation of PPAR<italic>γ</italic>2 modifies the A/B domain and reduces its ligand binding affinity [##REF##9845075##29##]. However,\nnot all phosphorylation events are inhibitory. For example, it has been found\nthat missense mutation which results in the conversion of proline to glutamine\nat position 115 can render PPAR<italic>γ</italic>2 constitutively active through modulation of the\nMAPK-dependent phosphorylation status of serine 114 [##REF##9753710##30##] while\nphosphorylation by protein kinase A (PKA) was shown to enhance its activity [##REF##11117527##31##].</p>", "<p>Until now, three molecular processes have been\nproposed for the termination and downregulation of PPAR<italic>γ</italic> signaling: the phosphorylation of Ser-84/112 of PPAR<italic>γ</italic>1/2 by ERKs [##REF##9030579##27##], the\nproteasomal degradation of ligand-activated PPAR<italic>γ</italic> [##REF##11733495##32##], and the interaction with MEKs, which promotes its expulsion\nfrom the nucleus [##REF##17101779##33##].</p>", "<title>3. PPAR<italic>γ</italic> IN CANCER</title>", "<p>Early studies portrayed\nPPAR<italic>γ</italic> as an important regulator of preadipocyte\ndifferentiation and glucose homeostasis. Later on, it was identified that PPAR<italic>γ</italic> \nregulates biological processes which are considered\nhallmarks of cancer such as cell differentiation, apoptosis, and angiogenesis. This\nknowledge, coupled with data showing that PPAR<italic>γ</italic> ligands could yield anticancer\neffects in several cell types, led researches postulate a role for PPAR<italic>γ</italic> in carcinogenesis [##UREF##2##11##, ##REF##14708026##34##, ##REF##16887936##35##].</p>", "<p>Apoptosis is\nbelieved to be a fundamental molecular mechanism through which PPAR<italic>γ</italic> activators\nexert their action against cells which undergo malignant transformation [##REF##17911009##36##–##REF##17582756##38##]. Moreover, apart from their direct inhibitory\neffects on cancerous transformed cells, PPAR<italic>γ</italic> can also inhibit angiogenesis\nwhich is a prerequisite for tumor formation and growth [##REF##17396134##39##–##REF##16082179##41##]. It is suggested that the antiangiogenic\nactivity of PPAR<italic>γ</italic> can be accomplished either by blocking the production the angiogenic\nELR+CXC chemokines by cancer transformed cells or by inducing expression of the\nthrombospondin-1 receptor CD36 in endothelial cells [##REF##15799829##42##–##REF##17548167##44##] In\naddition, latest exciting data, which showed that PPAR<italic>γ</italic> agonists were able to inhibit the\ncanonical WNT signaling in human colonic epithelium, raises hopes that such\nagents can possibly block cancer initiation at a stem cell level [##REF##17634527##45##].</p>", "<p>It must be\nunderlined herein that despite demonstration of cancer-preventive effects of\nPPAR<italic>γ</italic> ligands in vitro, clinical trials and animal\nmodels failed so far to show significant benefits [##REF##16916271##46##]. The fact\nthat PPAR<italic>γ</italic> ligands have been used in clinic\ntrials at concentrations above those needed to elicit receptor agonistic activity\nposes questions for receptor-independent off-target effects [##REF##17200362##47##].</p>", "<title>3.1. PPAR<italic>γ</italic> and gastrointestinal cancer</title>", "<p>PPAR<italic>γ</italic> are heterogeneously expressed throughout the\ngastrointestinal epithelium, showing significant differences in abundance,\ndistribution, and functions. This\nprotein is principally expressed in differentiated epithelial colonic cells,\npreferably in the proximal colon [##REF##10467224##48##]. Sarraf et al. showed that PPAR<italic>γ</italic> activation could stimulate a\nprogram that is characteristic of colonic cell differentiation [##UREF##5##49##].</p>", "<p>A functional\ngenomics analysis conducted for the identification of PPAR<italic>γ</italic> gene targets revealed that the\nmajority of these genes were transcribed throughout the colon, but their\nexpression varied in cells purified from the proximal colon and in those from\nthe distal colon. Metabolic functions of PPAR<italic>γ</italic> were elicited primarily in the proximal colon,\nwhereas signaling functions were recognized in the distal colon. Interestingly,\nTZDs transactivated the PPAR<italic>γ</italic> gene targets at the proximal colon but repressed them in the distal\ncolon. TSC22, a TGF<italic>β</italic> target gene known to inhibit colon cell proliferation, was also\nidentified as a PPAR<italic>γ</italic> target gene [##REF##12468551##50##]. It is\nworth mentioning that both TGF<italic>β</italic> and PPAR<italic>γ</italic> pathways attenuate during transition from adenoma to carcinoma [##REF##16574647##51##]. From a\npharmacological point of view, Yamazaki at al. showed that activation of the RXR/PPAR<italic>γ</italic>\nheterodimer by their respective ligands could be considered a useful chemopreventive\nstrategy for colorectal cancer. They found that a combination of the RXR alpha ligand 9-cis-retinoic acid with ciglitazone synergistically inhibited the cell growth and induced\napoptosis in Caco2 human colon cancer cells that expressed high levels of p-RXR\nalpha protein [##REF##17604322##52##].</p>", "<p>In\nthe most widely used preclinical model of sporadic colon carcinogenesis, the\nazoxymethane-treated mice, activation of PPAR<italic>γ</italic> suppressed carcinogenesis\nbut only before damage to the APC/beta-catenin pathway [##REF##12370429##53##]. However, two papers\npublished ten years ago reported that troglitazone and rosiglitazone increased occurrence\nof colon tumors in mice-caring mutations in the <italic>APC</italic> gene [##REF##10467224##48##, ##UREF##6##54##]. Moreover, although\npioglitazone was later reported to suppresses colon tumor growth in Apc+/− mice\n[##REF##14611672##55##], biallelic knockdown of\nPPAR<italic>γ</italic> in\ncolonic epithelial cells was associated with an increase of tumor incidence [##REF##16858678##56##]. It should be reminded, however, that\nalthough TZDs are considered pure PPAR agonists, they also wield off-target\neffects not mediated through linkage to PPAR receptors. An in-depth analysis of the role of TZDs against\ncolon cancer can be facilitated through development of tissue-specific PPAR<italic>γ</italic> knockout mice [##REF##17978568##57##]. Interestingly, a\nsmall phase II clinical trial using \ntroglitazone failed to document tumor responses in patients with advance\nstage metastatic colon cancer [##UREF##7##58##].</p>", "<p>Overall, existing evidence indicates that PPAR<italic>γ</italic> agonists have a potential \nto inhibit cancer formation in the distal colon, but they are practically inactive in advanced \nstages of colon cancer.</p>", "<title>3.2. PPAR<italic>γ</italic> and lung cancer</title>", "<p>Lung cancer is a\nmajor global health problem because of its incidence and mortality. It remains\nthe top cancer killer worldwide to which early-detection strategies and\ndevelopment of new therapies failed so far to improve its lethal outcome [##REF##17873161##59##]. This\ntobacco-related cancer epidemic persists despite public implementation of\ntobacco control measures because the majority of tobacco-smoke users declare\npowerlessness to quit. Therefore, the search for potent chemopreventive agents\nand the development of effective chemoprevention strategies for lung cancer is\na viable pursuit highly justified [##REF##14602246##60##, ##REF##15886305##61##].</p>", "<p>Several studies\nhave shown that PPAR<italic>γ</italic> agonists can inhibit growth\nand induce changes associated with differentiation and apoptosis in lung cancer\n[##REF##10706135##62##–##REF##12391285##64##]. TZDs\ninduced upregulation of PTEN and p21, downregulation of cyclins D and E, and\nreduced expression of fibronectin and its receptor integrin <italic>α</italic>5<italic>β</italic>1 in human lung\ncarcinoma cell lines [##REF##16386327##65##–##REF##15677767##68##].</p>", "<p>A first evidence\nof clinical efficacy of PPAR<italic>γ</italic> agonists as\ncancer chemopreventives in lung cancer was recently published. A retrospective analysis of a database from\nten Veteran Affairs medical centers revealed a significant reduction (33%) in\nlung cancer risk in diabetic patients who were treated with TZDs compared with\nnonusers of TZDs [##REF##17442990##69##]. However,\nother studies damped early this enthusiasm by showing that diabetic patients\ntreated with TZDs were at increased risk for cardiovascular complications [##REF##17517853##70##].</p>", "<p>It is critical to understand that cancer-protecting effects of PPAR<italic>γ</italic> agonists in lung cancer can be PPAR<italic>γ</italic> dependent but also PPAR<italic>γ</italic> independent [##REF##17975488##71##]. Characteristically, TZDs suppressed the expression of antiapoptotic\nmediator prostaglandin E(2) in NCLC cells through induction of\n15-hydroxyprostagladin dehydrogenase [##REF##17412838##72##] and enhanced TRAIL-induced apoptosis through\nupregulation of death receptor 5 DR5 and downregulation of c-FLIP in human lung\ncancer cells [##REF##17172826##73##].</p>", "<p>The combination\nof PPAR<italic>γ</italic> agonists with other chemopreventive\nagents emerges as a challenging issue in lung cancer chemoprophylaxis. Notably,\nan amazing synergy of clinically achievable concentrations of lovastatin (an\nHMG-CoA reductase inhibitor) and troglitazone was recently shown against lung cancer\ncells [##REF##16094629##74##]. This effect\nwas accompanied by synergistic modulation of E2F-1, p27∧Kip1, CDK2,\ncyclin A and RB. In another study, a combination of low-doses of MK886 (5-lipoxygenase\nactivating protein-directed inhibitor), ciglitazone and 13-cis-retinoic acid,\nalso demonstrated synergistic inhibitory activity against lung cancer cells [##REF##15899809##75##]. These\nstudies provide a framework for the development of rationally designed drug\ncombinations aimed to target simultaneously the PPAR<italic>γ</italic> and other cofactors.</p>", "<title>3.3. PPAR<italic>γ</italic> and other malignancies</title>", "<p>Epidemiological studies suggested that high consumption\nof carotenoids (known PPAR<italic>γ</italic> activators) could protect women\nfrom the development of breast cancer [##REF##9486468##76##, ##REF##11415946##77##]. These findings are also\nsupported by experiments which show that activation of PPAR<italic>γ</italic> can induce terminal\ndifferentiation, cell cycle arrest, or apoptosis of preneoplastic and cancerous\nmammary epithelial cells [##REF##9660931##78##–##REF##9671760##80##]. Unfortunately, this is not the case for advanced breast\ncancer: a phase II trial of troglitazone in patients\nwith breast cancer metastases failed recently to prove clinical benefits [##REF##12846423##81##].</p>", "<p>Prostate cancer appears to be an attractive\ntumor target for PPAR<italic>γ</italic> agonists because cancerous prostate\ncells express higher levels of PPAR<italic>γ</italic> compared with their normal counterparts [##REF##10984506##82##]. Moreover,\nit has been shown that PPAR<italic>γ</italic>1/2 activation suppressed the high level of\nendogenous COX-2 in normal\nprostate epithelial cells [##UREF##8##83##] while TZDs mediated\napoptosis in prostate cancer cells through inhibition of Bcl-xL/Bcl-2 functions\n[##REF##15735046##84##]. In the\nclinical setting, reduction and prolonged stabilization of prostate-specific antigen\nlevels were demonstrated\nin patients treated with troglitazone [##REF##10984506##82##, ##REF##11034093##85##]. The\nabove data provide a rationale to consider investigating PPAR<italic>γ</italic> ligands for their role in preventive and possibly therapeutic management\nof prostate cancer.</p>", "<p>In gynecological cancer, Wu et al. reported that rosiglitazone could\nblock or delay the development of hyperplasia and subsequent endometrial\ncancer. This PPAR<italic>γ</italic> agonist induced apoptosis in both PTEN intact\nand PTEN null cancer cell lines and decreased proliferation of the endometrial\nhyperplastic lesions in a PTEN(+/−)\nmurine model [##REF##18334011##86##].</p>", "<p>In human\npancreatic cancer cell lines, treatment with TZDs was found to induce cell\ncycle arrest and increase expression of pancreatic differentiation markers [##REF##11078801##87##, ##REF##11034103##88##]. Moreover,\nactivation of PPAR<italic>γ</italic> together with RXR resulted\nin suppression of pancreatic cancer cell growth through suppression of cyclin\nD1 [##REF##11895107##89##].</p>", "<p>Among sarcoma\ntumors, it is liposarcomas which are considered targets for PPAR<italic>γ</italic> agonists because they show a high expression of this nuclear receptor [##REF##8990192##90##]. However,\nalthough pioglitazone was found capable to terminally differentiate human\nliposarcoma cells in vitro, it failed an early phase II trial despite induced\nchanges in relevant target genes [##REF##14562008##91##].</p>", "<p>In thyroid\ncancer, a functional chromosomal translocation of part of <italic>PAX</italic>8 gene which encodes the DNA-binding domain to the activation\ndomain of the PPAR<italic>γ</italic> gene has been\ndetected in patients with follicular type carcinoma [##REF##10958784##92##]. This chimeric\nfusion protein is resistant to PPAR<italic>γ</italic> ligands, invalidating any anticancer effects of PPAR<italic>γ</italic> ligands in this setting. However,\nit has been suggested that PPAR<italic>γ</italic> ligands could\nhave activity in combination with retinoids and/or histone deacetylase\ninhibitors in thyroid tumors which express both PPAR<italic>γ</italic> and also RXR<italic>γ</italic> [##REF##16029127##93##, ##REF##15299084##94##].</p>", "<title>4. PPAR<italic>γ</italic> AS A MEDIATOR TO CANCER PROTECTING\nNATURAL PRODUCTS</title>", "<p>Evidence has\naccumulated which affirms that bioactive natural compounds can play an important role in cancer\nchemoprevention through modulation of PPAR<italic>γ</italic>. Preclinical studies and\nepidemiological data support that tumor growth and metastasis can be restrained\nor delayed by several herbal products [##REF##12195161##95##–##REF##17487840##98##].\nMoreover, it is believed that novel agents derived from bioactive\nphytochemicals can be used as adjuncts to enhance therapeutic efficacy of\nstandard treatments [##REF##17258704##99##, ##REF##16551868##100##]. Among natural products, triterpenoids,\nflavononoids, carotenoids, and linoleic acid are the most extensively studied\nas cancer chemopreventives and have invariably been found to operate as PPAR<italic>γ</italic> activators.</p>", "<p>Terpenoids of\nplant origin have shown antitumor activity which indicates a potential role for\nthese compounds as cancer chemopreventives [##REF##16551868##100##–##REF##17125231##102##]. Specifically,\n2-cyano-3,12-dioxooleana-1,9-dien-28-oic\nacid (CDDO), a synthetic triterpenoid, which was shown to activate PPAR<italic>γ</italic> and induce\ngrowth arrest and apoptosis in treated\nbreast cancer cells\n[##REF##14522919##103##]; also, glycyrrhizin\nthe major triterpene gycoside phytochemical in licorice root and the\ntriterpenoid acid betulinic acid which is found in the bark of several species\nof plants, both have shown pro-PPAR<italic>γ</italic> activities in\ncancer cells. These phytochemicals were found to induce expression of\nproapoptotic protein caveolin-1 and the tumor-suppressor gene Kruppel-like\nfactor-4 (KLF-4) in colon and pancreatic cancer cells [##REF##17513608##104##, ##REF##17724373##105##]. It\nshould though be noted that although caveolin-1 is generally considered a proapoptotic\nmolecule, it has also been associated with drug resistance and possibly metastasis\n[##REF##12414512##106##]. It is believed that some PPAR-<italic>γ</italic> agonists\ninduce whilst others repress caveolin-1 [##REF##12813462##107##].</p>", "<p>Isoflavones are well\nknown to function as phytoestrogens. They bind to the estrogen-related\nreceptors but also to PPAR<italic>α</italic>\nand PPAR<italic>γ</italic> [##REF##15936643##108##]. As a\nresult, their biological effects are determined by the balance between\nactivated ERs and PPAR<italic>γ</italic> [##REF##12421816##109##]. Liang et al. investigated apigenin, chrysin,\nand kaempferol in mouse macrophages and found that these flavonoids stimulated PPAR<italic>γ</italic> transcriptional activities as allosteric effectors rather than pure\nagonists [##REF##11343698##110##]. In the\nclinical setting, purified isoflavones have only been investigated for safety,\nbioavailability, and pharmacokinetics in men with early-stage prostate cancer [##REF##15231450##111##–##UREF##10##114##].</p>", "<p>Carotenoids are another class of phytochemicals found\nto activate PPAR<italic><italic>γ</italic></italic> in cancer cells. Hosokawa\net al. reported that the edible carotenoid fucoxanthin, when combined with\ntroglitazone, induced apoptosis of Caco-2 cells [##REF##15535974##115##]. Moreover, in epidemiological studies, consumption of carotenoids was shown to protect against breast cancer [##REF##9486468##76##, ##REF##11415946##77##].\nInterestingly, Cui et al. unveiled recently the molecular mechanisms which underlie the chemopreventive\nactivity of <italic>β</italic>-carotene against breast cancer. They found that <italic>β</italic>-carotene\nsignificantly increased PPAR<italic>γ</italic> mRNA and protein levels in\na time-dependent fashion, while 2-chloro-5-nitro-N-phenylbenzamide (GW9662), an\nirreversible PPAR<italic>γ</italic> antagonist, attenuated apoptosis caused by <italic>β</italic>-carotene in cancer-transformed cells [##REF##17911009##36##].</p>", "<p>Linoleic acid, a naturally occurring omega-6\nfatty acid which is abundant in many vegetable oils, has been studied\ncomprehensively for its prophylactic effects against cancer formation [##REF##15316938##116##]. Conjugated linoleic acid, which is found especially in eggs and in the\nmeat and dairy products of grass-fed ruminants, was shown to modulate cell-cell\nadhesion and invasiveness of MCF-7 cells through regulation of PPAR<italic>γ</italic> expression [##REF##17354222##117##]. Moreover\n<italic>α</italic>-eleostearic acid (ESA), a linolenic acid isomer, induced apoptosis in\nendothelial cells and inhibited angiogenesis, also through activation of PPAR<italic>γ</italic> [##REF##18174233##118##]. More recent studies brought up additional\nevidence and provided insights into molecular mechanisms of the protective\neffects of linoleic acid against colon cancer. Yasui et al. reported that\n9trans-11trans-conjugated linoleic acid inhibited the development of\nazoxymethane-induced colonic aberrant crypt foci in rats at preinitiation and\npostinitiation level through activation of PPAR<italic>γ</italic> and downregulation of\ncyclooxygenase-2 and cyclin D1 [##REF##17927506##119##]. In\naddition, Sasaki at al.\nshowed that linoleic acid was capable to inhibit azoxymethane-induced\ntransformation of intestinal cells and tumor formation [##REF##16094650##120##]. In most studies,\nthe differentiation-promoting and carcinogenesis-blocking effects were mostly attributed\nto activation of PPAR<italic>γ</italic> by linoleic acid products [##REF##12949056##121##]. Finally,\napart from its direct action as a PPAR<italic>γ</italic> activator, linoleic acid\nwas found to modulate interactions between PPAR<italic>β</italic>/<italic>δ</italic> and PPAR<italic>γ</italic> isoforms [##REF##16288226##122##].</p>", "<p>Finally, in the class of capsaicinoids, capsaicin, the\nspicy component of hot peppers, was shown to induce apoptosis of melanoma as\nwell as colon and prostate cancer cells, and was associated with activation of the PPAR<italic>γ</italic> in the case of colon cancer [##REF##15383218##123##–##REF##17306913##125##]. However, controversy exists regarding\ncancer-preventing and cancer-promoting effects of capsaicin [##REF##8621114##126##, ##REF##11604990##127##].</p>", "<p>It must be noted that besides their PPAR<italic>γ</italic>-mediated effects, natural products can also induce\ntranscription of detoxification enzymes glutathione S-transferases (GST) which are known to protect cells from\nchemical-induced carcinogenesis [##REF##9815533##128##, ##REF##11350917##129##]. Recently, Park et al. examined GSTA2 gene\ninduction by thiazolidinedione and 9-cis-retinoic acid and investigated the\nmolecular basis of PPAR<italic>γ</italic>/RXR-mediated GSTA2 induction in the\nH4IIE hepatocytes. They found that both PPAR<italic>γ</italic> and RXR agonists could increase the expression of GSTA2 but treatment of cells\nwith a combination of PPAR<italic>γ</italic> and RXR agonists\nproduced synergistic increase [##REF##15150131##130##]. This data\nsuggest that cancer-preventive functions of PPAR<italic>γ</italic> activators may be related to some\nextent to a parallel induction of GSTA2.</p>" ]
[]
[ "<fig id=\"fig1\" position=\"float\"><label>Figure 1</label><caption><p>\n<italic>Peroxisome\nproliferator-activated receptor-<italic>γ</italic> and ligands: pathways\nand functions.</italic> PPAR<italic>γ</italic> protein exhibits a structural organization\nconsisting of three functional domains: an N-terminal domain, a DNA-binding\ndomain (DBD) and a carboxy-terminal ligand binding domain (LBD). PPAR<italic>γ</italic> forms heterodimers with a second member of the\nnuclear receptor family, the retinoic X receptor (RXR). Unliganded PPAR<italic>γ</italic> suppresses transcription (pathway A) either by\ninterfering with key transcription factors (pathway A1) or through recruitment\nof corepressors (CoRep) on a PPRE element (pathway A2). Ligand binding to PPAR<italic>γ</italic> (pathway B) triggers conformational changes that lead\nto dissociation of corepressors (CoRep) and subsequent association of\ncoactivators (CoAct). The complex is binding to PPREs and triggers\ntranscription (pathway B). PPARs ligands can also\nexert their action through PPAR<italic>γ</italic>-independent mechanisms\nalso (pathway C). For instance in NSCLC cell lines activation of TNF-TRAIL\ninduce apoptosis, while PGE<sub>2</sub> degradation, trough 15-hydroxyprostagladin dehydrogenase induction, results in enhanced epithelial\ndifferentiation. In endothelial cells PPAR<italic>γ</italic>\nligands can markedly boost expression of CD36 which\nfunctions as the receptor of endogenous antiangiogenic molecule\nthrombospondin-1, thereby potentiating the apoptotic response. (PFAs: polyunsaturated\nfatty acids, TZDs: thiazolidinediones, PPRE: peroxisome proliferator response\nelement, TNF: tumor necrosis factor, TRAIL: TNF-related apoptosis-inducing\nligand, NSCLC: non-small cell lung carcinoma).</p></caption></fig>" ]
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[ "<graphic xlink:href=\"PPAR2008-436489.001\"/>" ]
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[{"label": ["6"], "surname": ["Lock", "Mitchell", "Elcombe"], "given-names": ["EA", "AM", "CR"], "article-title": ["Biochemical mechanisms of induction of hepatic peroxisome proliferation"], "italic": ["Annual Review of Pharmacology and Toxicology"], "year": ["1989"], "volume": ["29"], "fpage": ["145"], "lpage": ["163"]}, {"label": ["8"], "surname": ["Corton", "Anderson", "Stauber"], "given-names": ["JC", "SP", "A"], "article-title": ["Central role of peroxisome proliferator-activated receptors in the actions of peroxisome proliferators"], "italic": ["Annual Review of Pharmacology and Toxicology"], "year": ["2000"], "volume": ["40"], "fpage": ["491"], "lpage": ["518"]}, {"label": ["11"], "surname": ["Martinasso", "Oraldi", "Trombetta"], "given-names": ["G", "M", "A"], "article-title": ["Involvement of PPARs in cell proliferation and apoptosis in human colon cancer specimens and in normal and cancer cell lines"], "italic": ["PPAR Research"], "year": ["2007"], "volume": ["2007"], "fpage": ["9 pages"], "comment": ["Article ID 93416."]}, {"label": ["14"], "surname": ["Berger", "Moller"], "given-names": ["J", "DE"], "article-title": ["The mechanisms of action of PPARs"], "italic": ["Annual Review of Medicine"], "year": ["2002"], "volume": ["53"], "fpage": ["409"], "lpage": ["435"]}, {"label": ["40"], "surname": ["Giaginis", "Tsantili-Kakoulidou", "Theocharis"], "given-names": ["C", "A", "S"], "article-title": ["Peroxisome proliferator-activated receptor-"], "italic": ["\u03b3", "PPAR Research"], "year": ["2008"], "volume": ["2008"], "fpage": ["12 pages"], "comment": ["Article ID 431763."]}, {"label": ["49"], "surname": ["Sarraf", "Mueller", "Jones"], "given-names": ["P", "E", "D"], "article-title": ["Differentiation and reversal of malignant changes in colon cancer through PPAR"], "italic": ["\u03b3", "Nature Medicine"], "year": ["1998"], "volume": ["4"], "issue": ["9"], "fpage": ["1046"], "lpage": ["1052"]}, {"label": ["54"], "surname": ["Saez", "Tontonoz", "Nelson"], "given-names": ["E", "P", "MC"], "article-title": ["Activators of the nuclear receptor PPAR"], "italic": ["\u03b3", "Nature Medicine"], "year": ["1998"], "volume": ["4"], "issue": ["9"], "fpage": ["1058"], "lpage": ["1061"]}, {"label": ["58"], "surname": ["Kulke", "Demetri", "Sharpless"], "given-names": ["MH", "GD", "NE"], "article-title": ["A phase II study of troglitazone, an activator of the PPAR"], "italic": ["\u03b3", "Cancer Journal"], "year": ["2002"], "volume": ["8"], "issue": ["5"], "fpage": ["395"], "lpage": ["399"]}, {"label": ["83"], "surname": ["Sabichi", "Subbarayan", "Llansa", "Lippman", "Menter"], "given-names": ["AL", "V", "N", "SM", "DG"], "article-title": ["Peroxisome proliferator-activated receptor-"], "italic": ["\u03b3", "Cancer Epidemiology Biomarkers & Prevention"], "year": ["2004"], "volume": ["13"], "issue": ["11, part 1"], "fpage": ["1704"], "lpage": ["1709"]}, {"label": ["97"], "surname": ["Lagiou", "Rossi", "Lagiou", "Tzonou", "La Vecchia", "Trichopoulos"], "given-names": ["P", "M", "A", "A", "C", "D"], "article-title": ["Flavonoid intake and liver cancer: a case-control study in Greece"], "italic": ["Cancer Causes & Control"], "year": ["2008"], "fpage": ["1"], "lpage": ["6"]}, {"label": ["114"], "surname": ["Takimoto", "Glover", "Huang"], "given-names": ["CH", "K", "X"], "article-title": ["Phase I pharmacokinetic and pharmacodynamic analysis of unconjugated soy isoflavones administered to individuals with cancer"], "italic": ["Cancer Epidemiology Biomarkers & Prevention"], "year": ["2003"], "volume": ["12"], "issue": ["11, part 1"], "fpage": ["1213"], "lpage": ["1221"]}]
{ "acronym": [], "definition": [] }
130
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2022-01-13 03:12:57
PPAR Res. 2008 Sep 3; 2008:436489
oa_package/8f/54/PMC2528242.tar.gz
PMC2528254
18802455
[ "<title>Introduction</title>", "<p>Blood-vessel development is essential for myriad biological phenomena in healthy and diseased individuals, including wound healing and tumor growth ##REF##11001068##[1]##,##REF##16355210##[2]##. Blood vessels form either de novo, via <italic>vasculogenesis</italic> or by sprouting or splitting of existing blood vessels via <italic>angiogenesis</italic>.</p>", "<p>In vasculogenesis, dispersed <italic>endothelial cells</italic> (<italic>ECs</italic>; the cells lining the inner walls of fully-formed blood vessels) organize into a primary vascular plexus of solid cords which then remodel into a vascular network. ECs elongate parallel to the cords, with final aspect ratios of tens to one. Because the early stages of vasculogenesis depend on a single cell type, vasculogenesis is relatively easy to reproduce in vitro. When cultured in vitro on <italic>Matrigel</italic>, a commercial product mimicking the extracellular matrix (<italic>ECM</italic>; the mixture of proteins, growth-factors and carbohydrates surrounding cells in vivo), even in the absence of other cell types or positional cues, ECs organize into cords which form large-scale, honey-comb like patterns, with cords of ECs surrounding regions devoid of ECs. This network slowly reorganizes, with the size of the polygonal, cell-free <italic>lacunae</italic>, gradually increasing. This observation suggests that ECs have <italic>autonomous</italic> patterning ability, rather than following morphogen pre-patterns.</p>", "<p>The sprouting or splitting of existing blood vessels during <italic>angiogenesis</italic> is more complex. In the first step of angiogenesis, a vessel dilates and releases plasma proteins that induce a series of changes in EC behavior. The ECs which will form the sprout next detach from each other and from the surrounding smooth-muscle cells, destabilizing the vessel. These detached ECs proliferate, migrate out of the vessel and organize into a sprout. EC proliferation continues in the sprout and is fastest just behind the leading <italic>tip cell</italic>, which is selected using a lateral-inhibition mechanism mediated by Dll4 and Notch1 ##REF##17259973##[3]##. Finally, the sprout forms a lumen, secretes a basal lamina and associates with pericytes that stabilize the sprout to form a mature new vessel ##REF##10742145##[4]##.</p>", "<p>Two fundamental questions concerning vasculogenesis and angiogenesis and their relation to each other are: 1) Does blood-vessel formation require external patterning cues (<italic>pre-patterns</italic> of morphogens) to define the precise position of the ECs, or can ECs organize into vascular patterns autonomously, with external cues merely initiating and fine-tuning vascular morphogenesis? 2) Do vasculogenesis and angiogenesis require the same or different cell behaviors, molecular signals and biomechanics?</p>", "<title>Experimental Background</title>", "<p>Despite the biomedical importance of angiogenesis and vasculogenesis, existing experiments are sufficiently ambiguous that even the fundamental mechanisms guiding patterning are uncertain. Experiments suggest a central role for chemotaxis in both de novo and sprouting blood-vessel growth ##REF##10742145##[4]##–##REF##16355211##[6]##. ECs respond to, and often produce, a wide range of chemoattractants and chemorepellants, including the many isoforms of vascular-endothelial growth factor A (<italic>VEGF-A</italic>) ##REF##16355211##[6]##, the chemokine SDF-1 ##REF##11929756##[7]##,##REF##16840724##[8]##, which ECs secrete ##REF##11929756##[7]##, fibroblast growth factor 2 (<italic>FGF-2</italic>), which induces ECs in developing vessels to secrete VEGF ##REF##9647657##[9]##, Slit-2, which can act either as a chemoattractant or a chemorepellant depending on the receptor to which it binds ##REF##15664389##[10]##, and the chemorepelling semaphorins ##REF##15664389##[10]##.</p>", "<p>Which of these molecules (if any) govern vascular patterning is still unclear. The Torino Group (e.g., ##REF##12682010##[11]##,##REF##12688968##[12]##) argued that a VEGF-A was the short-range autocrine chemoattractant that their chemotaxis-based blood-vessel-growth model required, since ECs express receptors for VEGF (<italic>VEGFR-2</italic>), chemotax towards sources of VEGF under favorable conditions, and secrete VEGFs. However, experiments suggest that cell-autonomous secretion of VEGF is essential only for vascular maintenance, not for angiogenesis <italic>per se</italic>: mice genetically-engineered to lack the VEGF gene only in their ECs have normal vascular density and patterning, but impaired vascular homeostasis and EC survival ##REF##17719546##[13]##. A plausible, alternative cell-autonomous chemoattractant to guide EC aggregation is the chemokine SDF-1/CXCL12, which ECs both secrete and respond to ##REF##16840724##[8]##.</p>", "<p>However, based on experiments that suggest that ECs can follow stresses in the ECM (see, e.g., ##REF##7573362##[14]## for review), Manoussaki and Murray ##REF##8953213##[15]##, and Namy et al. ##REF##14969709##[16]## proposed that mechanical interactions rather than, or in addition to, chemical interactions govern vasculogenesis. Further complicating this picture, Szabo and coworkers ##REF##17358734##[17]## showed that non-vascular, glia or muscle cells cultured on rigid, plastic culture dishes in continuously-shaken medium can form linear structures. Such culture conditions should reduce both the formation of chemoattractant gradients or migration along stress lines in the ECM. In the absence of ECM, they hypothesized that cells preferentially move towards elongated structures. Szabo and coworkers ##REF##17358734##[17]## proposed two mechanisms for such cell behavior: cells would align to surrounding cells, or they would mechanotactically follow stress fields in the cytoskeleton of neighboring cells. The molecular mechanisms of such cell behavior remains unclear as is the relevance of these results to ECs.</p>", "<p>Angiogenesis and vasculogenesis also require a number of local, contact-dependent (<italic>juxtracrine</italic>) signals: Tip-cell selection during angiogenic sprouting depends on Delta-notch signaling ##REF##17259973##[3]##, while Eph receptor-ephrin ligand binding amplifies ECs' response to SDF-1 ##REF##16840724##[8]##. All ECs express vascular-endothelial-cadherin (<italic>VE-cadherin</italic>), a homophilic, trans-membrane cell-adhesion molecule, which appears to play a crucial role in vascular patterning ##REF##10207135##[18]##,##REF##18062955##[19]##. Besides its role in cell-cell adhesion, VE-cadherin has a signaling function that determines how ECs respond to VEGF-A. When ECs bind to other ECs through their VE-cadherin, VEGF-A reduces their motility and proliferation. In the absence of VE-cadherin binding, VEGF-A activates pathways related to actin polymerization and the cell cycle, enhancing cell motility and proliferation in sub-confluent monolayers, and causes preferential extension of pseudopods in directions with higher VEGF-A concentrations ##REF##15071551##[20]##. We hypothesize that VE-cadherin-binding acts locally to prevent extension of pseudopods in the direction of cell-cell contacts for all critical chemoattractants, not only to VEGF-A. VE-cadherin −/− double-knock-out mice develop abnormal vascular networks in the yolk sac ##REF##10207135##[18]##, with ECs forming isolated vascular islands instead of wild-type polygonal vascular networks. These mice also have defective angiogenic sprouting, suggesting that both vasculogenesis and angiogenesis require VE-cadherin. VE cadherin −/− ECs still form strong adhesive junctions, so loss of VE-cadherin-mediated signaling rather than loss of intercellular adhesion seems to be responsible for the knock-out phenotype ##REF##10207135##[18]##.</p>", "<title>Computational Background</title>", "<p>A number of models and simulations replicate features of in vitro vascular patterning and can help partially reconstruct minimal sets of behaviors ECs require to self-organize into polygonal, vascular patterns ##REF##12682010##[11]##, ##REF##12688968##[12]##, ##REF##8953213##[15]##–##REF##17358734##[17]##, ##UREF##1##[21]##–##REF##19526066##[23]##. Because of the experiments we discussed above, and others which have demonstrated that sprouting angiogenesis and vasculogenesis both require chemotaxis (see, e.g., ##REF##11929756##[7]##,##REF##16840724##[8]##,##REF##12810700##[24]##), most models of vasculogenesis assume that intercellular signaling occurs via a diffusible chemoattractant. Using continuum models deriving from the fluid-dynamic Burgers' equation, Preziosi and coworkers (called the <italic>Torino Group</italic> in this paper) showed that simulated ECs secreting a chemoattractant that attracts surrounding ECs, could self-organize into polygonal patterns similar to the patterns in EC cultures and in vivo ##REF##12682010##[11]##,##REF##12688968##[12]##,##UREF##2##[25]##,##UREF##3##[26]##. However, their work assumed that endothelial cells accelerate in chemical gradients, which is not plausible in the highly viscous, non-inertial environment of the ECM. Microfluidic evidence indicates that mammalian cells (HL60) rapidly reach a flow-dependent, constant velocity ##REF##15915253##[27]## in chemoattractant gradients rather than continuously accelerating. We have previously suggested that ##REF##16325173##[22]## a linear force-velocity relation is the most appropriate model of ECs' experimental response, with the velocity of ECs proportional to the strength of the gradient of the chemoattractant. However, in simulations of this simple model, isotropic ECs form well-separated rounded clusters instead of networks. We have shown that adding one of a number of mechanisms (including cell adhesion ##UREF##1##[21]## and cell elongation ##REF##16325173##[22]##) to chemotactic aggregation suffices to produce quasi-polygonal networks. Section “<xref ref-type=\"sec\" rid=\"s2\">Results</xref>” discusses these mechanisms in more detail.</p>", "<p>In the mechanical models of Manoussaki and Murray ##REF##8953213##[15]##, and Namy et al. ##REF##14969709##[16]## ECs pull on the elastic ECM and aggregate by <italic>haptotactically</italic> migrating along the resulting ECM stress lines. Surprisingly, the mathematical form of the chemical and mechanical models is practically identical. Because these mechanical models assume that ECs exert radially-symmetric stresses on the ECM, modeling stress fields and EC haptotaxis or EC secretion and response to a chemoattractant, results in the same cell movement. Since simulations of the two mechanisms are identical, distinguishing between the effects of chemical and mechanical mechanisms will require additional experiments (such experiments are currently underway in the Glazier laboratory (Shirinifard, Alileche and Glazier, <italic>preprint, 2008</italic>)).</p>", "<p>A separate set of simulations addresses angiogenesis. Many models of sprouting blood-vessel growth introduce blood-vessel-level phenomenology by hand through high-level rules for branching ##UREF##4##[28]##–##UREF##5##[30]##. Attempts to derive blood-vessel sprouting and splitting from the underlying behavior of ECs include Levine and coworkers' ##REF##11315313##[31]## model of the onset of angiogenic sprouting as a reinforced random walk, where the ECs degrade the ECM, which locally enhances EC motility and produces paths of degraded ECM, and Bauer and Jiang's ##REF##17277180##[32]## cell-based model of blood-vessel sprouting along externally generated morphogen gradients, which assumed that branch splitting results from ECM inhomogeneities. Neither model can explain both EC assembly and blood-vessel sprouting.</p>", "<p>Could the behavior of the individual ECs also explain aspects of blood-vessel sprouting? Because the same genetic machinery regulates both angiogenesis and vasculogenesis ##REF##10742145##[4]##, a common set of mechanisms is plausible. Manoussaki ##UREF##6##[33]## extended her mechanical model of vasculogenesis to describe angiogenesis by adding long-range, chemotactic guidance cues. In her simulations, ECs migrated from an aggregate towards a chemoattractant source and cell-traction-driven migration contracted the sprout into a narrow, vessel-like cord.</p>", "<p>In this paper we present an alternative chemotaxis-based mechanism that can produce networks both from dispersed ECs and EC clusters without requiring long-range guidance cues. Instead, in our model long-range signals would only steer the self-organized vessels, a more biologically-realistic mechanism. Extending simulations that we have briefly introduced elsewhere ##REF##19526066##[23]##, we show that VE-cadherin-mediated contact inhibition of chemotactic pseudopod projections, in combination with secretion of a diffusing, rapidly decaying chemoattractant by ECs, suffices to reproduce aspects of both de novo and sprouting blood-vessel growth. In our simulations ECs: a) secrete a chemoattractant and b) preferentially extend pseudopods up gradients of the chemoattractant, unless, c) contact inhibition locally prevents chemotactic pseudopod extension. Thus, cell-cell binding suppresses the extension of chemotactic pseudopods, while unbound cell surfaces in contact with the ECM continue to extend pseudopods towards sources of chemoattractant ##REF##12810700##[24]##. We compare two biologically-plausible scenarios for chemotaxis, one in which ECs actively extend and retract pseudopods along chemoattractant gradients, and one in which the pseudopods' retractions are chemotactically neutral. The second scenario suggests a sprouting mechanism where a secreted autocrine factor acts both as a long-range chemoattractant and a local inhibitor of pseudopod sprouting.</p>" ]
[ "<title>Materials and Methods</title>", "<title>The Glazier–Graner–Hogeweg (GGH) Model</title>", "<p>The GGH represents biological cells as patches of identical lattice indices on a square or triangular lattice, where each index uniquely identifies, or labels a single biological cell. Connections (<italic>links</italic>) between neighboring lattice sites of unlike index represent bonds between apposing cell membranes, where the <italic>bond energy</italic> is , assuming that the types and numbers of adhesive cell-surface proteins determine <italic>J</italic>. A penalty increasing with the cell's deviation from a designated target volume <italic>A</italic>\n<sub>target</sub>(<italic>σ</italic>) imposes a <italic>volume constraint</italic> on the simulated ECs. We define the pattern's <italic>effective energy</italic>:where and are neighboring lattice sites (up to fourth-order neighbors), <italic>a</italic> is the current area of cell <italic>σ</italic>, <italic>A</italic>\n<sub>target</sub>(<italic>σ</italic>) is its target area, <italic>λ</italic> represents a cell's resistance to compression, and the Kronecker delta is <italic>δ</italic>(<italic>x</italic>,<italic>y</italic>) = {1,<italic>x</italic> = <italic>y</italic>; 0,<italic>x</italic>≠<italic>y</italic>. Each lattice site represents an area of 2 µm×2 µm. Since we assume that ECs do not divide or grow during patterning, we set <italic>A</italic>\n<sub>target</sub>(<italic>σ</italic>) = 50 lattice sites, corresponding to a cell diameter of about 16 µm, and <italic>λ</italic> = 25 for all cells. The ECs reside in a very thin layer of extracellular fluid, which is a generalized cell without a volume constraint and with <italic>σ</italic> = 0. We assume that the ECs and fluid sit on top of a rigid ECM through which the chemoattractant diffuses, but we do not represent this ECM in the GGH lattice. We also assume that the presence of the fluid does not disturb the chemoattractant distribution in the ECM. Unless we specify otherwise, we use a bond energy <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 40 between the ECs, and <italic>J</italic>(<italic>c</italic>,<italic>M</italic>) = 20 between the ECs and the ECM. For these settings the ECs do not adhere without chemotaxis. We define a special, high <italic>cell-border energy J</italic>(<italic>c</italic>,B) = 100 to prevent ECs from adhering to the lattice boundaries. We use fixed boundary conditions.</p>", "<p>To mimic cytoskeletally-driven pseudopod extensions and retractions, we randomly choose a source lattice site , and attempt to copy its index into a randomly-chosen neighboring lattice site . For better isotropy we select the source site from the twenty, first- to fourth-nearest neighbors ##REF##9905332##[46]##. During a <italic>Monte Carlo Step</italic> (<italic>MCS</italic>) we carry out <italic>N copy attempts</italic>, with <italic>N</italic> the number of sites in the lattice. We set the experimental time per MCS to 30 s; for this setting the simulated ECs move with nearly their experimental velocity ##REF##16325173##[22]##. We calculate how much the effective energy would change if we performed the copy, and accept the attempt with probability , where <italic>T</italic> defines the <italic>intrinsic cell motility</italic>. All our simulations, except those in ##FIG##10##Figures 11##–\n##FIG##12##13##, use <italic>T</italic> = 50.</p>", "<p>In experiments, cells respond to chemoattractant gradients by executing a more-or-less-strongly biased random walk up or down the gradient, where, over times short enough to allow us to neglect adaptation, the velocity of the drift depends on the gradient strength and the absolute concentration. We therefore define a set of extensions to the basic GGH model which reproduce these empirical behaviors due to preferential extension and retraction of pseudopods up chemoattractant gradients ##REF##12810700##[24]## by including a <italic>chemical effective-energy change</italic> at each copy attempt ##UREF##1##[21]##,##UREF##8##[36]##,where <italic>c</italic> is the concentration of the chemoattractant, which we assume is present everywhere in a layer of ECM <italic>under</italic> the ECs, is the target site, the source site, and <italic>s</italic> regulates the saturation of the chemotactic response. Unless we specify otherwise, we set <italic>s</italic> = 0, in which case chemotaxis depends linearly on the chemoattractant gradient only, independent of the chemoattractant concentration. Δ<italic>H</italic>\n<sub>chemotaxis</sub> → 0 for large values of <italic>s</italic> and, for <italic>s</italic>≠0, for high chemical concentrations. The <italic>chemotaxis coefficient</italic> is <italic>μ</italic> = <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) at cell-ECM interfaces and <italic>μ</italic> = <italic>Χ</italic>(<italic>c</italic>,<italic>c</italic>) at cell-cell interfaces respectively. Setting <italic>Χ</italic>(<italic>c</italic>,<italic>c</italic>) = 0 <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 500 ensures that chemotactic extensions occur only at cell-ECM interfaces, reflecting VE-cadherin's suppression of pseudopods. Both extending and retracting pseudopods contribute to the chemical effective-energy change. To implement <italic>pseudopod-extension-only chemotaxis</italic> (see ##FIG##10##Figures 11##–\n##FIG##12##13##), where only <italic>extending</italic> pseudopods at the cell-ECM interface respond to the chemoattractant, cells experience a chemical effective-energy change only if the source lattice site belongs to an EC, i.e.,\n</p>", "<p>For a more detailed discussion of chemotaxis in the GGH model see ##UREF##11##[47]##. We solve the partial-differential equation for chemoattractant diffusion and degradation (Eq. 1) numerically using a finite-difference scheme on a lattice matching the GGH lattice. We use 15 diffusion steps per MCS, with Δ<italic>t</italic> = 2 s. For these parameters, the chemoattractant diffuses more rapidly than the ECs, enabling us to ignore advection in the medium as the cells push the fluid.</p>", "<p>Source code and parameters for the simulations in this paper are available online in ##SUPPL##1##Protocol S1## from the supporting material, and from <ext-link ext-link-type=\"uri\" xlink:href=\"http://sourceforge.net/projects/tst\">http://sourceforge.net/projects/tst</ext-link>. Parameter files for the simulations in this paper are included in ##SUPPL##0##Dataset S1##.</p>", "<title>Allantois Culture and Immunolabeling</title>", "<p>We dissected allantoides from mouse embryos at embryonic stages 7.5–8.0. We washed the explants in fresh, cold ePBS and pipetted them into fibronectin-coated (5 mg/ml) Delta-T culture dishes (Bioptechs, Butler, PA) containing high-glucose, phenol-red-free Dulbecco's modified Eagles' medium (<italic>DMEM</italic>) supplemented with 10% fetal bovine serum (<italic>FBS</italic>), 1% penicillin-streptomycin, and 1% L-glutamine (GibcoBRL, Grand Island, NY). We maintained the allantoic explants using standard culture conditions (37°C and 5% CO<sub>2</sub>/95% air atmosphere) in a custom-designed culture chamber for 12–24 hours in the presence of an endothelial-specific marker, CD34 monoclonal antibody (BD PharMingen, San Diego, CA) directly conjugated to Cy3 (Amersham Biosciences). We fixed the allantoides in 3% paraformaldehyde for 20 minutes at room temperature, followed by an ePBS wash. For VE-cadherin antibody perturbations, we added anti-VE-cadherin monoclonal antibody (BD PharMingen, San Diego, CA) at 25 µg/ml to the culture medium.</p>", "<title>Image Acquisition</title>", "<p>We observed the cultures with a 10× objective (0.30 N.A.) on an inverted, automated, wide-field, epifluorescence/differential-interference-contrast (<italic>DIC</italic>) microscope (Leica DMIRE2, Leica Microsystems, Germany). We recorded images (608×512 pixel spatial and 12-bit intensity resolution) with a cooled Retiga 1300 camera (QImaging, Burnaby, British Columbia) in 2×2 binned acquisition mode, using 100–300 ms exposures. Image acquisition and microscope settings used software described in ##REF##12067365##[44]##.</p>" ]
[ "<title>Results</title>", "<p>We modeled endothelial cell behavior at a mesoscopic level using the Glazier-Graner-Hogeweg (GGH) model, also known as the Cellular Potts Model (CPM) ##REF##10046374##[34]##–##UREF##9##[37]##. The GGH is a lattice-based Monte-Carlo approach that describes biological cells as spatially extended patches of identical lattice indices. Intercellular junctions and cell junctions to the ECM determine adhesive (or binding) energies. The GGH algorithm, which we describe in more detail in the section <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>, models pseudopod protrusions by iteratively displacing cell interfaces, with a preference for displacements which reduce the local <italic>effective energy</italic> of the configuration. Cells reorganize to favor stronger rather than weaker cell-cell and cell-ECM bonds and shorter rather than longer cell boundaries. In addition to interface displacements that reduce the effective energy, active cell motility also allows displacements that increase the effective energy. The likelihood of these active displacements increases with the <italic>cell-motility</italic> parameter <italic>T</italic>. Further constraints regulate cell volumes, surface areas, and chemotaxis. To model chemotaxis, we use the Savill and Hogeweg ##UREF##8##[36]## algorithm that favors extensions and retractions of pseudopods up concentration gradients of a chemoattractant (see Eq. 3 in the section <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>). In the simplest implementation of chemotaxis in the GGH, cell velocity is proportional to the strength of the chemical gradient, in general agreement with experiments; see, e.g., ##REF##16325173##[22]## (we discuss the details of chemotaxis implementation below in the subsections Sensitivity analysis and A dissipative sprouting mechanism and in <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>; see especially Eq. 3).</p>", "<p>The advantage of the GGH over alternative cell-based modeling approaches ##UREF##10##[38]## that represent cells as point particles or fixed-sized spheres or ellipsoids is that we can differentiate between bound and unbound regions of cell membrane. The GGH naturally represents the stochastic, exploratory behavior of migrating cells, modeling it as the biased extension and retraction of pseudopods, instead of a biologically-implausible single force acting on cells' centers of mass as in some cell-based simulations.</p>", "<p>We described chemoattractant diffusion and degradation macroscopically, using a continuum approximation. In analogy to the Torino Group's continuum model of de novo blood-vessel growth ##REF##12688968##[12]##,##UREF##2##[25]##, ECs secrete a diffusing chemoattractant at a rate <italic>α</italic>, which degrades in the ECM at a rate <italic>ε</italic> (e.g., due to proteolytic enzymes or by binding to ECM components), obeying:where inside cells and is in the ECM. Because we wish to compare our simulations to experimental yolk-sac cultures, where the vascular patterns are essentially monolayers, we use a two-dimensional GGH.</p>", "<p>We set the chemoattractant's secretion rate by cells <italic>α</italic> = 10<sup>−3</sup>\n<italic>s</italic>\n<sup>−1</sup>, its decay rate <italic>ε</italic> = <italic>α</italic>, and its diffusion constant in ECM to a slow <italic>D</italic> = 10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>. These parameter values produce steeper gradients than those for VEGF-A<sub>165</sub>, the chemoattractant which Gamba et al. suggested was responsible for vasculogenesis, which has a diffusion coefficient of <italic>D</italic>∼10<sup>−11</sup> m<sup>2</sup> s<sup>−1</sup>\n##REF##12688968##[12]##. The diffusion coefficient of SDF-1/CXCL12 is in the range of 1.7×10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>\n##REF##17066171##[39]##. However, the phenomena we observe in our simulations hold over a large range of diffusion coefficients.</p>", "<title>EC Aggregation and Vasculogenesis in the Absence of Contact Inhibition</title>", "<p>In in vitro cultures of mouse allantois explants, ECs (fluorescently labeled in red) organized into polygonal patterns (##FIG##0##Figure 1A–1C##). When we blocked VE-cadherin receptors with anti-VE-cadherin antibodies, thus preventing VE-cadherin receptors from binding to those on apposing cells, the mouse ECs formed isolated vascular islands (##FIG##0##Figure 1D–1F##). We hypothesize that anti-VE-cadherin's antibody blockage of VE-cadherin signaling prevents contact inhibition of chemotactic motility, sensitizing the endothelial cells to the chemoattractant at cell–cell interfaces.</p>", "<p>In our corresponding simulations (##FIG##1##Figure 2A–2C## and ##SUPPL##4##Video S1##), we randomly distributed 1,000 ECs, each with an area of ∼200 µm<sup>2</sup> over an area of ≈700 µm×700 µm (333×333 lattice sites, or <italic>pixels</italic>, of 2 µm×2 µm each), which we positioned inside a larger lattice of 1,00 µm×1,00 µm to minimize boundary effects. In this cell-based simulation of the Torino Group's continuum model ##REF##12682010##[11]##,##REF##12688968##[12]##, without endothelial-cell acceleration in chemoattractant gradients our cells form disconnected, vascular islands rather than a vascular network. We would expect this result, because with the more realistic chemotactic response we employ, the Torino Group's model reduces to the classic Keller-Segel equations ##REF##5462335##[40]## of chemotactic aggregation ##UREF##2##[25]##, which, like our simulations, form isolated vascular islands. Apparently, the basic Torino-Group model of chemotactic cell aggregation misses a biological mechanism essential for vasculogenesis. We have previously suggested a number of additional mechanisms, any one of which, together with cell aggregation, suffices to induce vasculogenesis-like patterning. For example, when we gave the ECs the elongated shapes observed in later stages of experiments, neighboring cells aligned with each other, causing cell clusters to elongate and interconnect, creating a vascular network, in a mechanism similar to Szabo's ##REF##17358734##[17]##. These vascular networks remodel gradually, with dynamics resembling those of in vitro vascular networks. The causes of cell elongation in experiments are not clear. ECs could elongate either cell-autonomously (e.g., by remodeling their cytoskeletons), or non-cell-autonomously, by maximizing their contact areas with surrounding cells or by aligning to morphogen gradients in the ECM ##REF##16325173##[22]##. Unless we state otherwise, in this paper we neglect cell-autonomous elongation.</p>", "<p>Even without strong cell-cell adhesion the ECs can form vascular-like structures in simulations of vasculogenesis if the <italic>diffusion length</italic> of the chemoattractant (the length <italic>L</italic> over which the concentration drops to half its value at the EC membrane) is short enough, because the ECs align with the chemical gradients ##REF##19526066##[23]##. This length scale <italic>L</italic> depends on the diffusion coefficient <italic>D</italic> and the chemoattractant decay rate <italic>ε</italic> as \n##REF##12688968##[12]##.</p>", "<title>Sprouting Angiogenesis in the Absence of Contact-Inhibition</title>", "<p>To investigate whether the Torino-Group Model could reproduce sprouting angiogenesis, we started our simulations with rounded clusters of simulated ECs representing a blood vessel's surface after degradation of the ECM, keeping the simulation parameters unchanged from ##FIG##1##Figure 2##. As in vasculogenesis, cell-elongation sufficed to drive angiogenesis-like sprouting (see ##FIG##2##Figure 3A–3C##), where we used a length constraint, see ##REF##16325173##[22]##). EC clusters also produced sprouts for strong cell-cell adhesion (i.e., for values of <italic>J</italic>(<italic>c</italic>,<italic>c</italic>)&lt;10); ##FIG##2##Figure 3D–3F##), via a mechanism similar to the cell-elongation-dependent mechanism for vasculogenesis ##REF##16325173##[22]##. Adhesion-independent sprouting occurred only for a narrow range of very small diffusion constants of the chemoattractant, between <italic>D</italic>&lt;2·10<sup>−14</sup> m<sup>2</sup> s<sup>−1</sup> and <italic>D</italic>&gt;2·10<sup>−14</sup> m<sup>2</sup> s<sup>−</sup> (see ##FIG##2##Figure 3G–3I##). The allowable range of <italic>D</italic> increased for bigger cells ##REF##19526066##[23]##. We also systematically screened for sprouting in the absence of contact-inhibited chemotaxis. We present the results of these screens in the section Sensitivity analysis and in ##SUPPL##2##Figure S1##, but we defer an in-depth study of these phenomena to our future work.</p>", "<title>Contact-Inhibited Chemotaxis in De Novo Blood Vessel Growth</title>", "<p>In this paper, we focus on the role of contact-inhibited chemotaxis in sprouting blood-vessel growth. We hypothesize that VE-cadherin's local inhibition of chemotaxis-induced pseudopod extensions at EC-EC boundaries, may be responsible for ECs' self-organization into vascular-like networks.</p>", "<p>We modeled contact inhibition of chemotaxis in our simulations by suppressing chemotaxis at cell-cell interfaces. Thus, only interfaces between cells and ECM respond to the chemoattractant. ##FIG##1##Figure 2D## and ##SUPPL##5##Video S2## and ##SUPPL##6##Video S3## show typical simulations of de novo blood-vessel growth with contact inhibition. The ECs assemble into a structure resembling a capillary plexus: cords of cells enclose lacunae, which grow slowly. Smaller lacunae shrink and disappear, while larger lacunae subdivide via vessel sprouting as, for example, in the quail yolk sac ##REF##9109485##[41]##.</p>", "<title>Contact-Inhibited Chemotaxis in Blood Vessel Sprouting</title>", "<p>To investigate the role of contact-inhibited chemotaxis in blood vessel sprouting, we ran a set of simulations with a large cluster of endothelial cells representing a blood vessel's surface after degradation of the ECM, keeping all simulation parameters the same as those in ##FIG##1##Figure 2D##. The surface of the cluster first roughens, with some cells protruding from the surface, then digitates into a structure reminiscent of a primary vascular plexus (##FIG##3##Figure 4A–4C## and ##SUPPL##7##Video S4## and ##SUPPL##8##Video S5##), the first type of structure to develop in both de novo and sprouting blood-vessel growth ##REF##9109485##[41]##. The sprouting instability requires contact inhibition of chemotaxis. Without it, the clusters remained rounded and compact (##FIG##3##Figure 4D##). Thus our simulations suggest that a process operating at the level of individual cells—chemotaxis with contact inhibition—may drive in vitro blood-vessel growth both sprouting and de novo.</p>", "<p>What drives blood vessel sprouting in our model? At equilibrium, the chemoattractant has a quasi-Gaussian profile across the cluster. It levels off towards the cluster's center, while its inflection point is at the cluster boundary. Chemotaxis produces a continuous, inward, normal force at the cluster boundary, creating a buckling instability (see, e.g., ##REF##10990656##[42]##); chemotactic forces also compress small initial bumps laterally, producing sprouts. Since contact inhibition of chemotaxis leaves the interior cells insensitive to the chemoattractant, ingressing surface cells easily push them aside. When we omit contact inhibition of motility to mimic anti-VE-cadherin-antibody-treated allantois cultures, the interior cells also feel the inward-directed chemotactic forces and resist displacement (##FIG##3##Figure 4D## and ##SUPPL##9##Video S6##).</p>", "<p>To explore this idea, we varied the ratio of the chemotactic response at cell–cell interfaces relative to the chemotactic response at cell-ECM interfaces (<italic>Χ</italic>(<italic>c</italic>,<italic>c</italic>)/<italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>)), where <italic>Χ</italic>(<italic>c</italic>,<italic>c</italic>)is the ECs' sensitivity to the chemoattractant at cell-cell interfaces and <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) the sensitivity at cell-ECM interfaces (see the section <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref> for details). We looked for sprouting in clusters of 128 cells, each of area ∼200 µm<sup>2</sup>, placed in a 400 µm×400 µm lattice, keeping all other parameters unchanged from their values in ##FIG##3##Figure 4##.</p>", "<p>We defined the clusters' compactness after 10,000 Monte Carlo Steps (the time unit of the simulation, see the section <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>, with 1 MCS equivalent to about 30 s) to be <italic>C</italic> = <italic>A</italic>\n<sub>cluster</sub>/<italic>A</italic>\n<sub>hull</sub>, the ratio between the cluster's area, <italic>A</italic>\n<sub>cluster</sub>, and the area of its convex hull (that is the tightest possible “gift wrapping” around the cluster), <italic>A</italic>\n<sub>hull</sub>. The compactness <italic>C</italic> = 1 for a perfectly circular cluster, whereas <italic>C</italic> → 0 for highly branched or dispersed clusters of cells.</p>", "<p>We found a phase transition at (<italic>Χ</italic>(<italic>c</italic>,<italic>c</italic>)/<italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>))≈0.5 separating sprouting from non-sprouting clusters (##FIG##4##Figure 5##), suggesting that the sprouting instability only occurs when the core of the cluster behaves as a fluid: because each cell's volume is nearly conserved (apart from small fluctuations around its target volume), the core cells can only release the pressure the ingressing cells exert on them by moving outwards as sprouts. Our ongoing work characterizes this instability mathematically, proving that the cluster self-organizes into a network structure with fixed cord width (A. Shirinifard and J. A. Glazier, preprint 2008).</p>", "<p>To validate our model against published EC tracking experiments ##REF##18062955##[19]##, we compared the trajectories of cells in sprouting and non-sprouting clusters. ##FIG##5##Figure 6A–6D## shows the trajectories of ten cells in a sprouting cluster (with contact-inhibition; ##FIG##5##Figure 6A–6B##), and ten cells in a non-sprouting cluster (without contact-inhibition; ##FIG##5##Figure 6C–6D##). In non-sprouting clusters, cells followed random-walk trajectories, while in sprouting clusters, they followed biased random-walk trajectories. To further characterize cell motility, we measured cells' average displacements and velocities over 10 independent simulations of 128 cells each. In sprouting clusters, the cells moved further during a given interval than in non-sprouting clusters. Thus, the cell velocity \n##REF##18062955##[19]## is larger during sprouting if the interval Δ<italic>t</italic> between subsequent cell positions is sufficiently large (here we use Δ<italic>t</italic> = 2.5 h as in Perryn et al. ##REF##18062955##[19]##); for shorter intervals (e.g., 30 s) the cell velocity is highest in non-sprouting clusters (not shown), indicating that ECs in sprouting clusters moved faster, but had a somewhat slower random motility.</p>", "<p>Our simulations agree with recent experiments tracking ECs in embryonic mouse allantoides ##REF##18062955##[19]## that measured the cell-autonomous motility of ECs cells in allantoides relative to the motility of the surrounding mesothelium in which the ECs reside. Administration of anti-VE-cadherin antibodies reduced both cell-autonomous motion and net displacement of ECs. Thus, our simulations suggest that VE-cadherin's role as a contact-dependent inhibitor of cell motility suffices to explain the reduced cell motility observed in anti-VE-cadherin-treated allantoides cultures.</p>", "<title>Sensitivity Analysis</title>", "<p>Contact-inhibited sprouting occurs for a wide range of parameter values. In most of our simulations we set the EC-EC adhesion equal to the EC-ECM adhesion (i.e., <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = <italic>2J</italic>(<italic>c</italic>,<italic>M</italic>); the factor of 2 arises because we model the ECM as a single large generalized cell), which is equivalent to setting the surface tension of the cluster to zero ##UREF##7##[35]##. Zero surface tension clarifies the role of contact inhibition in sprouting, but real ECs adhere strongly to each other via <italic>adherens junctions</italic>\n##REF##10207135##[18]##. In ##FIG##6##Figure 7## and in ##SUPPL##10##Video S7##, ##SUPPL##11##S8##, ##SUPPL##12##S9##, ##SUPPL##13##S10##, ##SUPPL##14##S11##, ##SUPPL##15##S12##, ##SUPPL##16##S13##, ##SUPPL##17##S14##, ##SUPPL##18##S15##, ##SUPPL##19##S16##, ##SUPPL##20##S17##, ##SUPPL##21##S18##, ##SUPPL##22##S19##, ##SUPPL##23##S20##, ##SUPPL##24##S21##, ##SUPPL##25##S22##, we studied the effect of cell-cell adhesion on sprouting in clusters of 128 cells (256 cells in the Videos). For stronger EC-EC adhesion, equivalent to positive surface tension, <italic>J</italic>(<italic>c</italic>,<italic>c</italic>)&lt;<italic>2J</italic>(<italic>c</italic>,<italic>M</italic>), the sprouts are longer and thinner and the network less compact than for zero surface tension. For very weak EC-EC adhesion <italic>J</italic>(<italic>c</italic>,<italic>c</italic>)≫2<italic>J</italic>(<italic>c</italic>,<italic>M</italic>), equivalent to strong negative surface tension, the ECs separate from each other, so contact-inhibition no longer occurs, and the clusters do not sprout. For small negative surface tensions, with values of <italic>J</italic>(<italic>c</italic>,<italic>c</italic>)&gt;2<italic>J</italic>(<italic>c</italic>,<italic>M</italic>), chemotaxis overcomes the negative surface tension, so ECs still touch each other and sprouting occurs as for zero surface tension, producing thickened sprouts and elongated clusters. The insets to ##FIG##6##Figure 7## and ##SUPPL##23##Video S20##, ##SUPPL##24##S21##, ##SUPPL##25##S22## show the results for 50≤<italic>J</italic>(<italic>c</italic>,<italic>c</italic>)≤70.</p>", "<p>We also investigated how sprouting depends on the chemotactic strength <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) (##FIG##7##Figure 8## and ##SUPPL##26##Video S23##, ##SUPPL##27##S24##, ##SUPPL##28##S25##, ##SUPPL##29##S26##, ##SUPPL##30##S27##\n##SUPPL##31##S28##, ##SUPPL##32##S29##, ##SUPPL##33##S30##, ##SUPPL##34##S31##, ##SUPPL##35##S32##, ##SUPPL##36##S33##). For <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 500, most vascular cords are two cells wide (##SUPPL##27##Video S24##), while for For <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>)&gt;500 the cords become thinner and longer, with cords only one cell wide (##SUPPL##28##Videos S25##, ##SUPPL##29##S26##, ##SUPPL##30##S27##, ##SUPPL##31##S28##, ##SUPPL##32##S29##, ##SUPPL##33##S30##, ##SUPPL##34##S31##, ##SUPPL##35##S32##, ##SUPPL##36##S33##). For higher chemotactic forces, the cells intercalate, moving to the chemical gradients' peak. We have derived the conditions for this <italic>folding instability</italic> in our ongoing work (A. Shirinifard and J. A. Glazier, preprint, 2008). Higher chemotactic strengths increase ruffling of the cluster boundary, reducing the cluster's compactness in the absence of contact inhibition (##FIG##7##Figure 8##).</p>", "<p>We assumed that ECs extend or retract pseudopods depending on the difference in chemoattractant concentration between the retracted and extended positions, independent of the absolute chemoattractant concentrations. However, at higher chemoattractant concentrations, most chemoattractant receptors will saturate with chemoattractant and become insensitive to chemoattractant levels. To study the effect of saturated chemotactic response ##UREF##1##[21]## on angiogenic sprouting, we varied the saturation parameter s (see Eq. 3 in <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>) leaving all other parameters unchanged. For <italic>s</italic> = 0, the chemotactic response is linear; for <italic>s</italic>&gt;0, the response to the chemoattractant gradient vanishes at high concentrations (see <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>). For small positive <italic>s</italic>, the clusters sprout normally (see ##FIG##8##Figure 9## and ##SUPPL##37##Videos S34##, ##SUPPL##38##S35##, ##SUPPL##39##S36##); however, for large <italic>s</italic>, the chemotactic response weakens at the chemoattractant levels present at the edge of the cell cluster; thus cells no longer chemotact towards the cluster's interior and the sprouting instability disappears (##SUPPL##40##Videos S37##, ##SUPPL##41##S38##, ##SUPPL##42##S39##). We could test this prediction experimentally by partially inactivating the ECs' chemoattractant receptors. We observed the same effect when we increased the chemoattractant secretion rate for moderate response saturation (<italic>s</italic> = 0.05; see ##SUPPL##3##Figure S2##, bottom panel) leading to higher overall chemoattractant concentrations. We could test this situation experimentally by overexpressing the chemoattractant in ECs. Since for unsaturated chemotactic response (<italic>s</italic> = 0), multiplying the chemoattractant concentrations is equivalent to multiplying the chemotactic strength (<italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>)) by the same factor, increasing the secretion rate first thins and lengthens the cords by increasing the chemotactic strength, then eventually prevents sprouting as the chemotactic response saturates. This effect is most apparent for <italic>s</italic> = 0.01 (##SUPPL##3##Figure S2##, top panel).</p>", "<p>In the Torino Group's continuum model, the separation between the cords increases with the diffusion length <italic>L</italic> of the chemoattractant, ##FIG##9##Figure 10## and ##SUPPL##43##Videos S40##, ##SUPPL##44##S41##, ##SUPPL##45##S42##, ##SUPPL##46##S43##, ##SUPPL##47##S44##, ##SUPPL##48##S45##, ##SUPPL##49##S46## show sprouting clusters for a range of diffusion lengths. In agreement with the Torino Group's model, longer diffusion lengths produce thicker cords with larger intercord spaces. The clusters do not sprout well when <italic>L</italic> approaches the EC-cluster diameter. Clusters consisting of 1,024 cells sprout for <italic>D</italic>&gt;3·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup> (<italic>L</italic>&gt;17.3 µm), while 128-cell clusters do not (##FIG##9##Figure 10## and ##SUPPL##50##Video S47##, ##SUPPL##51##S48##, ##SUPPL##52##S49##, ##SUPPL##53##S50##, ##SUPPL##54##S51##, ##SUPPL##55##S52##, ##SUPPL##56##S53##). If the diffusion length is shorter than the ECs' diameter, the clusters dissociate: the ECs perform random walks with long persistence lengths, moving up the chemoattractant gradients they leave behind themselves (##SUPPL##43##Video S40## and ##SUPPL##50##S47##).</p>", "<title>A Dissipative Sprouting Mechanism</title>", "<p>In our simulations, the trailing edges of the ECs retract actively in response to the chemoattractant and exert an inward-normal, compressive force on the EC cluster. To check if sprouting requires this compressive force, we also simulated a situation in which only <italic>extending</italic> pseudopods at cell-ECM interfaces respond to the chemoattractant, while retraction is chemotactically neutral. Both sprouting-angiogenesis and vasculogenesis occurred, but required higher intrinsic cell motilities (larger values of the parameter <italic>T</italic>). ##FIG##10##Figure 11## shows the motilities required under both assumptions. We looked for sprouting after 5000 MCS (∼40 h) in clusters of 128 cells, each of area ≈200 µm<sup>2</sup>, placed in a 400 µm×400 µm lattice, with all other parameters the same as in ##FIG##3##Figure 4##. For <italic>T</italic>&lt;100, our original chemotaxis assumptions produced sprouts, while no sprouting occurred if pseudopods responded to the chemoattractant only during extension. For 100&lt;<italic>T</italic>&gt;400, both mechanisms produced sprouts. For <italic>T</italic>&gt;400, the ECs broke up into small pieces, a well-characterized, non-biological artifact of the GGH ##UREF##7##[35]##. With extension-only chemotaxis, sprouting was slightly slower than for standard, extension-retraction Savill-Hogeweg ##UREF##8##[36]## chemotaxis, as a plot of the time evolution of the clusters' compactness shows (##FIG##11##Figure 12## and ##SUPPL##57##Video S54##, ##SUPPL##58##S55##, ##SUPPL##59##S56##). However, at long times (<italic>t</italic>&gt;2500 MCS) the compactness of clusters decreased at identical rates for both methods.</p>", "<p>These results suggest an additional mechanism for blood-vessel sprouting: at the cluster surface, <italic>all</italic> pseudopod extensions increase the effective energy slightly, so the chemoattractant <italic>inhibits</italic> pseudopod extension. A recent experimental study ##REF##17038622##[43]## found that autocrine secretion of the sprouting inhibitor TGF-β1 enhances branching in mammary epithelial tubes. Our model suggests a mechanism by which an autocrine, secreted chemical can act <italic>both</italic> as a chemoattractant and as an inhibitor. The rates of pseudopod extensions and retractions are critical to pattern evolution (##FIG##10##Figure 11##). Cells in growing tips see a shallower gradient than do those in valleys between the tips (see, e.g., ##FIG##3##Figure 4B##), so pseudopod extensions at growing tips are more frequent than in the valleys between tips because they have a lower effective-energy cost. During sprouting, conservation of cell area requires that the cells in the valleys must retract, while those in the tips protrude. In the Savill-Hogeweg algorithm, retraction is energetically favorable, while it is energetically neutral in our pseudopod-extension-only chemotaxis algorithm, making the net change in effective energy positive with a rate depending on the cell motility. The effective-energy change is negative in the Savill-Hogeweg algorithm and thus nearly independent of <italic>T</italic> (##FIG##12##Figure 13##, where <italic>H</italic>\n<sub>0</sub> is the initial effective energy).</p>" ]
[ "<title>Discussion</title>", "<p>We have shown that a single set of cell behaviors, i.e., contact-inhibited chemotaxis to an autocrine, secreted chemoattractant can explain aspects of both de novo and sprouting blood-vessel growth. Our results suggest that branching in aggregates of chemotacting ECs could result from two separate effects of the same mechanism. For low cell motilities <italic>T</italic>, i.e., a low probability for active, dissipative cellular protrusion, the branching resembles a buckling instability (see, e.g., ##REF##10990656##[42]##), in which the surface cells exert a surface-normal force on the cluster's inner core. For larger cell motilities, the shallower chemoattractant gradients at protrusions make the ECs there more likely to extend outward-directed pseudopods than cells in the valleys between the protrusions.</p>", "<p>While we have adopted the Torino Group's assumption that ECs chemotax in response to gradients of a diffusible, autocrine, secreted chemoattractant ##REF##12688968##[12]##,##UREF##2##[25]##, our simulation also reproduces continuum models that assume that ECs stress the ECM ##REF##8953213##[15]##, which either pulls on the surrounding ECs, provides haptotactic cues for active EC migration ##REF##14969709##[16]##, or both ##UREF##3##[26]##. Because these models assume that ECs exert radially-symmetric stresses on the ECM, the underlying mathematical descriptions of the chemotactic and haptotactic mechanisms are equivalent. In both cases, contact inhibition should still operate and the patterning mechanism we have proposed should still apply, with traction or haptotaxis replacing chemotaxis and the mechanical screening length replacing the diffusion length. Our simulation may also apply to the formation of linear structures by non-vascular, glia or muscle cells cultured on rigid, plastic culture dishes in continuously-shaken medium ##REF##17358734##[17]## in which cells explore their environment using long filopodia, then move towards their neighbors by pulling themselves along bound filopodia. Thus, the combination of cell aggregation and contact-inhibition that drives patterning in our model, could also occur without chemical gradients and even without ECM.</p>", "<p>Our simulations also allow us to clarify a number of subtleties concerning the interpretation of our own and others' experiments in which blocking VE-cadherin interfered with normal vascular patterning. In our in vitro experiments, anti-VE-cadherin treatment caused ECs to round, in addition to its hypothesized effect on contact inhibition, so our experiments cannot rule out the possibility that the anti-VE-cadherin treatment inhibited vascular patterning because of its effect on EC shape. A further complication is that anti-VE-cadherin treatment could conceivably reduce the adhesion between ECs. As we noted above, In VE-cadherin −/− knock-out mice, ECs still form strong adhesive junctions ##REF##10207135##[18]##, suggesting that VE-cadherin is not required for EC-EC binding.</p>", "<p>Our simulations show that the contact-inhibition patterning mechanism operates over a wide range of cell-cell adhesions, suggesting that changes in adhesivity are not significant provided that contact-inhibition persists, and independent of cell shape ##REF##19526066##[23]##, suggesting that the shape change is not significant. However, we have also shown that strong cell-cell adhesion plus chemotaxis can produce vascular-like patterns in simulations ##UREF##1##[21]##. Fortunately, the three vascular patterning mechanisms (contact-inhibition, cell-elongation and cell-cell adhesion) have vastly different kinetics ##REF##16325173##[22]##. Thus time-lapse microscopy experiments ##REF##18062955##[19]##,##REF##12067365##[44]## quantifying the kinetics of capillary-plexus development (see, e.g., ##REF##16325173##[22]##), will allow us to definitively distinguissh among these three patterning mechanisms. Already, we can say that adhesion-driven patterning is so slow and requires such strong adhesion that it appears incompatible with the available qualitative data from experiments.</p>", "<p>To further test if VE-cadherin-mediated, contact-dependent signaling to VEGF-R2 ##REF##15071551##[20]##, rather than VE-cadherin's function as a cell-adhesion molecule is responsible for the effects of anti-VE-cadherin treatment in mouse yolk sacs, we could experimentally block signal transduction from VE-cadherin to VEGFR-2, specifically interfering with VE-cadherin's signaling function, while leaving its role as an adhesion molecule intact. A possible target would be CD148, which phosphorylates VEGFR-2 after VE-cadherin binding ##REF##15071551##[20]##,##REF##12588999##[45]##. Embryonic vascularization and angiogenic sprouting are severely deficient in CD148 −/− knock-out mice ##REF##12588999##[45]##, further supporting our hypothesis that VE-cadherin's contact-dependent intercellular signaling is crucial to vasculogenesis and angiogenesis.</p>", "<p>Perryn et al. ##REF##18062955##[19]## showed that anti-VE-cadherin treatment reduced sprout extension in murine allantois cultures by 70%, while it reduced cell-autonomous motility along sprout segments by 50%. Based on these results, they postulated that VE-cadherin is required for the motility of ECs along sprouts towards the tip. However, our simulations show that the observed cell slow-down after anti-VE-cadherin administration may be an indirect effect of a reduction of sprouting. Furthermore, our simulations suggest that even substantially reduced cell motility may not prevent patterning, though it does slow it down.</p>", "<p>In our simulations, branching and pattern formation require only experimentally-observed cell-level mechanisms, instead of the blood-vessel-level phenomenology in some other angiogenesis models ##UREF##4##[28]##–##UREF##5##[30]##. However, by starting with a cluster of endothelial cells, our simulations ignore many events preceding sprout formation, including the release of plasma proteins by the vessel, the breakdown of the basal lamina, the detachment of the ECs from surrounding ECs and smooth muscle cells, and cell proliferation. They also ignore subsequent processes consolidating outgrowth of the sprout, including tip-cell selection, any long-range chemoattractants and chemorepellants that guide the vessel to its target, the formation of new basal lamina, the sprout's association with stabilizing cells including pericytes, lumen formation within the sprout, and flow-induced remodeling of the developed vasculature. The mechanism for sprouting and network formation we have proposed forms a firm basis for future, more complete models of angiogenesis which include basal lamina and pericytes. We are currently studying the formation of directed sprouts with proliferating ECs in response to additional chemoattractants or chemorepellants and analyzing the role of cell elongation during sprouting. We are also studying the effect of additional, cell-cell contact-dependent signaling mechanisms, including delta-notch tip-cell selection ##REF##17259973##[3]## and chemoattractant-response amplifying Eph receptor-ephrin ligand interactions ##REF##16840724##[8]##.</p>" ]
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[ "<p><bold>¤a:</bold> Current address: Netherlands Institute for Systems Biology and CWI (Center for Mathematics and Computer Science), Amsterdam, The Netherlands</p>", "<p><bold>¤b:</bold> Current address: Krumlauf Laboratory, Stowers Institute for Medical Research, Kansas City, Missouri, United States of America</p>", "<p>Conceived and designed the experiments: RMHM EDP JG. Performed the experiments: RMHM EDP. Analyzed the data: RMHM AS. Contributed reagents/materials/analysis tools: RMHM EDP AS. Wrote the paper: RMHM JG.</p>", "<p>Blood vessels form either when dispersed <italic>endothelial</italic> cells (the cells lining the inner walls of fully formed blood vessels) organize into a vessel network (<italic>vasculogenesis</italic>), or by sprouting or splitting of existing blood vessels (<italic>angiogenesis</italic>). Although they are closely related biologically, no current model explains both phenomena with a single biophysical mechanism. Most computational models describe sprouting at the level of the blood vessel, ignoring how cell behavior drives branch splitting during sprouting. We present a cell-based, Glazier–Graner–Hogeweg model (also called Cellular Potts Model) simulation of the initial patterning before the vascular cords form lumens, based on plausible behaviors of endothelial cells. The endothelial cells secrete a chemoattractant, which attracts other endothelial cells. As in the classic Keller–Segel model, chemotaxis by itself causes cells to aggregate into isolated clusters. However, including experimentally observed VE-cadherin–mediated contact inhibition of chemotaxis in the simulation causes randomly distributed cells to organize into networks and cell aggregates to sprout, reproducing aspects of both de novo and sprouting blood-vessel growth. We discuss two branching instabilities responsible for our results. Cells at the surfaces of cell clusters attempting to migrate to the centers of the clusters produce a buckling instability. In a model variant that eliminates the surface–normal force, a dissipative mechanism drives sprouting, with the secreted chemical acting both as a chemoattractant and as an inhibitor of pseudopod extension. Both mechanisms would also apply if force transmission through the extracellular matrix rather than chemical signaling mediated cell–cell interactions. The branching instabilities responsible for our results, which result from contact inhibition of chemotaxis, are both generic developmental mechanisms and interesting examples of unusual patterning instabilities.</p>", "<title>Author Summary</title>", "<p>A better understanding of the mechanisms by which <italic>endothelial</italic> cells (the cells lining the inner walls of blood vessels) organize into blood vessels is crucial if we need to enhance or suppress blood vessel growth under pathological conditions, including diabetes, wound healing, and tumor growth. During embryonic development, endothelial cells initially self-organize into a network of solid cords via blood vessel growth. The vascular network expands by splitting of existing blood vessels and by sprouting. Using computer simulations, we have captured a small set of biologically plausible cell behaviors that can reproduce the initial self-organization of endothelial cells, the sprouting of existing vessels, and the immediately subsequent remodeling of the resulting networks. In this model, endothelial cells both secrete diffusible <italic>chemoattractants</italic> and move up gradients of those chemicals by extending and retracting small <italic>pseudopods</italic>. By itself, this behavior causes simulated cells to accumulate to aggregate into large, round clusters. We propose that endothelial cells stop extending pseudopods along a given section of cell membrane as soon as the membrane touches the membrane of another endothelial cell (<italic>contact inhibition</italic>). Adding such contact-inhibition to our simulations allows vascular cords to form sprouts under a wide range of conditions.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>We gratefully acknowledge discussions with Stuart A. Newman, András Czirók, and Charles D. Little, and helpful comments of the anonymous referees. The simulation software is based on codes RM developed during work with Paulien Hogeweg at Utrecht University, The Netherlands.</p>" ]
[ "<fig id=\"pcbi-1000163-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g001</object-id><label>Figure 1</label><caption><title>Anti-VE-cadherin antibody treatment inhibits de novo blood-vessel growth in mouse allantois cultures.</title><p>Endothelial cells fluorescently labeled in red with endothelium-specific CD34-Cy3 antibody. DIC/fluorescent image overlays. (A–C) Control. (D–F) Anti-VE-cadherin-treated cell cultures.</p></caption></fig>", "<fig id=\"pcbi-1000163-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g002</object-id><label>Figure 2</label><caption><title>Endothelial cell aggregation; simulation initiated with 1000 scattered cells.</title><p>(A) After 10 Monte Carlo steps (<italic>MCS</italic>) (∼5 min). (B) After 1000 MCS (∼8 h). (C) After 10,000 MCS (∼80 h). (D) Contact-inhibited chemotaxis drives formation of vascular networks. Scale bar: 50 lattice sites (≈100 µm). Contour levels (green) indicate ten chemoattractant levels relative to the maximum concentration in the simulation. Grey shading indicates absolute concentration on a saturating scale.</p></caption></fig>", "<fig id=\"pcbi-1000163-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g003</object-id><label>Figure 3</label><caption><title>Sprout formation in the absence of contact inhibition.</title><p>(A–C) Cell-autonomous cell elongation; (A) <italic>Λ</italic> = 22 µm; (B) <italic>Λ</italic> = 24 µm; (C) <italic>Λ</italic> = 32 µm; (D–F) Adhesion-driven sprouting. (D) <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 1; (E) <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 5; (F) <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 10; (G–I) Passive cell elongation at short diffusion lengths; (G) <italic>D</italic> = 1·10<sup>−14</sup> m<sup>2</sup> s<sup>−1</sup>; (H) <italic>D</italic> = 2·10<sup>−14</sup> m<sup>2</sup> s<sup>−1</sup>; (I) <italic>D</italic> = 3·10<sup>−14</sup> m<sup>2</sup> s<sup>−1</sup>.</p></caption></fig>", "<fig id=\"pcbi-1000163-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g004</object-id><label>Figure 4</label><caption><title>Sprouting instability in a simulation initiated with a cluster of endothelial cells.</title><p>(A) After 10 MCS (5 min). (B) After 1,000 MCS (∼8 h). (C) After 10,000 MCS (∼80 h). (D) No sprouting in a simulation without contact inhibition of chemotaxis (<italic>Χ</italic>(<italic>c</italic>,<italic>c</italic>)/<italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 1) at 10,000 MCS (∼80 h). Scale bar: 50 lattice sites (≈100 µm).</p></caption></fig>", "<fig id=\"pcbi-1000163-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g005</object-id><label>Figure 5</label><caption><title>Compactness (<italic>C</italic> = <italic>A</italic>\n<sub>cluster</sub>/<italic>A</italic>\n<sub>hull</sub>) of 128-cell clusters after 10,000 MCS (∼80 h) as a function of the relative chemotactic response at cell-cell <italic>vs.</italic> cell-ECM interfaces.</title><p>Error bars show standard deviations over ten simulations.</p></caption></fig>", "<fig id=\"pcbi-1000163-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g006</object-id><label>Figure 6</label><caption><title>Cell trajectories of simulated endothelial cells in 128-cell clusters in a contact-inhibited, sprouting cluster (A,B) and in a non-contact-inhibited, non-sprouting cluster (C,D).</title><p>(A,C) Cell trajectories during initial sprouting, indicating the cells' centers of mass at 100 MCS (∼50 min) intervals from 100 to 5,000 MCS (∼1–40 h). (B,D) Cell trajectories after initial sprouting, indicating the cells' centers of mass at 1,000 MCS (∼8 h) intervals from 4,000 to 20,000 MCS (∼30–170 h). Closed circles indicate initial cell positions; open circles indicate final cell positions. Colors identify individual cells; brightness increases from dark (initial positions) to bright (final positions). Outlines of clusters shown at 1000 MCS (∼8 h) intervals (A,C) or 4000 MCS (∼33 h) intervals (B,D). (E) Average displacement of cells from original positions over time in 10 simulations with 128 cells each, in contact-inhibited (solid curves) and non-contact-inhibited simulations (dashed curves). Grey curves indicate standard deviations. (F) Cell velocity \n##REF##18062955##[19]## with Δ<italic>t</italic> = 300 MCS (∼2.5 h) for contact-inhibited (solid curves) and non-contact-inhibited (dashed curves) simulations.</p></caption></fig>", "<fig id=\"pcbi-1000163-g007\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g007</object-id><label>Figure 7</label><caption><title>Compactness (<italic>C</italic> = <italic>A</italic>\n<sub>cluster</sub>/<italic>A</italic>\n<sub>hull</sub>) of 128-cell clusters on 200×200-pixel lattices (∼400 µm×400 µm) after 5,000 MCS (∼40 h) for standard chemotaxis, as a function of the adhesion between endothelial cells, <italic>J</italic>(<italic>c</italic>,<italic>c</italic>).</title><p>For <italic>J</italic>(<italic>c</italic>,<italic>c</italic>)&lt;40 (i.e., <italic>J</italic>(<italic>c</italic>,<italic>c</italic>)&lt;2 <italic>J</italic>(<italic>c</italic>,<italic>M</italic>) the cells adhere without chemotaxis. Insets: Representative configurations after 5000 MCS (∼40 h).</p></caption></fig>", "<fig id=\"pcbi-1000163-g008\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g008</object-id><label>Figure 8</label><caption><title>Compactness (<italic>C</italic> = <italic>A</italic>\n<sub>cluster</sub>/<italic>A</italic>\n<sub>hull</sub>) of 128-cell clusters on 200×200-pixel lattices (∼400 µm×400 µm ) after 5000 MCS (∼40 h) for standard chemotaxis as a function of absolute chemotactic strength, <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>).</title><p>Insets: Representative configurations after 5000 MCS (∼40 h).</p></caption></fig>", "<fig id=\"pcbi-1000163-g009\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g009</object-id><label>Figure 9</label><caption><title>Compactness (<italic>C</italic> = <italic>A</italic>\n<sub>cluster</sub>/<italic>A</italic>\n<sub>hull</sub>) of 128-cell clusters on 200×200-lattices (∼400 µm×400 µm) after 5000 MCS (∼40 h) for standard chemotaxis as a function of the saturation of the chemotactic response, <italic>s</italic>.</title><p>Insets: representative configurations after 5000 MCS (∼40 h).</p></caption></fig>", "<fig id=\"pcbi-1000163-g010\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g010</object-id><label>Figure 10</label><caption><title>Compactness (<italic>C</italic> = <italic>A</italic>\n<sub>cluster</sub>/<italic>A</italic>\n<sub>hull</sub>) of 128-cell clusters (solid curve) on 200×200-pixel lattices (∼400 µm×400 µm) and 1,024-cell clusters (dashed-dotted curve) on 400×400-pixel lattices (∼800 µm×800 µm) after 5000 MCS (∼40 h) for Savill-Hogeweg chemotaxis as a function of the chemoattractant diffusion constant <italic>D</italic>.</title><p>Larger diffusion constants have longer diffusion lengths, . Dashed curve shows the compactness of VE-cadherin-inhibited 128-cell clusters. Insets: Representative configurations after 5,000 MCS (∼40 h) of the 128-cell clusters (left panels) and 1,024-cell clusters (right panels; not to scale).</p></caption></fig>", "<fig id=\"pcbi-1000163-g011\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g011</object-id><label>Figure 11</label><caption><title>Compactness (<italic>C</italic> = <italic>A</italic>\n<sub>cluster</sub>/<italic>A</italic>\n<sub>hull</sub>) of 128-cell clusters on 400×400-pixel lattices (∼400 µm×400 µm) after 5,000 MCS (∼40 h) as a function of the cell motility <italic>T</italic>, for standard Savill–Hogeweg ##UREF##8##[36]##\n<italic>extension–retraction</italic> chemotaxis (solid curve), and for <italic>extension-only</italic> chemotaxis (dashed curve).</title><p>Black curves show the mean over 100 simulations for each <italic>T</italic> (with a <italic>T</italic>-increment of 10). Dotted grey curves indicate one standard deviation. Insets: Representative configurations after 5,000 MCS (∼40 h).</p></caption></fig>", "<fig id=\"pcbi-1000163-g012\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g012</object-id><label>Figure 12</label><caption><title>Evolution of the compactness (<italic>C</italic> = <italic>A</italic>\n<sub>cluster</sub>/<italic>A</italic>\n<sub>hull</sub>) of 256-cell clusters on 500×500-pixel lattices (∼1,000 µm×1,000 µm) <italic>vs.</italic> time for standard Savill-Hogeweg ##UREF##8##[36]##\n<italic>extension-retraction</italic> chemotaxis (solid and dashed curves, for <italic>T</italic> = 50 and <italic>T</italic> = 200 respectively), and for <italic>extension-only</italic> chemotaxis (dash-dotted curve, <italic>T</italic> = 200), with only extending pseudopods responding to the chemoattractant.</title><p>Black curves show the mean of 100 simulations. Dotted grey curves mark one standard deviation. Insets: Representative configurations after 1000 (∼8 h) and 5000 MCS (∼40 h). Videos available online.</p></caption></fig>", "<fig id=\"pcbi-1000163-g013\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000163.g013</object-id><label>Figure 13</label><caption><title>Cumulative energy differences for standard Savill–Hogeweg ##UREF##8##[36]##\n<italic>extension</italic>–<italic>retraction</italic> chemotaxis (solid and dashed curves, for <italic>T</italic> = 50 and <italic>T</italic> = 200, respectively), and for <italic>extension-only</italic> chemotaxis (dash-dotted curve, <italic>T</italic> = 200) as a function of time.</title><p>Black curves show the mean of 100 simulations. Dotted grey curves mark one standard deviation.</p></caption></fig>" ]
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[ "<disp-formula><label>(1)</label></disp-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<disp-formula><label>(2)</label></disp-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<disp-formula><label>(3)</label></disp-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<disp-formula><label>(4)</label></disp-formula>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s001\"><label>Dataset S1</label><caption><p>Parameter files for the simulations shown in ##FIG##1##Figures 2##, ##FIG##3##4##, ##FIG##10##11##, and ##FIG##11##12##, packed as a tar.gz archive To use, unpack the parameter-file archive and install the Tissue Simulation Toolkit (##SUPPL##1##Protocol S1##). Run the simulations from the command line using the command “vessel [parameter-file]”. Reproduce the other simulations by editing the parameter files using a standard text editor to set the values specified in the text.</p><p>(4 KB ZIP)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s002\"><label>Protocol S1</label><caption><p>Tissue Simulation Toolkit v0.1.3. The source code for the software used for the simulations presented in this paper is also available from <ext-link ext-link-type=\"uri\" xlink:href=\"http://sourceforge.net/projects/tst\">http://sourceforge.net/projects/tst</ext-link>. Installation: Unpack and compile according to the instructions given in the INSTALL file The code is written in C++ using the cross-platform (Windows, Mac, or Unix/Linux) library Qt (available from <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.trolltech.com\">www.trolltech.com</ext-link>).</p><p>(332 KB ZIP)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s003\"><label>Figure S1</label><caption><p>Compactness (<italic>C</italic> = <italic>A</italic>\n<sub>cluster</sub>/<italic>A</italic>\n<sub>hull</sub>) of 128-cell clusters after 5000 MCS (∼40 h) as a function of the cell length, in the presence (solid curve) or absence (dashed curve) of contact inhibition. Lengths given in terms of the <italic>target length Λ</italic> as defined in Merks et al. 2006 ##REF##16325173##[22]##. Grey curves show standard deviations over ten simulations.</p><p>(1278 KB EPS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s004\"><label>Figure S2</label><caption><p>Configurations of 128-cell clusters after 5,000 MCS (∼40 h) for increasing chemoattractant secretion rates for low (<italic>s</italic> = 0.01) and high (<italic>s</italic> = 0.05) chemoattractant receptor saturations.</p><p>(3536 KB EPS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s005\"><label>Video S1</label><caption><p>Endothelial cell aggregation without contact-inhibited chemotaxis. Simulation initiated with 1,000 scattered cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame.</p><p>(1902 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s006\"><label>Video S2</label><caption><p>Endothelial cell aggregation in with contact-inhibited chemotaxis. Simulation initiated with 1000 scattered cells. 0 MCS to 2000 MCS (∼0–20 h), 10 MCS per frame.</p><p>(4534 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s007\"><label>Video S3</label><caption><p>Same simulation as ##SUPPL##5##Video S2##. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame.</p><p>(2000 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s008\"><label>Video S4</label><caption><p>Sprouting instability in a simulation with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 2,000 MCS (∼0–20 h), 10 MCS per frame.</p><p>(4554 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s009\"><label>Video S5</label><caption><p>Same simulation as ##SUPPL##7##Video S4##. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame.</p><p>(1895 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s010\"><label>Video S6</label><caption><p>Simulation with non-contact–inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 2,000 MCS (∼0–20 h), 10 MCS per frame.</p><p>(1640 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s011\"><label>Video S7</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 5.</p><p>(699 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s012\"><label>Video S8</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 10.</p><p>(685 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s013\"><label>Video S9</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 15.</p><p>(691 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s014\"><label>Video S10</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 20.</p><p>(669 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s015\"><label>Video S11</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 25.</p><p>(677 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s016\"><label>Video S12</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 30.</p><p>(669 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s017\"><label>Video S13</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 35.</p><p>(679 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s018\"><label>Video S14</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 40.</p><p>(648 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s019\"><label>Video S15</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 45.</p><p>(383 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s020\"><label>Video S16</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c,c</italic>) = 50.</p><p>(557 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s021\"><label>Video S17</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 55.</p><p>(528 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s022\"><label>Video S18</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 60.</p><p>(516 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s023\"><label>Video S19</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 65.</p><p>(457 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s024\"><label>Video S20</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c,c</italic>) = 70.</p><p>(439 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s025\"><label>Video S21</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 75.</p><p>(411KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s026\"><label>Video S22</label><caption><p>Effect of cell adhesion on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##6##Figure 7##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>J</italic>(<italic>c</italic>,<italic>c</italic>) = 80.</p><p>(383 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s027\"><label>Video S23</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 0.</p><p>(238 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s028\"><label>Video S24</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 500.</p><p>(653 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s029\"><label>Video S25</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 1,000.</p><p>(707 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s030\"><label>Video S26</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 1,500.</p><p>(714 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s031\"><label>Video S27</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 2,000.</p><p>(727 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s032\"><label>Video S28</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 2,500.</p><p>(765 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s033\"><label>Video S29</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 3,000.</p><p>(797 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s034\"><label>Video S30</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 3,500.</p><p>(805 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s035\"><label>Video S31</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 4,000.</p><p>(835 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s036\"><label>Video S32</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 4,500.</p><p>(853 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s037\"><label>Video S33</label><caption><p>Effect of the chemotactic strength on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##7##Figure 8##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>Χ</italic>(<italic>c</italic>,<italic>M</italic>) = 5,000.</p><p>(858 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s038\"><label>Video S34</label><caption><p>Effect of chemotaxis saturation on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##8##Figure 9##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>s</italic> = 0.0.</p><p>(655 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s039\"><label>Video S35</label><caption><p>Effect of chemotaxis saturation on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##8##Figure 9##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>s</italic> = 0.05.</p><p>(577 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s040\"><label>Video S36</label><caption><p>Effect of chemotaxis saturation on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##8##Figure 9##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>s</italic> = 0.1.</p><p>(409 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s041\"><label>Video S37</label><caption><p>Effect of chemotaxis saturation on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##8##Figure 9##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>s</italic> = 0.15.</p><p>(224 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s042\"><label>Video S38</label><caption><p>Effect of chemotaxis saturation on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic> Figure 39). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>s</italic> = 0.2.</p><p>(213 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s043\"><label>Video S39</label><caption><p>Effect of chemotaxis saturation on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##8##Figure 9##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>s</italic> = 0.25.</p><p>(215 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s044\"><label>Video S40</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 4·10<sup>−14</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(1053 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s045\"><label>Video S41</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 5·10<sup>−14</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(717 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s046\"><label>Video S42</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 1·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(631 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s047\"><label>Video S43</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 2·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(501 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s048\"><label>Video S44</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 3·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(418 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s049\"><label>Video S45</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 4·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(371 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s050\"><label>Video S46</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 256 endothelial cells. 0 MCS to 20,000 MCS (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 5·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(316KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s051\"><label>Video S47</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 1024 endothelial cells. MCS 0 to 20,000 (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 1·10<sup>−14</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(1376 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s052\"><label>Video S48</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 1024 endothelial cells. MCS 0 to 20,000 (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 5·10<sup>−14</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(1011 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s053\"><label>Video S49</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 1024 endothelial cells. MCS 0 to 20,000 (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 1·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(1003 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s054\"><label>Video S50</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 1024 endothelial cells. MCS 0 to 20,000 (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 2·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(994 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s055\"><label>Video S51</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 1024 endothelial cells. MCS 0 to 20,000 (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 3·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(976 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s056\"><label>Video S52</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 1024 endothelial cells. MCS 0 to 20,000 (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 4·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(978 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s057\"><label>Video S53</label><caption><p>Effect of the diffusion constant <italic>D</italic> on the proposed sprouting-angiogenesis mechanism (<italic>cf.</italic>\n##FIG##9##Figure 10##). Simulations with contact-inhibited chemotaxis, initiated with a cluster of 1024 endothelial cells. MCS 0 to 20,000 (∼0–170 h), 100 MCS per frame. <italic>D</italic> = 5·10<sup>−13</sup> m<sup>2</sup> s<sup>−1</sup>.</p><p>(982 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s058\"><label>Video S54</label><caption><p>Sprouting of a 256-cell cluster on a 500×500-pixel lattice (∼1 mm×1 mm) with standard Savill–Hogeweg, extension-retraction chemotaxis at <italic>T</italic> = 50, as in ##FIG##11##Figure 12##.</p><p>(2366 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s059\"><label>Video S55</label><caption><p>Sprouting of a 256-cell cluster on a 500×500-pixel lattice (∼1 mm×1 mm) with standard Savill–Hogeweg, extension–retraction chemotaxis at <italic>T</italic> = 200, as in ##FIG##11##Figure 12##.</p><p>(2372 KB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000163.s060\"><label>Video S56</label><caption><p>Sprouting of a 256-cell cluster on a 500×500-pixel lattice (∼1 mm×1 mm) with extension-only chemotaxis at <italic>T</italic> = 200, as in ##FIG##11##Figure 12##.</p><p>(2381 KB AVI)</p></caption></supplementary-material>" ]
[ "<fn-group><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p>This work received support from NIH grants NIGMS 1R01 GM077138-01A1 and 5R01 GM076692 (JAG; AS), the Indiana University Biocomplexity Institute (RM), the VIB Department of Plant Systems Biology (RM), the Indiana University AVIDD program (JAG; RM) and an American Heart Association predoctoral fellowship 0410084Z (EDP). None of these funding agencies has had any role in the design and conduct of the study, in the collection, analysis, and interpretation of the data, or in the preparation, review, or approval of the manuscript.</p></fn></fn-group>" ]
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xlink:href=\"pcbi.1000163.s009.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s010.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s011.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s012.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s013.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s014.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s015.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s016.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s017.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s018.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s019.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s020.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s021.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s022.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s023.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s024.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s025.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s026.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s027.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s028.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s029.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s030.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s031.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s032.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s033.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s034.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s035.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s036.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s037.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s038.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s039.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s040.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s041.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s042.avi\"><caption><p>Click 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"<media xlink:href=\"pcbi.1000163.s051.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s052.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s053.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s054.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s055.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s056.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s057.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000163.s058.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media 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[{"label": ["5"], "element-citation": ["\n"], "surname": ["Helmlinger", "Endo", "Ferrara", "Hlatky", "Jain"], "given-names": ["G", "M", "N", "L", "RK"], "year": ["2000"], "article-title": ["Growth factors - formation of endothelial cell networks."], "source": ["Nature"], "volume": ["405"], "fpage": ["139"], "lpage": ["141"]}, {"label": ["21"], "element-citation": ["\n"], "surname": ["Merks", "Newman", "Glazier", "Sloot", "Chopard", "Hoekstra"], "given-names": ["RMH", "SA", "JA", "PMA", "B", "AG"], "year": ["2004"], "article-title": ["Cell-oriented modeling of in vitro capillary development."], "source": ["Cellular Automata: 6th International Conference on Cellular Automata for Research and Industry"], "publisher-loc": ["Berlin"], "publisher-name": ["Springer Verlag, Lect. Notes Comput. Sc. 3305,"], "fpage": ["425"], "lpage": ["434"]}, {"label": ["25"], "element-citation": ["\n"], "surname": ["Ambrosi", "Gamba", "Serini"], "given-names": ["D", "A", "G"], "year": ["2004"], "article-title": ["Cell directional persistence and chemotaxis in vascular morphogenesis."], "source": ["B Math Biol"], "volume": ["66"], "fpage": ["1851"], "lpage": ["1873"]}, {"label": ["26"], "element-citation": ["\n"], "surname": ["Tosin", "Ambrosi", "Preziosi"], "given-names": ["A", "D", "L"], "year": ["2006"], "article-title": ["Mechanics and chemotaxis in the morphogenesis of vascular networks."], "source": ["B Math Biol"], "volume": ["68"], "fpage": ["1819"], "lpage": ["1836"]}, {"label": ["28"], "element-citation": ["\n"], "surname": ["Anderson", "Chaplain"], "given-names": ["ARA", "MAJ"], "year": ["1998"], "article-title": ["Continuous and discrete mathematical models of tumor-induced angiogenesis."], "source": ["B Math Biol"], "volume": ["60"], "fpage": ["857"], "lpage": ["899"]}, {"label": ["30"], "element-citation": ["\n"], "surname": ["Sun", "Wheeler", "Obeyesekere", "Patrick"], "given-names": ["SY", "MF", "M", "CW"], "year": ["2005"], "article-title": ["A deterministic model of growth factor-induced angiogenesis."], "source": ["B Math Biol"], "volume": ["67"], "fpage": ["313"], "lpage": ["337"]}, {"label": ["33"], "element-citation": ["\n"], "surname": ["Manoussaki"], "given-names": ["D"], "year": ["2003"], "article-title": ["A mechanochemical model of angiogenesis and vasculogenesis."], "source": ["ESAIM-Mathematical Modelling and Numerical Analysis"], "volume": ["37"], "fpage": ["581"], "lpage": ["599"]}, {"label": ["35"], "element-citation": ["\n"], "surname": ["Glazier", "Graner"], "given-names": ["JA", "F"], "year": ["1993"], "article-title": ["Simulation of the differential adhesion driven rearrangement of biological cells."], "source": ["Phys Rev E"], "volume": ["47"], "fpage": ["2128"], "lpage": ["2154"]}, {"label": ["36"], "element-citation": ["\n"], "surname": ["Savill", "Hogeweg"], "given-names": ["NJ", "P"], "year": ["1997"], "article-title": ["Modelling morphogenesis: from single cells to crawling slugs."], "source": ["J Theor Biol"], "volume": ["184"], "fpage": ["229"], "lpage": ["235"]}, {"label": ["37"], "element-citation": ["\n"], "surname": ["Merks", "Glazier"], "given-names": ["RMH", "JA"], "year": ["2005"], "article-title": ["A cell-centered approach to developmental biology."], "source": ["Phys A"], "volume": ["352"], "fpage": ["113"], "lpage": ["113"]}, {"label": ["38"], "element-citation": ["\n"], "surname": ["Anderson", "Chaplain", "Rejniak"], "given-names": ["ARA", "MAJ", "RA"], "year": ["2007"], "source": ["Single-cell-based models in biology and medicine"], "publisher-loc": ["Basel, Boston and Berlin"], "publisher-name": ["Birkha\u00fcser Verlag"]}, {"label": ["47"], "element-citation": ["\n"], "surname": ["Glazier", "Balter", "Pop\u0142awksi", "Anderson", "Chaplain", "Rejniak"], "given-names": ["JA", "A", "NJ", "ARA", "MAJ", "RA"], "year": ["2007"], "article-title": ["Magnetization to morphogenesis: A history of the GGH model."], "source": ["Single-cell-based models in biology and medicine"], "publisher-loc": ["Basel Boston Berlin"], "publisher-name": ["Birkha\u00fcser, Mathematics and Biosciences in Interaction"], "fpage": ["79"], "lpage": ["106"]}]
{ "acronym": [], "definition": [] }
47
CC BY
no
2022-01-13 00:55:15
PLoS Comput Biol. 2008 Sep 19; 4(9):e1000163
oa_package/9e/78/PMC2528254.tar.gz
PMC2528255
18779871
[ "<title>1. INTRODUCTION</title>", "<p>PPAR<italic>γ</italic> is one of the three known peroxisome\nproliferator-activated receptors and is a member of the nuclear receptor (NR)\nsuperfamily. Since it has a\npredominantly nuclear location, regardless of whether cognate ligands are\npresent, it is classified as a type II NR. \nIt functions as a transcription factor by heterodimerizing with the\nretinoid X receptor (RXR), after which this complex binds to specific DNA\nsequence elements called peroxisome proliferator response elements\n(PPREs) [##REF##9312141##1##]. In order to become fully\nactive as a transcription factor, PPAR<italic>γ</italic> must be bound by ligand. RXR can be affected\nby binding its own cognate ligands, usually resulting in incremental increases\nin transcriptional activity. After the PPAR<italic>γ</italic>/RXR heterodimer binds to PPREs in promoter\nregions of target genes, coactivator proteins, such as p300 (CBP),\nSRC-1, and Drip205 (or TRAP220) family members, are recruited to this complex\nto modulate gene transcription [##REF##9121466##2##–##REF##9653119##4##]. Different\nPPAR<italic>γ</italic> ligands appear to be able to recruit different coactivators, which may explain differences in the biological activity\nbetween ligands [##REF##10944516##5##].</p>", "<p>The cardinal biologic activity of PPAR<italic>γ</italic> is the induction of differentiation of\nadipocytes, the cell type that expresses the highest levels of PPAR<italic>γ</italic> amongst normal tissues. Lower levels of PPAR<italic>γ</italic> are, however, found in other normal tissues\nand cell types such as skeletal muscle, liver, breast, prostate,\ncolon, type 2 alveolar pneumocytes, some endothelial cells as well as monocytes,\nand B-lymphocytes. There are three <italic>PPAR<italic>γ</italic></italic> mRNA isoforms (<italic>γ</italic>1, <italic>γ</italic>2, and <italic>γ</italic>3) and two major protein species (<italic>γ</italic>1 and <italic>γ</italic>2). The\nmRNA isoforms are generated by alternate promoter usage, resulting in an\nadditional 28 amino acids at the N-terminus of PPAR<italic>γ</italic>2 compared with PPAR<italic>γ</italic>1. Most tissues express PPAR<italic>γ</italic>1, whereas the PPAR<italic>γ</italic>2 isoform is expressed mostly by adipocytes.\nThe longer N-terminal domain of PPAR<italic>γ</italic>2 may affect function, since this isoform was\nshown to confer a higher level of ligand-independent transcriptional activity,\nwhich was further increased by physiologic concentrations of insulin [##REF##9242701##6##]. High\nlevels of PPAR<italic>γ</italic> expression by fat and its role in adipogenesis\nled to the recognition that agonistic PPAR<italic>γ</italic> ligands have antidiabetic effects. The\nchemical class of PPAR<italic>γ</italic> agonists known as thiazolidinediones (TZDs)\ndemonstrated high-affinity binding to PPAR<italic>γ</italic> [##REF##7768881##7##] as well as favorable therapeutic\nproperties, and such drugs were eventually registered for the treatment of type\nII diabetes mellitus (T2DM). Three TZD\ndrugs have been registered in the U.S.: rosiglitazone (Avandia), pioglitazone (Actos), and troglitazone(Rezulin). \nSubsequent to its marketing and widespread use, troglitazone was\nassociated with idiosyncratic and, in rare cases, fatal hepatic toxicity, and,\nthus, was withdrawn from the market. The former two drugs, however, have remained\nas safe and effective therapeutic options for the management of T2DM.</p>", "<p>Not long after reports of the cloning of PPAR<italic>γ</italic> and its expression in normal tissues [##REF##8262913##8##, ##REF##8240342##9##],\nPPAR<italic>γ</italic> expression was observed in an array of primary\ncancers and derivative cell lines. Its expression was reported initially in\nliposarcoma [##REF##8990192##10##], and soon thereafter in colon, breast, and prostate carcinomas\nand additional cancer types [##REF##9472692##11##–##REF##11431375##14##]. In addition\nto the in vitro and preclinical in vivo anticancer effects of TZDs, pilot clinical studies using\ntroglitazone showed antitumor activity in patients with liposarcoma and\nprostate cancer [##REF##10097144##15##, ##REF##10984506##16##]. Compounds from other chemical classes were also shown\nto bind PPAR<italic>γ</italic> and to have antiproliferative effects in\ncancer models, such as the naturally occurring eicosanoid, 15-deoxy-Δ<sup>12,14</sup>-prostaglandin J<sub>2</sub>(15-d-PGJ<sub>2</sub>), the N-aryl tyrosine derivative, GW1929 [##REF##11205925##17##], and the\ntriterpenoid, 2-cyano-3,12-dioxooleana-1,9-diene-28-oic acid, CDDO [##REF##9927043##18##]. While\ncompounds that exhibit PPAR<italic>γ</italic> agonist activity, such as TZDs, have PPAR<italic>γ</italic>-dependent antiproliferative effects, they have\nalso been shown to have antiproliferative effects in cell types that are\ngenetically PPAR<italic>γ</italic>-null [##REF##11507074##19##]. Also, uncertainty about mechanisms\nof anticancer effects of PPAR<italic>γ</italic> ligands has resulted from variability in the\nclassification of some compounds (e.g., bisphenol A diglycidyl ether [BADGE], which has been\nshown to have both agonist and antagonist activities) [##REF##10636887##20##, ##REF##11030710##21##].</p>" ]
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[ "<p>Recommended by Dipak Panigrahy</p>", "<p>PPAR<italic>γ</italic> is a therapeutic target that has been exploited for\ntreatment of type II diabetes mellitus (T2DM) with agonist drugs.\nSince PPAR<italic>γ</italic> is expressed by many hematopoietic, mesodermal and\nepithelial cancers, agonist drugs were tested and shown to have\nboth preclinical and clinical anticancer activities. While\npreclinical activity has been observed in many cancer types,\nclinical activity has been observed only in pilot and phase II\ntrials in liposarcoma and prostate cancer. Most studies address\nagonist compounds, with substantially fewer reports on anticancer\neffects of PPAR<italic>γ</italic> antagonists. In cancer model systems, some\neffects of PPAR<italic>γ</italic> agonists were not inhibited by PPAR<italic>γ</italic> antagonists,\nsuggesting noncanonical or PPAR<italic>γ</italic>-independent mechanisms. In\naddition, PPAR<italic>γ</italic> antagonists, such as T0070907 and GW9662, have\nexhibited antiproliferative effects on a broad range of\nhematopoietic and epithelial cell lines, usually with greater\npotency than agonists. Also, additive antiproliferative effects\nof combinations of agonist plus antagonist drugs were observed.\nFinally, there are preclinical in vivo data showing that\nantagonist compounds can be administered safely, with favorable\nmetabolic effects as well as antitumor effects. Since PPAR<italic>γ</italic>\nantagonists represent a new drug class that holds promise as a\nbroadly applicable therapeutic approach for cancer treatment, it\nis the subject of this review.</p>" ]
[ "<title>2. EFFECTS OF PPAR<italic>γ</italic> ANTAGONIST COMPOUNDS\nIN EPITHELIAL CANCER MODEL SYSTEMS: CELL\nGROWTH AND APOPTOSIS</title>", "<p>The initial report of Fehlberg et al. [##REF##11879183##22##] showed an inhibitory effect of this class of agents on\na colon cancer and a lymphoma cell line using the compound, BADGE, which as\nnoted has been classified as both an agonist and antagonist. This initial study\ndid not examine effects on proliferation, but showed that apoptotic effects, such\nas increases in annexin-V binding and reductions in DNA content as assessed by propidium\niodide staining, required 50–100 <italic>μ</italic>M concentrations of BADGE, which\nwould tend to increase off-target effects. Subsequently, Seargent et al. [##REF##15533890##23##] showed that a\nhigher affinity, selective PPAR<italic>γ</italic> antagonist, GW9662, had direct antiproliferative\neffects on three breast cancer cell lines of differing phenotypes (ER+, ER−,\nand p53-null). This antagonist compound was somewhat more potent in its effects\nthan an agonist (rosiglitazone). In this report, the role of PPAR<italic>γ</italic> in mediating growth inhibition was addressed,\nbut not fully elucidated. All three cell lines expressed it and the predicted,\ncanonical PPAR<italic>γ</italic>-related transactivation effects were\ndemonstrated, with the agonist inducing transactivation and the antagonist suppressing\nit, thus excluding PPAR<italic>γ</italic>-mediated transactivation as the mechanism of\nthis effect. There are data, however, that\nsuggest that antagonist-type compounds may also act via other PPAR<italic>γ</italic>-dependent pathways. Lea et al. reported similar results using\na range of agonist and antagonist compounds on both murine and human cell lines\n[##REF##15517883##24##]. Schaefer et al. showed\nthat the antiproliferative effect of the PPAR<italic>γ</italic> antagonist, T0070907, on\nhepatocellular carcinoma cell lines was attenuated by knockdown of PPAR<italic>γ</italic> by siRNA [##REF##15781638##25##]. \nThese data are consistent with a PPAR<italic>γ</italic>-mediated transrepression mechanism, which has\nbeen demonstrated with respect to anti-inflammatory effects of PPAR<italic>γ</italic> ligands mediated by the NF-<italic>κ</italic>B signaling pathway. Pascual et al. showed similar\neffects of a pure agonist (rosiglitazone) and a mixed agonist/antagonist (GW0072)\non the repression of a NF-<italic>κ</italic>B-regulated gene, <italic>iNOS</italic>, suggesting that pure antagonists may also be capable of\nmediating this effect [##REF##16127449##26##].</p>", "<p>There are also data that PPAR<italic>γ</italic> ligands (both agonist and antagonist) exert PPAR<italic>γ</italic>-independent effects suggesting other cellular\ntargets of these compounds. This was demonstrated clearly by Palakurthi\net al., who demonstrated in\nvitro and in vivo growth\ninhibition of two agonist compounds, troglitazone and ciglitazone, in\nexperiments utilizing PPAR<italic>γ</italic>\n<sup>−/−</sup> and PPAR<italic>γ</italic>\n<sup>+/+</sup> embryonic stem cell lines (ES),\nboth of which exhibited very similar sensitivity to these compounds [##REF##11507074##19##]. This\neffect was shown to be mediated in part by the inhibition of the initiation of\nprotein translation, since these TZD compounds increased the phosphorylation\nand consequent inactivation of elongation-initiation factor 2 (eIF2) both in\ncells that expressed and were null for PPAR<italic>γ</italic>. The\neffect of antagonist compounds on this pathway has not been reported. As noted, BADGE had similar proapoptotic\neffects in a colon cancer line expressing PPAR<italic>γ</italic> and a T-lymphoma line that showed no\ndetectable expression of it (by immunoblotting and RT-PCR) of this target [##REF##11879183##22##].\nBut, given the variable classification of this compound as both an antagonist\nand agonist, the mechanism underlying this effect and its attribution are unclear.</p>", "<title>3. OTHER EFFECTS OF PPAR<italic>γ</italic> ANTAGONIST COMPOUNDS</title>", "<p>PPAR<italic>γ</italic> antagonist compounds have also\nbeen shown to affect cell shape, adhesion, and invasiveness of cancer cell\nlines. Masuda et al. evaluated\nthe effects of the PPAR<italic>γ</italic> antagonists, BADGE, GW9662 and\nT0070907, on four squamous carcinoma cell lines derived from tumors of the oral\ncavity. Antiproliferative effects were\nshown for the three antagonists, but not for the agonist, pioglitazone [##REF##15930335##27##]. Effects of these agents on adhesion and\nanoikis were also evaluated. Antagonists\nwere found to inhibit adhesion and induce cell death related to loss of\nadhesion (known as anoikis) under normal tissue culture conditions on untreated\nplastic dishes. T0070907 induced similar inhibition of adhesion to\nfibronectin-coated plates, and this was significantly reversed by coincubation\nof cells with this antagonist and the agonist, pioglitazone, suggesting a PPAR<italic>γ</italic>-dependent effect. Since adhesion\nand detachment are related to cytoskeletal structure and function, this was\nassessed by fluorescent staining of F-actin. \nUsing confocal microscopy, T0070907 was shown to cause dose-dependent\ndisruption of F-actin, associated with rounding of the cells. Additional\nexperiments showed inhibition of FAK and MEK-ERK signaling pathways, as well as\ndecreased expression of integrin <italic>α</italic>5 and CD151, both of which are adhesion\nproteins that have been implicated in cancer cell invasion and metastasis. Schaefer\net al. showed similar effects\nof PPAR<italic>γ</italic> antagonists on hepatocellular\ncarcinoma cell lines including inhibition of adhesion, induction of anoikis, and\ninhibition of phosphorylation and activation of FAK [##REF##15781638##25##]. These effects were shown to be dependent on\nthe degree of PPAR<italic>γ</italic> inhibition, and could be mediated\nby the antagonist or knockdown of PPAR<italic>γ</italic> via specific, cognate siRNA.\nT0070907 was also shown to have substantially greater growth inhibitory effects\non the HepG2 line compared with the agonist drugs, troglitazone, and\nrosiglitazone. Takahashi et al. demonstrated anti-invasive and\ngrowth inhibitory effects of the antagonists, GW9662 and\nT0070907, on\nesophageal cancer cell lines. The anti-invasive effects were observed at levels\nsubstantially lower than those required for growth inhibition [##REF##16805824##28##]. In summary, all of these studies addressing\nanticancer effects of PPAR<italic>γ</italic> antagonist compounds have show\neffects on cell growth, adhesion, and invasion in multiple epithelial cancer\nmodels.</p>", "<p>Some of these effects\nare PPAR<italic>γ</italic>-dependent, but the potential role of other\ntargets is suggested by the similar effects of BADGE on a PPAR<italic>γ</italic>+ colon cancer line and a PPAR<italic>γ</italic>-negative T-lymphoma line. Also, the\nsubstantially different concentrations of PPAR<italic>γ</italic> antagonists needed to induce anti-invasive\neffects versus growth inhibition in esophageal cancer lines suggest different\nmechanisms with differing degrees of PPAR<italic>γ</italic> dependence or lack of involvement of the PPAR<italic>γ</italic>-signaling pathway for some effects. A PPAR<italic>γ</italic>-independent effect of antagonists on\ncolorectal cancer cell lines and in an in\nvivo tumor xenograft derived from one of the lines was shown in a more\nrecent report by Schaefer et al.\n[##REF##17096328##29##]. A decrease in tubulin levels was observed\nthat was independent of PPAR<italic>γ</italic>, PPAR<italic>δ</italic>,\nand proteasome function. This downregulation of tubulins <italic>α</italic> and <italic>β</italic> may explain\nthe antimigratory, anti-invasive, and antimetastatic effects that were\nobserved. Thus, in summary, PPAR<italic>γ</italic> antagonist compounds with varying chemical\nstructures (though GW9662 and T0070907 are similar) have several significant\nanticancer effects in vitro and\nin vivo in epithelial cancer\nmodel systems including breast, colon, aerodigestive squamous cell, and\nhepatocellular.</p>", "<title>4. EFFECTS OF PPAR<italic>γ</italic> ANTAGONISTS IN\nHEMATOPOIETIC CANCER MODEL SYSTEMS</title>", "<p>Studies were conducted\nin our lab to assess the effects of PPAR<italic>γ</italic> antagonists on hematopoietic cell lines. Initial screening showed that several\nmyeloma (MM) cell lines had the greatest sensitivity to the antiproliferative\neffects of the antagonists, GW9662 and T0070907. Thus multiple MM lines were\ntested, including one that is IL-6-dependent, for sensitivity to these\ncompounds as well as to the agonist, pioglitazone. MM lines as well as\nnon-Hodgkin lymphoma (NHL) lines showed significantly greater sensitivity to\nthe growth inhibitory effects of the two antagonist drugs compared with the\nagonist [##REF##17414621##30##]. As a group, the MM lines were more sensitive than the other\ngroups of cancer cell lines to the antiproliferative effects of the\nantagonists, particularly T0070907. Other goals were to directly compare the\nsensitivity of previously tested epithelial cancer types (breast and colon) to\nhematopoietic lines (MM and NHL) as well as to evaluate a chemoresistant\nepithelial cancer type (renal cell). These experiments showed that in all the\nepithelial and hematopoietic cell lines tested, the antagonists were\nsignificantly more potent in their growth inhibitory effects compared with the\nagonist drug.</p>", "<p>The IC<sub>50</sub> values for the panel of 16 cell lines\ntested in these studies are shown in ##TAB##0##Table 1##. For each of the cell lines in the panel, significant\ndifferences in the IC<sub>50</sub> values of the antagonist\ncompounds and the agonist drug, pioglitazone, were observed (<italic>P</italic> values ranging from &lt;.04 to\n&lt;.001, with 12 of 16 lines at &lt;.001). While the MM lines showed the\ngreatest sensitivity to the antagonists, similar degrees of sensitivity to the\nantagonists were also seen in the subset of breast cancer lines, which included\ntwo lines that are estrogen receptor-negative. Though not quite as sensitive as\na subset, significant differences between the antagonists and the agonist were\nalso observed in the renal cell lines, which are among the most chemoresistant\nepithelial lines. The differential sensitivities within and across cell lines\ndid not appear to be related to the levels of PPAR<italic>γ</italic> expression. Also, neither the agonist nor the\nantagonist induced significant upregulation of PPAR<italic>γ</italic> as has been reported in some studies with PPAR<italic>γ</italic> ligands. Consistent with prior reports, combinations\nof the agonist and with each of the antagonists did not result in attenuation\nof growth inhibitory effects. In fact, schedule-dependent increases in growth\ninhibition were observed, particularly when the antagonists were added to cells\n24 hours prior to the agonist. Aspects of the mechanisms of cytotoxicity of the\nantagonists and agonists were also compared. It was shown that both classes of PPAR<italic>γ</italic> ligand-induced apoptotic effects, but this\neffect was found to be caspase-independent for the agonist, pioglitazone [##REF##17414621##30##].</p>", "<p>Another question that\nwas addressed was the impact of IL-6 on the responses of the MM lines to PPAR<italic>γ</italic> antagonists, since this is a cytokine that\nplays a central role in the pathogenesis and progression of MM, as well as\nother cancer types. For these studies, 4 of the 5 MM lines that were utilized\nwere IL-6-independent in order to follow up on a previous\nreport of Wang et al. that\nanalyzed the responses of three MM lines to the PPAR<italic>γ</italic> agonists, 15-d-PGJ<sub>2</sub> and\ntroglitazone. This report showed that growth\ninhibition and certain downstream signaling events were PPAR<italic>γ</italic>-dependent, and also\nthat two IL-6-dependent MM lines expressed PPAR<italic>γ</italic> while an IL-6-independent line did not [##REF##14975242##31##].\nAlso, GW9662 was reported to block the effects of the agonists, and had no antiproliferative activity on its own. We utilized five\ndifferent MM lines, of which four are IL-6 independent (CAG, KMS12-BM, KMS12-PE,\nand OPM-6) as well as a fifth that is dependent on an IL-6 autocrine loop\n(U266B1). In contrast to the prior report cited above, of the lines analyzed,\nCAG expressed PPAR<italic>γ</italic>, while the autocrine IL-6-dependent line,\nU266B1, did not express PPAR<italic>γ</italic> by immunoblotting. Also, three of the four of IL-6-independent MM lines were more sensitive to the growth\ninhibitory effects of both of the two PPAR<italic>γ</italic> antagonist compounds compared with the\nIL-6-dependent line, U266B1 (see ##TAB##0##Table 1##).</p>", "<p>In MM cell lines, which\nare more often IL-6 dependent compared with other B cell lines, the strict dependence\non exogenous IL-6 is indicative of ongoing requirement for this signaling\npathway, which in pathophysiologic states, such as MM, usually depends on production\nof this cytokine by stromal cells. In\nMM, clinically more aggressive or treatment-resistant disease is associated\nwith production of IL-6 by the myeloma cell themselves as opposed to the bone\nmarrow stroma [##REF##11154226##32##]. MM lines show a spectrum of IL-6 dependence, with some\nbeing dependent on exogenous IL-6, others being dependent on its autocrine\nproduction, and yet others being IL-6-independent for their growth. Even those\nMM lines that are not strictly dependent on IL-6 for their growth (exogenous or\nautocrine) can still be affected by the addition of exogenous IL-6 [##REF##14500688##33##] (also\nshown in one of the lines tested, OPM-6, [##REF##10945640##34##]). Addition of IL-6 to such MM\nlines has been shown to induce either incremental stimulation of proliferation or\ninduction of resistance to various agents such as dexamethasone, standard chemotherapy\ndrugs such as melphalan and other agents. Thus the interaction of IL-6 and PPAR<italic>γ</italic> antagonist compounds were examined in two MM\nlines (KMS12-PE and OPM-6). MTT assays were performed in the\npresence and absence of exogenous IL-6 (5 ng/mL). For both of these MM lines,\naddition of IL-6 did not induce resistance, but instead appeared to increase\nthe sensitivity of these lines to T0070907, with a similar trend observed with\nGW9662 [##REF##17414621##30##].</p>", "<title>5. DOSE-RESPONSE EFFECTS OF PPAR<italic>γ</italic> ANTAGONIST COMPOUNDS AND\nINTERACTION WITH OTHER AGENTS</title>", "<p>The PPAR<italic>γ</italic> antagonist\ncompounds, GW9662 and T0070907, differ in their antiproliferative dose-response\neffects compared with the agonist as well as other agents. Not only are the corresponding IC<sub>50</sub> values for the\nantagonists significantly lower than the agonist, pioglitazone, but a greater\ndegree of growth inhibition (85–97% versus 50–80%) was observed\nwith the former compounds. Also, of note was that the maximal effects of these\nagents were seen at concentrations that were only 2- to 3-fold greater than the\nIC<sub>50</sub> across the entire panel of cell lines tested that included cell\nlines with relative and very high levels of chemoresistance (colon and renal\ncell, resp.). The dose-response curves were much steeper with the antagonist\ncompounds compared to the agonist, pioglitazone, and also much steeper than\nwhat is observed with most other agents, including standard chemotherapy drugs\nand other agents (see ##FIG##1##Figure 2##). This dose-response relationship suggests\neither a positive cooperative effect, potentially via increased, cooperative recruitment\nof corepresssors, thereby increasing transrepression. The alternate possibility\nis that different targets are being engaged with gradually increasing\nconcentrations, which together exhibit additive or supra-additive interactions.</p>", "<p>Since MM lines as a group were the most sensitive of\nthe cell lines we tested, interaction with other novel agents for therapy of MM\nwere evaluated. One such agent is anti-CD74 monoclonal antibody (mAb). CD74 was\nshown to be strongly expressed by the malignant plasma cells in the vast\nmajority of clinical MM specimens as well as the majority of MM lines [##REF##15475450##35##]. It\nwas also shown that this mAb in unlabeled (cold) form exhibited in vitro growth inhibitory effects on both NHL and MM lines [##REF##15297317##36##]. The\nanti-CD74 mAb used in these studies, LL1, also showed significant therapeutic\neffects in two preclinical murine NHL xenograft models. In preliminary in vitro studies, the humanized anti-CD74 mAb was combined with T0070907 in\ntwo MM lines. These studies also evaluated a sixth MM line (KMS11), which is\nIL-6 independent, expresses CD74 and is useful as a murine MM xenograft model. This line showed similar sensitivity to T0070907 as the other IL-6-independent\nlines, with an (unpublished observations, J Burton). Another IL-6-independent\nMM line that was used in initial studies, KMS12-PE, was also used to evaluate\ninteractions between T0070907 and the hLL1 mAb. While KMS11 line showed moderate\nsensitivity to hLL1 (maximum growth inhibition of 50–70%), the KMS12-PE line\nwas resistant to single-agent hLL1 (&lt;10% inhibition). However, in\ncombination with T0070907, there was a sizable shift to the left of the\ndose-response curve, as is shown in one representative experiment in ##FIG##1##Figure 2##.\nCurrent data indicate that the IC<sub>50</sub> value decreases by from a mean\nvalue of ~4.1 <italic>μ</italic>M for T0070907 alone versus ~3.0 <italic>μ</italic>M with T0070907 in combination\nwith hLL1, suggestive of a supra-additive effect (25–30% observed versus &lt;8%\nexpected based effect of hLL1 alone). This is a promising initial preclinical\nlead given that hLL1 is now being evaluated in several phase I/II clinical\ntrials in B-cell cancers such as NHL and MM, and appears to be safe and well\ntolerated.</p>", "<title>6. OVERVIEWOF MECHANISMS OF ACTION OF PPAR<italic>γ</italic> AGONIST AND ANTAGONIST COMPOUNDS</title>", "<p>The studies reviewed above have shown\nthat the effects of PPAR<italic>γ</italic> ligands are mediated by various mechanisms.\nSome studies show or suggest canonical PPAR<italic>γ</italic>-mediated effects (i.e., via transactivation),\nas exemplified by early in vitro studies with agonist compounds that\nshowed fat accumulation, a major PPAR<italic>γ</italic>-mediated effect, in both breast cancer and\nliposarcoma cell lines [##REF##8990192##10##, ##REF##9660931##12##]. This was\nalso demonstrated in liposarcoma patients in whom increased fat content within\ntumors was demonstrated by serial CT scanning before and after treatment with\nan agonist drug [##REF##8990192##10##]. The studies of Wang et al. showed that the growth-inhibitory effects of PPAR<italic>γ</italic> agonist compounds on MM lines was seen only in\nlines expressing PPAR<italic>γ</italic> and that these effects were reversed by\ncotreatment with an antagonist compound [##REF##14975242##31##]. In contrast, completely PPAR<italic>γ</italic>-independent effects were demonstrated for both\nagonist and antagonist compounds in reports from Palakurthi et\nal. [##REF##11507074##19##] and Schaefer et al. [##REF##17096328##29##]. This was clearly shown for the agonist\ncompounds, troglitazone and ciglitazone, which showed similar antiproliferative\neffects in PPAR<italic>γ</italic>-wild type and PPAR<italic>γ</italic>-null (knockout) embryonic stem cell lines,\nboth in vitro and in vivo [##REF##11507074##19##]. PPAR<italic>γ</italic>-independent growth inhibitory and\nantimetastatic effects of several antagonist compounds were shown in both in vitro and in vivo studies using three\ncolon carcinoma cell lines. These effects were associated with reductions in\ntubulin levels and were also shown to be independent of PPAR<italic>δ</italic> and proteasome function. The PPAR<italic>γ</italic>-independent effect of agonist compounds was\nshown to be associated with inhibitory effects on the protein translation\npathway. The mechanism of PPAR<italic>γ</italic>-independent effects of antagonist compounds on\ntubulin levels has not been elucidated.</p>", "<p>The\nmechanism of PPAR<italic>γ</italic>-mediated transrepression may explain some of\nthe effects of antagonist compounds. This was suggested by the attenuation of\nthe effects of antagonist compounds by PPAR<italic>γ</italic> knockdown by siRNA in hepatocellular carcinoma\ncell lines [##REF##15781638##25##]. Also, the observation that combinations of PPAR<italic>γ</italic> agonist and antagonist compounds result in\nadditive antiproliferative effects in various cancer cell lines [##REF##15517883##24##, ##REF##17414621##30##] is\nconsistent with this mechanism. This mechanism is plausible, as it has been\nshown to inhibit the NF-<italic>κ</italic>B signaling pathway, which is central to inflammation and to the proliferation \nand survival of multiple cancer types including hepatocellular and\ncolon carcinomas as well as multiple myeloma. The potential role of this and\nother mechanisms remain to be determined.</p>", "<title>7. SUMMARY OF PRECLINICAL STUDIES OF PPAR<italic>γ</italic> ANTAGONIST COMPOUNDS AND THEIR\nCLINICAL POTENTIAL</title>", "<p>The studies reviewed above have shown that PPAR<italic>γ</italic> antagonists have in vitro and preclinical in vivo anticancer effects that are as\nbroad and potent as agonist compounds. These effects have been demonstrated in\na wide range of epithelial cancer cell lines as well as hematopoietic cancer\ncell lines. Exploration of the\nunderlying mechanisms of action for antagonist compounds has shown either involvement\nof PPAR<italic>γ</italic> or a PPAR<italic>γ</italic>-independent effect. One study suggested the\ninvolvement of the canonical transactivation mechanism in that antagonist\neffects were antagonized by coincubation with an agonist compound, pioglitazone\n[##REF##15930335##27##]. In another study, where knockdown of PPAR<italic>γ</italic> affected responses to antagonist compounds,\nthe effect was not consistent with the canonical transactivation mechanism, but\nmay be consistent with a transrepressive mechanism [##REF##15781638##25##]. Another study showed\nthat anticancer effects were associated with reductions in tubulin levels (a\nvalidated cancer-related target), but this was not mediated by PPAR<italic>γ</italic>, PPAR<italic>δ</italic>, or the proteasome [##REF##17096328##29##].</p>", "<p>While there have been numerous preclinical in vivo studies in cancer models with\nPPAR<italic>γ</italic> agonists, there have been relatively few with\nantagonist compounds. Also agonists have been tested clinically. Some studies with\nantagonists have been conducted in noncancer models at low doses (≤1 mg/kg),\nwhich were not toxic and biologically active [##REF##16484917##37##, ##REF##16215386##38##]. A chemically distinct,\nbut selective PPAR<italic>γ</italic> antagonist, SR-202, has been synthesized and evaluated\nin preclinical models (##FIG##0##Figure 1##). It was given at a dose of 400 mg/kg for\nperiods of up to 10 weeks with favorable metabolic effects such protection\nagainst diet-induced hyperinsulinemia and reduction in hyperinsulinemia and\nhyperglycemia in genetically predisposed (ob/ob) mice [##REF##12403851##39##]. In pilot studies,\nwe have administered moderate doses of GW9662 (15 mg/kg) and T0070907 (7.5\nmg/kg) daily for 3 weeks by the intraperitoneal route to immunodeficient mice.\nThese doses and schedules were well tolerated and resulted in no signs of\ntoxicity (unpublished observations). \nThese data indicate that the doses of these antagonists that may be\nsufficient for anticancer therapy are well tolerated, paving the way for\nfurther development of these agents for treatment of cancer.</p>" ]
[ "<title>ACKNOWLEDGMENTS</title>", "<p>This\nwork was supported in part by a grant from the Thomas and Agnes Carvel\nFoundation. The authors wish to thank Mary\nEllen Castillo for her technical support and assistance.</p>" ]
[ "<fig id=\"fig1\" position=\"float\"><label>Figure 1</label><caption><p>The chemical structures of four PPAR<italic>γ</italic> antagonists:\n(1) GW9662, (2) T0070907, (3) SR-202, and (4) BADGE.</p></caption></fig>", "<fig id=\"fig2\" position=\"float\"><label>Figure 2</label><caption><p>Dose-response curves for the MM line,\nKMS12-PE, to T0070907, both in the presence and absence of the hLL1 mAb. Square symbols represent the dose-response\ncurve in the presence of hLL1, and diamond symbols represent the curve in the\nabsence of LL1. The ordinate shows percent growth inhibition values and the\nabscissa the concentration of T0070907 in micromolar.</p></caption></fig>" ]
[ "<table-wrap id=\"tab1\" position=\"float\"><label>Table 1</label><caption><p>Mean IC<sub>50</sub> values (<italic>μ</italic>M) for the PPAR<italic>γ</italic> ligands.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Cell lines</th><th align=\"center\" rowspan=\"1\" colspan=\"1\"> Pioglitazone </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">T0070907 </th><th align=\"center\" rowspan=\"1\" colspan=\"1\"> GW9662 </th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Colon </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Moser<sup>#</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.5 ± 2.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.9 ± 1.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.1 ± 0.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HT29<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53.0 ± 4.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.2 ± 0.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.1 ± 0.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">LS174T<sup>#</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38.7 ± 7.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.8 ± 1.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.5 ± 0.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCT-15<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53.1 ± 2.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.0 ± 0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.0 ± 0.8</td></tr><tr><td align=\"center\" colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> RCC </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">A498<sup>#</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38.9 ± 4.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24.3 ± 0.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29.1 ± 0.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ClearCa-2<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">56.4 ± 3.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.8 ± 1.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.5 ± 0.7</td></tr><tr><td align=\"center\" colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Breast </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ZR75-30<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">77.9 ± 7.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.9 ± 0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.6 ± 0.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">MCF7<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54.8 ± 3.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.2 ± 1.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.6 ± 2.4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">MDA-MB-231<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">78.7 ± 3.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.1 ± 1.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.8 ± 1.0</td></tr><tr><td align=\"center\" colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> MM </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CAG*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">62.4 ± 9.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.2 ± 1.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.8 ± 0.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KMS12-BM<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33.2 ± 5.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.2 ± 0.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.8 ± 1.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KMS12-PE<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">56.4 ± 1.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.3 ± 0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.5 ± 0.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">OPM6<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">48.9 ± 1.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.1 ± 0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.5 ± 0.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">U266B1<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">56.6 ± 1.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.9 ± 0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29.7 ± 1.5</td></tr><tr><td align=\"center\" colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> NHL </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ramos<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">66.5 ± 7.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.7 ± 0.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.1 ± 0.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SU-DHL6<sup>§</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53.1 ± 1.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.8 ± 0.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.8 ± 0.3</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><fn><p>Mean IC<sub>50</sub> values from replicate experiments with this panel of cells for each of\nthe three PPAR<italic>γ</italic> ligands are shown above, expressed in <italic>μ</italic>M ± SEM. Cell lines are grouped according to\ncancer type. IC<sub>50</sub> values from each cell line were compared by single\nfactor ANOVA analysis, with all lines showing significant differences as\nindicated: <sup>§</sup>\n<italic>P</italic> &lt; .0001; *<italic>P</italic> &lt; .005; <sup><italic>#</italic></sup>\n<italic>P</italic> &lt; .04.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"PPAR2008-494161.001\"/>", "<graphic xlink:href=\"PPAR2008-494161.002\"/>" ]
[]
[]
{ "acronym": [], "definition": [] }
39
CC BY
no
2022-01-13 03:12:57
PPAR Res. 2008 Sep 2; 2008:494161
oa_package/b4/f9/PMC2528255.tar.gz
PMC2528256
18779872
[ "<title>1. INTRODUCTION</title>", "<p>The regulation of the distribution of motile\ncells in both normal and disease situations depends upon a variety of peptide\nand nonpeptide mediators, which stimulate cell movement by both directed\n(chemotaxis) and nondirected (chemokinesis) mechanisms. Amongst these mediators\nare the chemokines, a class of peptide mediators that play critical roles in\nnormal development, regulation of the hematopoietic and immune systems in the\nadult, and in repair processes such as wound healing and inflammation. Among\nthe different chemokines is the stromal cell-derived factor-1 (SDF-1), which is\nnow known as CXCL12. CXCL12 binds principally to the receptor CXCR4, although\nit also acts through the more-recently-described receptor CXCR7 [##REF##16107333##1##]. This\nreview describes the roles of CXCL12 and CXCR4 in normal tissue functions and\nin cancer, and suggests that the regulation of CXCR4 expression by PPAR<italic>γ</italic> may\nemerge to be a unique avenue by which a key receptor involved in cancer cell\nmetastasis can be suppressed in a way that will assist with disease therapy.</p>" ]
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[ "<p>Recommended by Dipak Panigrahy</p>", "<p>Chemokines are peptide mediators involved in normal development,\nhematopoietic and immune regulation, wound healing, and\ninflammation. Among the chemokines is CXCL12, which binds\nprincipally to its receptor CXCR4 and regulates leukocyte\nprecursor homing to bone marrow and other sites. This role of\nCXCL12/CXCR4 is “commandeered” by cancer cells to facilitate the\nspread of CXCR4-bearing tumor cells to tissues with high CXCL12\nconcentrations. High CXCR4 expression by cancer cells predisposes\nto aggressive spread and metastasis and ultimately to poor patient\noutcomes. As well as being useful as a marker for disease\nprogression, CXCR4 is a potential target for anticancer therapies.\nIt is possible to interfere directly with the CXCL12:CXCR4 axis\nusing peptide or small-molecular-weight antagonists. A further\nopportunity is offered by promoting strategies that downregulate\nCXCR4 pathways: CXCR4 expression in the tumor microenvironment is\nmodulated by factors such as hypoxia, nucleosides, and\neicosanoids. Another promising approach is through targeting PPAR\nto suppress CXCR4 expression. Endogenous PPAR<italic>γ</italic> such as 15-deoxy-Δ<sup>12,14</sup>-PGJ<sub>2</sub> and synthetic agonists such as the\nthiazolidinediones both cause downregulation of CXCR4 mRNA and\nreceptor. Adjuvant therapy using PPAR<italic>γ</italic> agonists may, by\nstimulating PPAR<italic>γ</italic>-dependent downregulation of CXCR4 on cancer cells, slow the rate of metastasis and impact beneficially on\ndisease progression.</p>" ]
[ "<title>2. CHEMOKINES AND THEIR RECEPTORS IN\nCELL REGULATION</title>", "<p>Chemokines\nare low-molecular-weight peptide ligands involved in the trafficking of\nleukocytes and other motile cells [##REF##10699158##2##, ##UREF##0##3##]. \nThere are four major groups of chemokines, the CXC, CC, C and CX3C\nchemokines, categorized as such on the basis of their number and spacing of\nconserved cysteine residues [##REF##10699158##2##, ##UREF##1##4##]. The nomenclature\nof chemokines (e.g., “CXCL12\") is made up of their subclass (CXC, CC, etc.)\nfollowed by “L” for ligand, and a specific number [##REF##10699158##2##, ##UREF##0##3##].</p>", "<p>The receptors for\nchemokines are cell-surface, seven-transmembrane G protein-coupled receptors [##REF##10699158##2##].\nThe naming of these receptors (e.g., “CXCR4\") is based on the subclass of\nchemokine that the receptor recognizes, followed by “R” for receptor and a\nnumber (which need not correspond to the number assigned to its cognate ligand(s)). There are 19 well-recognized chemokine\nreceptors (e.g., CXCR1-6, CCR1-10, CX<sub>3</sub>CR1, and XCR1) [##REF##16107333##1##, ##REF##15914470##5##]. Many\nchemokine receptors have more than one known ligand, and many chemokines can\nactivate more than one receptor. Thus,\nthere is much promiscuity in chemokine/receptor signaling.</p>", "<p>Chemokines bind within\nthe extracellular domain of the chemokine receptor, which comprises the\nN-terminus and three extracellular loops [##UREF##0##3##]. \nThe intracellular domain, which consists of three loops and the\nC-terminus, associates with G proteins that, upon activation, lead to\ninhibition of adenylyl cyclase activity [##UREF##0##3##]. Typical cellular consequences of\nchemokine binding include changes in gene expression, cell polarization, and\nchemotaxis (directed cell migration) [##UREF##1##4##].</p>", "<p>Chemokines play a\nmajor role in regulating the migration of cells of the immune system, leading\nto the modulation of immune responses. \nTheir exact role depends on the expression pattern of receptors on\nspecific leukocyte subsets [##REF##10699158##2##] but encompasses the regulation of lymphocyte\ntrafficking, lymphoid tissue development, Th1/Th2 modulation, and the effecting\nof inflammatory reactions. Chemokine receptors are also found on other cell\ntypes, and play a part in stem cell recruitment and angiogenesis, in\ndevelopment and wound healing [##UREF##1##4##]. When such pathways are subverted in\nneoplastic cells, chemokines take over prominent roles in the metastatic\nprocess, both in terms of the dissemination of cells from primary tumors and in\ngrowth of the cancer at metastatic sites. As we will see, this is the case for\nCXCR4.</p>", "<title>3. THE CHEMOKINE RECEPTOR CXCR4 AND\nITS LIGAND CXCL12 (SDF-1)</title>", "<p>The receptor now\nknown as CXCR4 was cloned in 1994, and was originally given the name leukocyte-expressed\nseven-transmembrane domain receptor (LESTR) due to its abundant expression in\nseveral leukocyte populations [##REF##8276799##6##]. It\nwas independently cloned by others and named “fusin” because of its ability to\nact as a coreceptor for HIV fusion and entry [##REF##8629022##7##]. It further has the designation “CD184” as\npart of the cluster of differentiation antigens found on activated leukocytes. LESTR/fusin/CD184\nwas originally considered to be an orphan receptor. However, the chemokine CXCL12, originally termed\nstromal cell-derived factor 1 (SDF-1), was shown by two independent research\ngroups to be a ligand for LESTR/fusin/CD184, and the name CXCR4 was proposed [##REF##8752280##8##, ##REF##8752281##9##]. The <italic>CXCR4</italic> gene is constitutively expressed, and CXCR4 protein has been\ndetected on many leukocytes, including lymphocytes, monocytes, NK cells, and\ndendritic cells; as well as on vascular smooth muscle cells, endothelial cells,\ncells lining the gastrointestinal tract, microglia, neurons, and astrocytes [##REF##9599023##10##–##REF##15246052##13##].\nUntil recently, CXCR4 was considered to be the only receptor for CXCL12, but\nthe previous orphan receptor RDC1 is now recognized as an additional CXCL12 receptor,\nfor which the name CXCR7 has been given [##REF##16107333##1##]. \nCXCL12 itself is widely expressed at different levels in many tissues [##REF##7490086##14##].</p>", "<title>4. CXCL12 AND CXCR4 IN NORMAL TISSUE FUNCTION</title>", "<p>The interplay between CXCL12 and CXCR4 is\ncritical to normal development. Indeed (and unlike mice deficient in other\nchemokine/receptors) mice lacking CXCL12 or CXCR4 die in utero or shortly after birth [##REF##10699158##2##, ##REF##8757135##15##–##REF##9634238##17##]. CXCL12/CXCR4 signaling is required during the\ndevelopment of the hematopoietic, cardiac, vascular, and nervous systems. Absence\nof this axis in embryonic life leads to defects in bone marrow myeloid cell\nformation, cardiac dysfunction due to impaired ventricular septum formation,\nand developmental defects in the cerebellum and in the vasculature of the\ngastrointestinal tract [##REF##8757135##15##–##REF##9634238##17##].</p>", "<p>In the normal\nadult, CXCL12 and CXCR4 are involved in the homing and retention of\nhematopoietic progenitor cells in the bone marrow. These progenitor cells express high levels of\nCXCR4, and are attracted to CXCL12 produced by stromal cells in specialized bone\nmarrow niches [##REF##8996247##18##]. Activating mutations\nof the <italic>CXCR4</italic> gene lead to aberrant\nretention of myeloid cells within the bone marrow [##REF##12692554##19##]. CXCL12 also acts as a\nchemoattractant for stem cells and some differentiated cells in the pathological\ncontexts of inflammation and tissue regeneration/repair [##REF##15888687##20##–##REF##17572689##24##]. It is this\nfunction of controlling cell migration and homing that is subverted in cancer.</p>", "<title>5. CXCL12 AND CXCR4 IN CANCER\nMETASTASIS AND GROWTH</title>", "<p>In many ways, the\nprocess of metastasis is similar to leukocyte and stem cell trafficking,\nprocesses which involve the CXCL12/CXCR4 axis [##REF##15888687##20##]. Cancer cells that express CXCR4 exploit the\nsame signaling pathway, leading to homing and retention in tissues that are\nrich in CXCL12.</p>", "<p>The foundation for\nour appreciation of the role that CXCR4 and CXCL12 may play in cancer\nmetastasis was set in 2001, when a landmark study by Albert Zlotnik's\ngroup demonstrated the importance of the CXCL12/CXCR4 axis in site-specific\nmetastasis of breast cancer [##REF##11242036##25##]. In that study, it was found that CXCR4\nexpression was low or undetectable in normal epithelial cells, but consistently\nupregulated in breast cancer cell lines and primary breast cancer cells at both\nthe mRNA and protein level. Human breast\ncarcinoma cells that expressed high levels of CXCR4 underwent morphological\nchanges and migrated directionally in response to CXCL12, indicating that the\nCXCR4 receptor was active. Crucially,\nthe ligand CXCL12 was highly expressed in tissues taken from human organ sites\nto which breast cancer cells metastasize, including lymph nodes, lung, liver,\nand bone marrow, but expressed at low levels in tissues that represent rare\nsites of metastasis, including the kidney, skin, and muscle. The ability of MDA-MB-231 human breast cancer\ncells (a cell line that is metastastic in experimental models) to migrate\ntowards protein extracts of lung and liver, or to produce lung and lymph node\nmetastasis after tail-vein injection or orthotopic implantation, was inhibited by\nneutralizing anti-CXCR4 and/or anti-CXCL12 antibodies. These findings were the first to show the\nbiological importance of this chemokine/receptor pair in the evolution and\nspread of cancer.</p>", "<p>Since that time,\nthe CXCL12/CXCR4 axis has been shown to be important in the progression and\nspread of more than 25 different cancers. Our present knowledge is based on\n(i) studies in cellular and animal experimental models, (ii) surveys of human tissues at\ndifferent stages of cancer progression, and (iii) population-based studies of morbidity\nand survival. A summary of present data is shown in ##TAB##0##Table 1##.</p>", "<p>CXCR4 has been\nshown to be expressed at high levels on cells of all of the major adult solid\nepithelial cancers (breast, colorectal, lung, ovary, prostate, etc.). The\nability of the cells to colonize other tissues by gaining advantage from\nCXCR4-dependent mechanisms depends on the presence of CXCL12 in the tissue\nfluid. Various studies have shown significant CXCL12 concentrations in the\nfluid-filled cavities through which many cancers disseminate, and at tissue\nlocations in which metastases characteristically develop. Biologically,\nsignificant CXCL12 levels have been\nfound in peritoneal ascites from ovarian cancer patients [##REF##11431324##26##], pleural\neffusions in lung cancer [##REF##14672915##27##], lymph nodes, bone, and lungs as well as other\ntissues [##REF##11242036##25##, ##REF##12927045##28##, ##REF##11912162##29##].</p>", "<p>Detailed studies\nof the cellular interactions involved in the metastasis of prostate cancer\ncells to bone [##REF##11912162##29##] have shown that the interaction of CXCL12 with CXCR4 plays a\nmajor role in successive steps in the metastatic process. Human osteoblasts\nexpress CXCL12 mRNA and protein, whereas prostate cancer cells express CXCR4\nmRNA and receptor. Prostate cancer cells\nthat have become disseminated into the circulation respond to the CXCL12-CXCR4\npathway by enhanced adherence to the bone marrow endothelium and migration\nacross endothelial barriers and basement membranes, ultimately adhering to\ncomponents of the bone marrow in response to a CXCL12 gradient [##REF##11912162##29##]. CXCL12\nfrom osteoblasts has also been shown to act on CXCR4 to induce release of IL-6\nfrom human squamous cell carcinoma cells to promote osteoclastogenesis [##UREF##2##30##].</p>", "<p>As well as\npromoting the migration of cancer cells and their invasion through physical\nbarriers as well as adherence to target structures, CXCL12 can act upon CXCR4\non the cancer cells to promote cancer cell growth along with other mitogenic\nfactors. This has been shown in cells from colorectal [##REF##16823836##31##], prostate [##REF##12761880##32##], and\novarian [##REF##12384559##33##] cancers. Furthermore, CXCL12 can promote cancer\ndissemination indirectly by enhancing the vascular supply, since the\nCXCL12/CXCR4 axis may also promote tumor angiogenesis. Vascular endothelial growth factor (VEGF) and\nCXCL12 have been shown to increase angiogenesis synergistically in an in vivo Matrigel assay and to promote\nproliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro [##REF##15695388##34##].</p>", "<title>6. THE EFFECT OF CXCL12 ON CELLULAR PROCESSES</title>", "<p>Activation\nof CXCR4 produces specific cellular changes that are consistent with a\nmigratory and invasive cell phenotype. Exposure of cells to CXCL12 produces upregulation\nof matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9 [##REF##11242036##25##, ##REF##11431324##26##, ##REF##11912162##29##, ##REF##15059909##35##–##REF##18416455##39##].\nIn addition, CXCL12 enhances adhesion to components of the extracellular matrix\nsuch as fibronectin, laminin, and collagen types I/III [##REF##12239174##37##, ##REF##15592929##40##], or to other\ncell types (e.g., endothelial or bone marrow stromal cells) [##REF##11912162##29##, ##REF##11591806##41##, ##REF##15548713##42##]. These\nchanges are mediated in large part by integrin signaling [##REF##11912162##29##, ##REF##14583470##43##, ##REF##15806155##44##]. Many\nsignaling pathways are activated by CXCL12 downstream of CXCR4 in cancer\ncells. For example, CXCL12 has been\nshown to increase ERK1/2 phosphorylation [30, 31, 49, 0.70, 76.78, 79], Akt\nphosphorylation [50, 77.88], and PI3K activation [##REF##15235108##45##].</p>", "<title>7. CXCR4 IN BREAST CANCER</title>", "<p>CXCR4 is expressed\nat a low level in normal breast epithelium but becomes more strongly expressed\nin the early stages of carcinogenesis, showing both a more intense immunohistochemical\nstaining pattern and an altered cellular localization in studies of human\nductal carcinoma in situ (DCIS)\n[##REF##16344916##46##, ##REF##15026622##47##]. An extensive tissue microarray study of 1808 invasive breast\ncarcinomas and 214 pre-invasive breast samples linked to clinical data has\nshown that the level of CXCR4 expression can be linked to tumor progressivity\n(tumor grade and lymph node status) and to other prognostic factors such as\nHER2 expression and hormone receptor (ER and PR) negativity, as well as to\npatient survival [##REF##16344916##46##]. These observations in human tissues have led to the view\nthat CXCR4 provides a selective advantage to newly formed neoplastic cells in\nthe early primary breast tumor as well as being important to later invasion and\nmetastasis [##REF##15246052##13##, ##REF##16344916##46##–##REF##16671092##48##]. This is consistent with observations in mouse models of\nbreast cancer in which interventions affecting CXCR4 reduced both growth of the\nprimary tumor and metastasis [##REF##15574767##49##].</p>", "<p>Prominent CXCR4\nexpression is a feature of all major histological forms of invasive breast\ncancer, including ductal, lobular, mucinous [##REF##16344916##46##], and the distinctive and\nhighly aggressive inflammatory form of the disease [##REF##17351259##50##]. Several independent\nstudies have shown that the extent and pattern of CXCR4 expression is related\nto axillary lymph node involvement in different forms and stages of breast\ncancer [##REF##12927045##28##, ##REF##16239110##51##–##REF##17763975##53##]. CXCR4 positivity has also been noted as a key feature of\nbreast carcinoma metastasis to bone [##REF##17938257##54##] and brain [##REF##17717635##55##]. The power of CXCR4 as\na marker for lymph node metastasis can be greatly increased by concurrently\nexamining the expression of additional markers such as VEGF, MMP-9, and CCR7 [##REF##17306924##38##, ##REF##16115904##56##].\nFurthermore, CXCR4 is also one of a subset of markers (the others are uPAR,\nS100A4, and vimentin) that define highly aggressive and invasive breast\ncarcinoma cells that are associated with malignant pleural or peritoneal\neffusions in breast cancer patients [##REF##17450528##57##]. CXCR4 expression is therefore a general\nmarker for the spread of breast cancer to its secondary sites, and for aggressive\nstages of the disease.</p>", "<p>There is evidence\nnot only for the use of CXCR4 as a general marker for the progression and\nmetastasis of breast cancer, but also for the identification of individual tumor\ncells as they are homing from the primary tumor to secondary sites as patients\ndevelop metastatic disease. Individual CXCR4-expressing tumor cells have been\nfound in the peripheral blood of breast cancer patients [##REF##15914200##102##], and CXCR4\nexpression in breast cancer has been associated with the presence of individual\ntumor cells in the bone marrow of patients [##REF##16132577##103##].</p>", "<title>8. CXCR4 IN COLORECTAL CANCER</title>", "<p>CXCR4 is\nabundantly expressed by colorectal carcinoma cells [##REF##10525044##104##, ##REF##10419917##105##]. The involvement of CXCR4 expression in\ncolorectal cancer progression was first shown by Roos and colleagues [##REF##12839981##71##]. CT-26 mouse colon carcinoma cells were\ntransfected with CXCL12 extended with a Lys-Asp-Glu-Leu (KDEL) sequence. The KDEL receptor functions to retain\nresident endoplasmic reticulum (ER) proteins, which contain a C-terminal KDEL\nsequence, in the ER. With this\n“intrakine approach,\" CXCL12-KDEL binds to the KDEL receptor and is retained in\nthe ER, and CXCR4 protein which binds to CXCL12 is also retained in the ER,\npreventing its expression at the cell-surface [##REF##12839981##71##, ##REF##11457880##106##]. This approach was first developed as a\nstrategy to reduce HIV infection [##UREF##3##107##]. After intrasplenic injection, CXCL12-KDEL-transfected CT-26 cells, which had reduced cell-surface CXCR4\nprotein expression, did not form liver metastases, whereas control cells did [##REF##12839981##71##]. The incidence of lung metastasis was also\nreduced with CXCL12-KDEL-transfected cells, and survival was increased. Interestingly, unlike Zlotnik's group, who\nhad suggested that CXCR4 expression was necessary for the movement of tumor cells\nto secondary sites [##REF##11242036##25##], Zeelenberg and colleagues found that CXCR4 expression\nwas not required for migration of CT-26 colorectal tumor cells to the lungs,\nbut rather for tumor expansion at secondary sites [##REF##12839981##71##]. Therefore, these authors concluded that CXCR4\nis necessary for the outgrowth of colon cancer micrometastases.</p>", "<p>Ottaiano\nand colleagues found that CXCR4 was overexpressed in human colorectal carcinoma\ntissues compared to normal tissues [##REF##15592929##40##]. \nCell-surface CXCR4 protein was also expressed at high levels on SW620,\nSW48, and SW480 colorectal carcinoma cells, and at moderate levels on Caco-2\nand LoVo cells. CXCL12 enhanced the\nchemotaxis of SW480 cells as well as their adhesion to fibronectin and collagen\ntype I/III, and both effects were blocked with an anti-CXCR4 neutralizing\nantibody. CXCL12 also induced\ncytoskeletal changes, proliferation, and ERK1/2 phosphorylation in SW480 cells. Similarly, Schimanski and colleagues found that\nSW480, SW620, and HT-29 colorectal carcinoma cells expressed CXCR4 protein, as\ndid colorectal carcinoma tissue samples [##REF##15755995##72##]. \nCXCL12 induced the chemotaxis of SW480 and SW620 cells. Kim and colleagues found that in patients with colorectal cancer with liver\nmetastases, higher CXCR4 expression was found on metastatic tissues compared to\nthe primary tumor [##REF##15837989##73##]. Furthermore,\nelevated CXCR4 expression in colorectal cancer is associated with disease\nprogression and reduced survival [##REF##15592929##40##, ##REF##15755995##72##, ##REF##15837989##73##, ##REF##16675573##75##].</p>", "<title>9. THE UTILITY OF CXCR4 AS A MARKER OF\nTUMOR PROGRESSION</title>", "<p>CXCR4\nexpression has been associated with disease progression, increased recurrence,\nand reduced survival in many cancer types, as listed in ##TAB##0##Table 1##. As pointed out\nearlier, CXCR4 protein expression is detectable in the majority of cases of DCIS\nof the breast, whereas CXCR4 levels are very low in adjacent normal breast\nepithelium [##REF##16344916##46##]. This suggests that the acquisition\nof CXCR4 expression may occur very early in malignant transformation, suggesting\nits potential as a biomarker. As indicated earlier, it has been suggested that\nCXCR4 expression may be useful as an indicator of prognosis [##REF##16115904##56##, ##REF##15837989##73##].</p>", "<p>Although\nmutations in the <italic>CXCR4</italic> gene have not\nbeen reported in the context of cancer, patients with a single nucleotide\npolymorphism in the 3′ untranslated region of the <italic>CXCL12</italic> gene had reduced incidence of long distance metastasis of\nepidermoid non-small cell lung cancer (NSCLC) [##REF##16155757##108##].</p>", "<title>10. PRECLINICAL EFFICACY OF\nANTI-CXCR4 TREATMENTS</title>", "<p>Several studies have\ndemonstrated the efficacy of strategies designed to reduce CXCR4 expression or\ninhibit its activity in preclinical models of cancer development and metastasis. A neutralizing anti-CXCR4 antibody prevented\nmetastasis of MDA-MB-231 breast cancer cells in mice [##REF##11242036##25##] and in another study reduced\ntumor growth after intraperitoneal (IP) injection of Namalwa non-Hodgkin's lymphoma cells [##REF##12036921##86##]. Interestingly, a neutralizing antibody\nagainst CXCR4 also inhibited the growth of subcutaneous tumors derived from\npancreatic cancer cells that did not themselves express CXCR4, probably because\nof the ability of the antibody to block CXCR4 on tumor vasculature [##REF##15994964##109##].</p>", "<p>CXCR4 peptide\nantagonists have also proven effective in preclinical cancer models. The CXCR4 peptide antagonist 4F-benzoyl-TN14003\ninhibited lung metastasis of MDA-MB-231 breast cancer cells [##REF##12935890##110##], and\n4F-benzoyl-TE14011 reduced pulmonary metastasis of B16-BL6 melanoma cells [##REF##15207725##111##]. Murakami and colleagues assessed the\ncontribution of CXCR4 to the metastatic process by transducing B16 murine\nmelanoma cells with CXCR4, followed by IV injection in syngeneic B57BL/6 mice [##REF##12499276##112##]. CXCR4 expression in this context led to\nincreased pulmonary metastasis, which was reduced with the CXCR4 peptide\nantagonist T22. Liang and colleagues showed that TN14003 itself, which is a 14-mer peptide CXCR4 antagonist,\ninhibited in vitro invasion of\nMDA-MB-231 breast cancer cells and lung metastasis after tail vein injection of\nthese cells, without causing any toxicity [##REF##15205345##113##].</p>", "<p>Small molecule (nonpeptide)\ninhibitors of CXCR4 have also been tested in preclinical cancer models. Rubin and colleagues showed that the\nnoncompetitive CXCR4 antagonist AMD3100 inhibited tumor growth after\nintracranial implantation of Daoy medulloblastoma cells and U87 glioblastoma\ncells [##REF##14595012##63##] and also inhibited peritoneal carcinomatosis and ascites formation\nafter IP inoculation of NUGC4 human gastric carcinoma cells [##REF##16489019##78##]. In a different approach, blocking the\nmammalian target of rapamycin (mTOR) pathway downstream of CXCR4 was shown to\nsuppress processes involved in the peritoneal dissemination of gastric cancer [##REF##18375114##114##].</p>", "<p>Liang and\ncolleagues also showed the preclinical efficacy of anti-CXCR4 treatments using\nan RNA-silencing molecular approach [##REF##15705897##115##]. \nMDA-MB-231 breast cancer cells transfected with siRNA oligonucleotides\nto knock down CXCR4 were injected into the tail veins of SCID mice. Mice received twice-weekly IV injections of\nsiRNA oligonucleotides to maintain CXCR4 knockdown. The control mice all developed lung metastases,\nwhereas only one of six mice receiving CXCR4 siRNA-transfected cells and\nfollowup injections with CXCR4 siRNA developed metastases. Stable knockdown of\nCXCR4 expression in 4T1 murine breast carcinoma cells using short hairpin RNA\nreduced orthotopic tumor growth and lung metastasis [##REF##15574767##49##]. Similarly, MDA-MB-231\ncells that had undergone stable knockdown of CXCR4 did not form tumors or lung\nmetastases after orthotopic injection into mammary fat pads of SCID mice,\nwhereas CXCR4-positive cells were tumorigenic [##REF##15472715##116##]. NSCLC 95D lung cancer cells in which CXCR4\nwas knocked down using antisense technology also formed lung metastases in\nfewer mice after SC injection compared to CXCR4 positive cells [##REF##16322285##88##]. Finally, manipulations of CXCR4 expression\nhave become possible using microRNAs (miRNAs), which are endogenous short RNAs\nwith the ability to repress the translation of target mRNAs [##REF##12600936##117##–##REF##16713585##119##]. The\napproach of expressing a synthetic miRNA against CXCR4 mRNA to knock down CXCR4\nexpression has been used successfully in MDA-MB-231 breast cancer cells, HeLa\ncervical carcinoma cells, and U2-OS osteosarcoma cells [##REF##16287976##118##, ##REF##15937477##120##, ##REF##17889832##121##]. Reduced\nCXCR4 expression in the breast cancer model was accompanied by reduced\nmigration and invasion of the cells in\nvitro and fewer lung metastases in\nvivo [##REF##17889832##121##]. These studies show the importance of CXCR4 expression in\nboth primary and secondary tumor growth.</p>", "<title>11. CLINICAL ASSESSMENT OF\nCXCR4-TARGETED REAGENTS</title>", "<p>The bicyclam\ncompound AMD3100 was developed as a small molecule CXCR4 antagonist [##REF##12815382##122##]. Although this compound has not yet been fully\nassessed in clinical trials to determine its therapeutic potential in cancer, it\nhas been examined in small trials in the context of HIV treatment and\nhematopoietic progenitor cell mobilization [##REF##10817726##123##–##REF##15752146##128##]. One trial with AMD3100 reported one patient\nwith thrombocytopenia, two patients with premature ventricular contractions,\nand several patients with paresthesias [##REF##15385732##126##]. \nAMD3100 did not reduce viral load in HIV patients [##REF##12815382##122##], but did\neffectively increase hematopoietic progenitor cell mobilization [##REF##15020611##124##, ##REF##15890685##125##, ##REF##15900288##127##, ##REF##15752146##128##]. However, the mechanisms of action are under\ndebate and may be unrelated to inhibition of CXCR4 as was first presumed.</p>", "<title>12. REGULATION OF CXCR4 EXPRESSION BY\nFACTORS WITHIN THE TUMOR</title>", "<p>Zeelenberg and\ncolleagues found that CT-26 murine colon carcinoma cells grown in vitro expressed CXCR4 mRNA, but\ncell-surface protein levels were not detectable [##REF##12839981##71##]. When the same cells were freshly isolated\nfrom lung or liver metastases or from intrasplenic tumors, cell-surface\nexpression was strongly upregulated. \nThis elevated expression was lost after 2–4 days in culture, indicating\nthat it was not due to selection of a subpopulation of cells with a high CXCR4\nexpression. The authors concluded that\nCXCR4 expression was induced by the in\nvivo tumor microenvironment. Although\nothers have shown that metastatic cells maintain high CXCR4 expression when\ncultured in vitro [##REF##17671223##129##], and\nindeed CXCR4 has been suggested as a cancer stem cell biomarker [##REF##18371365##130##], as\ndiscussed below there is substantial evidence indicating that CXCR4\nexpression is nevertheless influenced by the tumor microenvironment. Additionally,\naberrant activation of signaling pathways within cancer cells, such as those\ninitiated through HER2, can also contribute to elevated CXCR4 expression [##REF##15542430##131##].</p>", "<p>Multiple features\nand factors present in the tumor microenvironment have been shown to regulate\nCXCR4 expression on tumor cells and other cell types. One such feature is hypoxia [##REF##13679920##97##, ##REF##14597738##132##]. Solid tumors tend to be hypoxic due to\nstructural abnormalities in their vasculature [##REF##15254862##133##]. Staller and colleagues were the first to\ndemonstrate the involvement of hypoxia in the regulation of CXCR4 expression [##REF##13679920##97##]. Their goal was to identify genes regulated by\nthe von Hippel-Lindau tumor suppressor protein\n(pVHL) in renal cell carcinoma cells. pVHL is often inactivated in renal cell\ncancer (RCC) leading to constitutive activation of hypoxia-inducible factor-1\n(HIF-1) target genes. In a microarray\nanalysis, they found that CXCR4 mRNA expression was suppressed by the reintroduction of functional pVHL into\npVHL-deficient A498 RCC cells, an effect that was due to inactivation of HIF-1. CXCR4 protein was also downregulated,\nresulting in reduced migration of RCC cells towards CXCL12. Hypoxia increased CXCR4 mRNA expression in\nHEK-293 human embryonic kidney cells and primary human proximal renal tubular\nepithelial cells, and a hypoxia response element (HRE) was identified within the\nCXCR4 promoter [##REF##13679920##97##]. The authors\nspeculated that intratumoral hypoxia may lead to increased CXCR4 expression in\ndiverse types of solid tumors, increasing metastasis to distant organs. Shioppa and colleagues found that hypoxia\nincreased CXCR4 mRNA and cell-surface protein expression in several cell types,\nincluding monocytes, human monocyte-derived macrophages, tumor-associated\nmacrophages, HUVECs, CAOV3 ovarian carcinoma cells, and MCF-7 breast carcinoma\ncells, leading to increased migration towards CXCL12 due to the activation of\nHIF-1 [##REF##14597738##132##].</p>", "<p>The hypoxic environment within tumors also\nleads to high extracellular levels of adenosine (adenine-9-<italic>β</italic>-D-ribofuranoside),\na nucleoside that is involved in energy metabolism and comprises the core\nstructure for adenine nucleotides. The concentration of adenosine in the\nextracellular fluid of solid tumors is about 100-fold that of adjacent normal\ntissue [##REF##9205063##134##]. Adenosine concentrations in tumors reach levels that can act on\nany of four subtypes of adenosine-selective, G-protein-coupled receptors: A1,\nA2a, A2b, and A3 [##UREF##4##135##]. Adenosine receptors of all four known subtypes are\nexpressed differentially on different cell types within the tumor, including\nstromal cells, endothelial cells, and infiltrating leukocytes. We have shown\nthat through such receptors, adenosine can have protumor effects directly on\ncancer cells and also indirectly via other\nsupporting/infiltrating cells [##REF##11992407##136##–##REF##16611738##139##]. Adenosine also acts through A2a and\nA2b adenosine receptors on human colorectal carcinoma cells to upregulate CXCR4\nmRNA expression up to 10-fold, and selectively increase cell-surface CXCR4\nprotein up to 3-fold [##REF##16823836##31##]. This increase in cell-surface CXCR4 enables the\ncarcinoma cells to migrate toward CXCL12 and enhances their proliferation in\nresponse to CXCL12.</p>", "<p>One of the further\nmajor factors that allows tumor expansion is vascular endothelial growth factor\n(VEGF), which is also produced in response to hypoxia and which promotes\nneovascularisation of the tumor. The angiogenic effect of VEGF increases the\nsupply of nutrients and blood-borne growth factors to allow growth of the\ntumor. There is significant interplay between the roles of VEGF and CXCR4 in\ntumor expansion. Concomitan high expression of CXCR4 and VEGF has been\nobserved in colorectal [##REF##17641542##74##, ##REF##16675573##75##], breast [##REF##17306924##38##], and ovarian [##REF##15695388##34##] cancers, as well\nas in glioma [##REF##17611402##140##] and osteosarcoma [##REF##16528367##91##], in each of which it has been linked\nto increased angiogenesis, invasion, and/or metastasis. Clinical studies have\nshown that although VEGF and CXCR4 both predispose to lymphatic involvement and\nnodal metastasis in colorectal cancer, they work through different regulatory\nstrategies [##REF##17641542##74##]. Their collaborative role in angiogenesis parallels a similar\njoint action in noncancer processes involving neovascularisation (e.g., [##REF##17522382##141##]), and it has been suggested in\nthe context of tumor angiogenesis that their actions may be synergistic [##REF##15695388##34##].\nIt is not surprising that these two entities are closely linked; VEGF receptors\nand CXCR4 have common regulatory pathways. For example, interference with Notch\nsignalling leads to downregulation of both VEGF receptor 2 and CXCR4 [##REF##18339870##142##].</p>", "<p>The\nrelationship between VEGF and CXCR4 is complex. Firstly, VEGF can promote CXCR4\npathways. VEGF is present in high levels in tumors and may upregulate CXCR4\nexpression on tumor cells, as has been demonstrated in glioma [##REF##15967572##143##] and breast\ncancer [##REF##12499259##144##]. In the case of tumor cells, this upregulation of CXCR4 by VEGF\ncan happen through an autocrine mechanism [##REF##12499259##144##]. VEGF can also upregulate CXCR4\non the endothelial cells that may be involved in angiogenesis during tumor\nexpansion [##REF##10233851##145##, ##REF##17075581##146##].</p>", "<p>Conversely,\nthe ability of CXCR4 to signal through PI3K/Akt and ERK1/2 provides a route\nthrough which VEGF expression may be regulated by CXCR4 [##REF##17559806##147##–##REF##16005185##149##]. Binding of CXCL12 to CXCR4 has been shown to\nincrease cellular secretion of VEGF in ovarian cancer [##REF##17697577##150##], breast cancer [##REF##17559806##147##],\nprostate cancer [##REF##16005185##149##, ##REF##15180966##151##], and malignant glioma [##REF##16084492##152##]. This phenomenon\nparallels the ability of the CXCL12/CXCR4 axis to stimulate VEGF secretion in\nnormal lymphohematopoietic cells [##REF##11553854##153##]. One might therefore expect a large part of the antitumor activity of\nCXCR4 antagonists to be mediated through reduced secretion of VEGF. Indeed,\ninterference with the CXCL12-CXCR4 pathway has been shown to cause\ndownregulation of expression of VEGF [##REF##18416455##39##]. However, blocking the CXCL12/CXCR4\naxis in vivo can inhibit tumor\ngrowth and angiogenesis without producing alterations in VEGF pathways [##REF##15994964##109##].</p>", "<p>Other\ngrowth factors whose levels are elevated in tumors may also enhance\nCXCR4-dependent mechanisms. Tumors have high levels of tumor necrosis factor-<italic>α</italic>\n(TNF-<italic>α</italic>), derived primarily from tumor-associated macrophages (TAMs) [##REF##8119765##154##–##REF##16951987##156##]. TNF-<italic>α</italic> itself, or macrophages that serve as a source of TNF-<italic>α</italic>, are\nable to increase CXCR4 mRNA and cell-surface protein expression on ovarian\ncancer cells [##REF##16288025##157##] and astroglioma cells [##REF##11160334##158##]. A significant correlation\nbetween TNF-<italic>α</italic> and CXCR4 expression was found in ovarian cancer biopsies [##REF##16288025##157##]. The increase in CXCR4 at a cellular level\nappears to be due to TNF-<italic>α</italic>-induced activation of NF-<italic>κ</italic>B signaling and is\nassociated with enhanced migration towards CXCL12 [##REF##16288025##157##]. Therefore, TAMs may contribute to increased\nCXCR4 expression on cancer cells via\nproduction of TNF-<italic>α</italic>.</p>", "<p>Finally,\npolypeptide growth factors that are associated with the extracellular matrix,\nand indeed components of the extracellular matrix itself, can upregulate CXCR4\non cancer cells. Transforming growth\nfactor-<italic>β</italic> (TGF-<italic>β</italic>) increases cell-surface CXCR4 protein expression on human\nmelanoma cells [##REF##15059909##35##] and we have recently found that FGF-2 upregulates CXCR4 on\nhuman colorectal cancer cells (Bseso B and Blay J, manuscript in preparation). Furthermore, type-I collagen and the\npreparation Matrigel, which is a secreted ECM rich in laminin [##REF##15975825##159##], also\nincrease levels of CXCR4 on melanoma cells [##REF##15059909##35##]. Therefore, interactions with matrix proteins\nwithin tumors may also increase CXCR4 expression.</p>", "<title>13. THE ROLE OF CYCLOOXYGENASE-2\nAND PGE<sub><bold>2</bold></sub> IN CANCER </title>", "<p>The shift to\nmalignancy in epithelia and indeed the progression to invasion and metastasis\nare associated with increased expression of the enzyme cyclooxygenase-2 (COX-2)\n[##REF##7926468##160##–##REF##15623626##163##]. High COX-2 expression is in\ncancer is often associated with reduced patient survival [##REF##15623626##163##]. The immediate\neffect of high COX-2 expression is increased prostaglandin synthesis, particularly\nprostaglandin E<sub>2</sub> (PGE<sub>2</sub>) [##REF##11278747##164##], which in experimental\nmodels is associated with the production of vascular loops and arches and\nevidence of abnormal vessel function [##REF##14688410##165##], a phenotype consistent with tumor\nangiogenesis. Observations of increased\nexpression of angiogenic regulatory genes, including VEGF, ang-1, and ang-2 are\nconsistent with this view [##REF##15930264##166##]. Furthermore, nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit\ncyclooxygenases, reduce both tumor incidence and microvessel density in\nCOX-2-expressing mice [##REF##15930264##166##] and reduce cancer progression in preclinical models\nand clinical trials [##REF##11900248##167##]. Indeed, NSAIDs\nand COX-2 inhibitors reduce the relative risk of developing colorectal cancer\nby 40–50% [##REF##11900248##167##–##REF##15887126##169##].</p>", "<p>Tumor-promoting\neffects of COX-2 overexpression appear to be due in large part to increased PGE<sub>2</sub> production [##REF##8228569##170##–##REF##12455057##173##]. Associated with the increase in COX-2, there is a\ndecreased expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an\nenzyme involved in the inactivation of PGE<sub>2</sub>, in cancer compared to normal tissues [##REF##15542609##174##], as well as\nupregulation of cytosolic PLA<sub>2</sub> (cPLA<sub>2</sub>), which increases the supply of arachidonic acid substrate\nfor COX-2 [##UREF##6##175##–##REF##12048163##177##]. In addition to promoting angiogenesis, PGE<sub>2</sub> also stimulates cancer cell proliferation [##REF##7548186##178##, ##REF##15753380##179##], promotes cell migration [##REF##14499627##180##],\nand causes transactivation of polypeptide growth factor receptors [##UREF##7##181##].</p>", "<title>14. OTHER PROSTAGLANDINS IN CANCER</title>", "<p>Prostaglandins\ntogether with the thromboxanes are classed as prostanoids, and belong to a larger group of compounds\nreferred to as eicosanoids [##REF##11729303##182##]. The main prostanoids apart from PGE<sub>2</sub> are prostaglandin F<sub>2<italic>α</italic></sub> (PGF<sub>2<italic>α</italic></sub>), prostaglandin D<sub>2</sub> (PGD<sub>2</sub>), prostaglandin I<sub>2</sub> (PGI<sub>2</sub> or\nprostacyclin), and thromboxane A<sub>2</sub> (TXA<sub>2</sub>). As well as\nreflecting changes in COX-2, cPLA<sub>2</sub>, and inactivating enzymes, the\nlevels of different prostanoids in tumors can be modulated by altered expression\nof specific prostaglandin synthases [##REF##12738712##183##]. Prostaglandins can also be metabolized\nnonenzymatically to form a range of products both in the body and in cell\nculture. PGD<sub>2</sub> can be\nconverted to cyclopentenone J-series prostaglandins, including prostaglandin J<sub>2</sub> (PGJ<sub>2</sub>), 9-deoxy-Δ<sup>9</sup>,Δ<sup>12‐13,14</sup>-dihydro-PGD<sub>2</sub> (Δ<sup>12</sup>-PGJ<sub>2</sub>), and 15-deoxy-Δ<sup>12,14</sup>-PGJ<sub>2</sub> (15dPGJ<sub>2</sub>); PGE<sub>2</sub> can be converted to prostaglandin A<sub>2</sub> (PGA<sub>2</sub>) [##REF##6578214##184##–##REF##1789996##186##]. The tumor microenvironment therefore has a rich\nand varied content of eicosanoid mediators.</p>", "<title>15. PROSTAGLANDIN EFFECTS ON CANCER CELLS</title>", "<p>Although the major\nfocus of attention has been on PGE<sub>2</sub>, a range of eicosanoids acts to\nrestrain tumor growth. Indeed the PGE<sub>2</sub> metabolite PGA<sub>2</sub> reduces\ncell number and induces apoptosis and cell cycle changes in both human breast\ncancer cells and human epithelial cervical carcinoma cells [##REF##12618334##187##].</p>", "<p>More notably, PGD<sub>2</sub> and its series of derivatives have anticancer effects. PGD<sub>2</sub> itself can reduce the growth of \ncarcinoma cells [##REF##9697269##188##]. However, other studies have shown that the nonenzymatic\nbreakdown of PGD<sub>2</sub> to sequential metabolites (##FIG##0##Figure 1##) may be required\nfor growth inhibition and that the latter metabolites are the active\neicosanoids [##REF##3857041##189##–##REF##15498850##194##]. PGD<sub>2</sub> therefore can act independently of its DP\nreceptors by its metabolism through a dehydration reaction to prostaglandin J<sub>2</sub> (PGJ<sub>2</sub>), Δ<sup>12</sup>-PGJ<sub>2</sub>, and then to\n15-deoxy-Δ<sup>12,14</sup>-prostaglandin J<sub>2</sub>(15dPGJ<sub>2</sub>) [##REF##6578214##184##].\nThis reaction occurs in cell culture media, both in the presence and absence of\nserum [##REF##6578214##184##, ##REF##3857041##189##, ##REF##11786541##195##]. Therefore, it is\npossible that many effects noted in\nvitro with PGD<sub>2</sub> are actually due to the formation of J-series\nprostaglandins. Frequent replacement\nwith fresh medium containing PGD<sub>2</sub> in such circumstances can\neliminate the response, while the addition of the metabolite(s) themselves\nleads to growth inhibition in a shorter timeframe than PGD<sub>2</sub> itself [##REF##3857041##189##].\nSome workers have proposed that Δ<sup>12</sup>-PGJ<sub>2</sub> is the key\nmetabolite [##REF##3857041##189##]; but in fact all of the successive J-series prostaglandins,\nthat is, PGJ<sub>2</sub>, Δ<sup>12</sup>-PGJ<sub>2</sub>, and 15dPGJ<sub>2</sub>,\nare able to reduce proliferation and induce apoptosis of cancer cells [##REF##10506103##190##]. Furthermore, the end metabolite 15dPGJ<sub>2</sub> is active against many cell types, including colorectal carcinoma cells [##REF##10076568##191##, ##REF##11950812##192##],\nprostate carcinoma cells [##REF##10984506##193##], and Burkitt lymphoma cells [##REF##15498850##194##], suggesting\nthat 15dPGJ<sub>2</sub> may be the crucial mediator.</p>", "<title>16. THE ROLE OF 15dPGJ<sub><bold>2</bold></sub> AND ITS ACTION ON PPAR<italic>γ</italic>\n</title>", "<p>15dPGJ<sub>2</sub> is an agonist for the nuclear receptor peroxisome proliferator-activated\nreceptor <italic>γ</italic> (PPAR<italic>γ</italic>) [##REF##8521497##196##, ##REF##8521498##197##], and activation of PPAR<italic>γ</italic> may account for the\ngrowth inhibitory effects of 15dPGJ<sub>2</sub>. PPAR<italic>γ</italic> activation results in\nits heterodimerization with the retinoid X receptor (RXR), binding to\nperoxisome proliferator response elements (PPREs) on DNA, and subsequent\nactivation of target gene expression [##REF##15629253##198##]. \nPPAR<italic>γ</italic> is aberrantly expressed in some cancer types [##REF##9472692##199##], and in many\ncases its activation leads to cell death or differentiation [##REF##10076568##191##, ##REF##9797355##200##, ##REF##9671760##201##]. This\naction of 15dPGJ<sub>2</sub>, and by extension its precursors PGD<sub>2</sub>,\nPGJ<sub>2</sub>, and Δ<sup>12</sup>-PGJ<sub>2</sub>, may underlie the\nmajor action of these eicosanoids on cell growth. For example, 15dPGJ<sub>2</sub> reduces the growth of PC-3 human prostate cancer cells through the activation\nof PPAR<italic>γ</italic> [##REF##16024620##202##]. However, in addition to\ndirect growth-inhibitory effects, 15dPGJ<sub>2</sub> may also exert anticancer\neffects by reducing expression of protumor proteins. For example, 15dPGJ<sub>2</sub> inhibits\nphorbol ester-induced VEGF and COX-2 expression in SW620 human colorectal\ncarcinoma cells [##REF##15289320##203##].</p>", "<title>17. 15dPGJ<sub><bold>2</bold></sub> CAUSES DOWNREGULATION OF\nCXCR4 ON CANCER CELLS</title>", "<p>In\nour studies of the possible effects of these different prostaglandins on CXCR4,\nwe focused upon the expression of the mature protein and furthermore restricted\nour quantitation exclusively to the receptor that is displayed to the external\nenvironment at the cell surface [##REF##16823836##31##]. Cell-surface CXCR4 reflects functional\nreceptor that is coupled to cellular responses [##REF##16823836##31##] rather than the very large\nintracellular pool of inaccessible receptor protein [##REF##15755995##72##].</p>", "<p>Although\nPGF<sub>2<italic>α</italic></sub> (to some extent) and PGE<sub>2</sub> (as well as its product\nPGA<sub>2</sub>) have some ability to modulate CXCR4 levels, by far the most\npotent prostaglandins in this regard are PGD<sub>2</sub> and its derivatives [##REF##17707368##204##]. Prostaglandin D<sub>2</sub> and the J-series\nprostaglandins used at low micromolar concentrations cause substantial loss of\nCXCR4 from the surface of HT-29 human colorectal carcinoma cells [##REF##17707368##204##]. In particular, 15dPGJ<sub>2</sub> completely\neliminates cell-surface CXCR4 at a concentration of 10<sup>−5</sup> M in vitro, and has significant effects\nafter a single dose of 300 nM, about 100-fold less than for PGF<sub>2<italic>α</italic></sub> [##REF##17707368##204##].\nThe time course of the decline in cell-surface CXCR4 protein is slow, reaching\na maximum only after 48–72 hours (##FIG##1##Figure 2##). The concentrations of prostaglandins\nthat are needed to cause downregulation after a single dose likely grossly\noverestimate the steady-state levels that would cause such a response, as we\nhave found in other studies with labile metabolites [##REF##16823836##31##, ##REF##15215186##138##]. We estimate that the\neffect of 15dPGJ<sub>2</sub> on CXCR4 is achievable with concentrations of\n15dPGJ<sub>2</sub> present in vivo.</p>", "<p>As\ncan be seen in ##FIG##1##Figure 2##, the response to 15dPGJ<sub>2</sub> occurs more rapidly\nthan that to PGJ<sub>2</sub>, which in turn has a more rapid onset than PGD<sub>2</sub>.\nWe further found that each of these prostaglandins does suppress CXCR4 mRNA\nexpression and that the effect of 15dPGJ<sub>2</sub> again occurs earlier than\nthat of PGD<sub>2</sub> [##REF##17707368##204##]. The different relative kinetics of the\ndownregulation of CXCR4 for the J-series prostaglandins are consistent with\ndata on the conversion of PGD<sub>2</sub> through to 15dPGJ<sub>2</sub> [##REF##3857041##189##] pointing\nto 15dPGJ<sub>2</sub> as the key factor in controlling the levels of functional\nCXCR4. PGD<sub>2</sub> produces similar downregulation of CXCR4 in other cell\ntypes such as the T47D human breast carcinoma cell line (Richard CL, Blay J,\nunpublished observations), suggesting that this may be a common phenomenon. The\ndownregulation of CXCR4 expression by 15dPGJ<sub>2</sub> differs from 15 dPGJ<sub>2</sub>-mediated\ndownregulation of other proteins, including cyclin D1 and estrogen receptor <italic>α</italic>,\nwhich has been shown to occur through protein degradation rather than through\nchanges in transcription [##REF##12615709##205##].</p>", "<title>18. 15dPGJ<sub><bold>2</bold></sub> DOWNREGULATES CXCR4\nPRIMARILY VIA PPAR<italic>γ</italic>\n</title>", "<p>The main target\nfor 15dPGJ<sub>2</sub> is the nuclear receptor PPAR<italic>γ</italic> [##REF##8521497##196##, ##REF##8521498##197##]. We found that\nthe ability of 15dPGJ<sub>2</sub> to downregulate CXCR4 occurred primarily\nthrough this pathway. The effect of 15dPGJ<sub>2</sub> was mimicked by PPAR<italic>γ</italic>\nagonists such as rosiglitazone (##TAB##1##Table 2##, [##REF##7768881##206##]), and antagonized or blocked by\nthe PPAR<italic>γ</italic> antagonists GW9662 and T0070907 [##REF##17707368##204##], which are irreversible\ninhibitors of PPAR<italic>γ</italic> [##REF##11877444##207##, ##REF##12022867##208##]. A minor part of the downregulatory activity of\n15dPGJ<sub>2</sub> was due to the inhibition of NF<italic>κ</italic>B since the 15dPGJ<sub>2</sub> analogue CAY10410 (9,10-dihydro-15-deoxy-Δ<sup>12,14</sup>-prostaglandin J<sub>2</sub>)\n[##REF##15755849##209##, ##REF##15695504##210##], which retains the ability to act on PPAR<italic>γ</italic> but lacks the ability of\n15dPGJ<sub>2</sub> to inhibit NF<italic>κ</italic>B, was less potent than 15dPGJ<sub>2</sub> [##REF##12022867##208##]. It is the cyclopentenone structure of 15dPGJ<sub>2</sub> (not present in CAY10410) that confers an ability to inhibit NF<italic>κ</italic>B [##REF##10781090##211##].\nConsistent with a role for this structure, cyclopentenone itself (but not\ncyclopentane or cyclopentene) caused downregulation of CXCR4 [##REF##17707368##204##]. Furthermore,\nsince PGA<sub>2</sub> possesses the cyclopentenone configuration [##REF##10638762##212##], this\nexplains the ability of PGA<sub>2</sub> (and that of PGE<sub>2</sub>) to\ndownregulate CXCR4, although it does not contain the <italic>α</italic>,<italic>β</italic>-unsaturated ketone\nmoiety necessary to activate PPAR<italic>γ</italic> signaling [##REF##15695504##210##].</p>", "<p>The\nexistence of a mechanism of 15dPGJ<sub>2</sub>-induced CXCR4 downregulation\nmay, in evolutionary terms, be an extension of the anti-inflammatory effects of\n15dPGJ<sub>2</sub>. Late in the inflammation process the prostaglandin profile\nshifts from a PGE<sub>2</sub>-rich state to a PGD<sub>2</sub>-rich (and\ntherefore 15dPGJ<sub>2</sub>-rich) state, leading to the resolution of\ninflammation [##REF##16084489##213##]. Reduced CXCR4\nexpression may be an additional mechanism by which 15dPGJ<sub>2</sub> attempts\nthe resolution of inflammation.</p>", "<p>It\nis clear that this mechanism is not operative in the context of metastatic\ntumors, because CXCR4 levels are characteristically high (##TAB##0##Table 1##). Unlike PGE<sub>2</sub> which is present in elevated concentration in tumors [##REF##8228569##170##–##REF##12455057##173##], 15dPGJ<sub>2</sub> levels are likely low in tumors compared to normal tissue. Levels of its precursor\nPGD<sub>2</sub> are low in tissues of familial adenomatous polyposis, a\ncondition that predisposes to colorectal cancer [##REF##9512123##172##], and have been negatively\ncorrelated with hepatic metastasis in tumor tissues taken from patients with\ncolorectal cancer [##REF##9697269##188##]. The enzyme involved in PGD<sub>2</sub> synthesis, PGD\nsynthase (PGDS), is decreased in cerebrospinal fluid of brain cancer patients\ncompared to patients without disease [##REF##9844724##214##]. There is a contested report of\nlevels of 15dPGJ<sub>2</sub> being decreased during breast cancer progression,\nwith the lowest levels being detected in metastatic disease [##REF##12455057##173##]. Finally,\nmechanisms to sequester or eliminate 15dPGJ<sub>2</sub> may be upregulated in\ncancer [##REF##12731885##215##, ##REF##14979731##216##]. Overall, it seems that the predominant prostaglandin within tumors\nis PGE<sub>2</sub>, and 15dPGJ<sub>2</sub> may not be present in high levels at\nall. Thus, 15dPGJ<sub>2</sub>-dependent suppression of CXCR4 seems to be a\nrestraint mechanism that is not operative in a cancer situation.</p>", "<title>19. SYNTHETIC PPAR<italic>γ</italic> AGONISTS DOWNREGULATE\nCXCR4 ON CANCER CELLS</title>", "<p>As indicated\nabove, the PPAR<italic>γ</italic> agonist rosiglitazone also decreased CXCR4 expression on human\ncolorectal cancer cells, congruent with an effect of 15dPGJ<sub>2</sub> through\nPPAR<italic>γ</italic>. This effect was seen at both the mRNA and protein level, and was more\ndurable than the effect of 15dPGJ<sub>2</sub>, as it would be expected for a\nmore chemically stable ligand [##REF##17390024##101##, ##REF##17707368##204##]. Moreover, we found that other\nglitazone agents also downregulate CXCR4, with a rank order of potency\n(rosiglitazone &gt; pioglitazone &gt; ciglitazone &gt; troglitazone) consistent\nwith their potencies for interaction with PPAR<italic>γ</italic> [##REF##7768881##206##, ##REF##9830009##217##, ##REF##10871190##218##]. Further confirming that these agents were\nacting through their expected target, PPAR<italic>γ</italic>, and that this target is linked to\nelimination or reduction of CXCR4 at the cell surface, we showed that the\nability of rosiglitazone to decrease CXCR4 was blocked by the PPAR<italic>γ</italic> antagonists\nGW9662 and T0070907 (##TAB##1##Table 2##), or by shRNA knockdown of PPAR<italic>γ</italic> expression in the\ncancer cells [##REF##17390024##101##].</p>", "<p>Therefore,\nrosiglitazone and its analogues act through PPAR<italic>γ</italic> to cause substantial and\npersistent suppression of CXCR4 on cancer cells. Since these agents are the\nsame chemicals as the thiazolidinedione (TZD) class of drugs that have been\nused clinically for the treatment of diabetes (although recent concerns\nregarding side effects have limited their utility), it opens up the possibility\nthat we may already have a means to manipulate CXCR4 levels in cancer. Given\nthat CXCR4 expression is linked to metastasis, judicious use of TZDs may allow\nus an opportunity to influence the metastatic process (##FIG##2##Figure 3##). Recent studies\nhave shown that a unique population of CXCR4+ stem cells may be crucial for\nexpansion of tumor cell populations [##REF##18371365##130##]. We suggest that TZD therapy, by\nstimulating PPAR<italic>γ</italic>-dependent downregulation of CXCR4 on cancer cells, may slow\nthe rate of metastasis and may impact beneficially on disease progression.</p>" ]
[ "<title>ACKNOWLEDGMENTS</title>", "<p>This work was supported by grants\nto Jonathan Blay from the Natural Sciences and Engineering Research Council of\nCanada (NSERC), and the Canadian Institutes for Health Research (CIHR); and by\nstudentship awards to Cynthia Lee Richard from NSERC, the Killam\nFoundation, and Cancer Care Nova Scotia.</p>" ]
[ "<fig id=\"fig1\" position=\"float\"><label>Figure 1</label><caption><p>Production of PGD<sub>2</sub> and conversion\nto its metabolites. Prostanoids follow an initial common pathway in which\narachidonic acid is released from membrane phospholipids by phospholipase A<sub>2</sub> and then converted to the short-term intermediates PGG<sub>2</sub> and PGH<sub>2</sub> by cyclooxygenases. Prostaglandin D synthase forms PGD<sub>2</sub> itself, but\nsubsequent nonenzymatic reactions in aqueous media lead to the sequential\nproduction of prostaglandin J<sub>2</sub> (PGJ<sub>2</sub>), 9-deoxy-Δ<sup>9</sup>,Δ<sup>12‐13,14</sup>-dihydro-PGD<sub>2</sub> (Δ<sup>12</sup>‐PGJ<sub>2</sub>),\nand 15-deoxy-Δ<sup>12,14</sup>‐PGJ<sub>2</sub> (15dPGJ<sub>2</sub>).</p></caption></fig>", "<fig id=\"fig2\" position=\"float\"><label>Figure 2</label><caption><p>Time course of changes in cell-surface CXCR4\nprotein expression on HT-29 cells by PGD<sub>2</sub> and its metabolites. HT-29 cells were treated with vehicle or\nwith 10 <italic>μ</italic>M PGD<sub>2</sub> (light gray bars), 10 <italic>μ</italic>M PGJ<sub>2</sub> (dark gray\nbars), or 3 <italic>μ</italic>M 15dPGJ<sub>2</sub> (hatched bars), and cell-surface CXCR4\nprotein expression was measured at the indicated time points. The data shown\nare expressed relative to the level of CXCR4 receptor on cells treated with\nvehicle alone at that time point. Values have also been corrected for any\npossible changes in cell number. The data are mean values ± SE \n(<italic>n</italic> = 4). Significant decrease due to prostaglandin, **<italic>P</italic> &lt; .01;*<italic>P</italic> &lt; .05. The figure is taken from\n[##REF##17707368##204##] with permission.</p></caption></fig>", "<fig id=\"fig3\" position=\"float\"><label>Figure 3</label><caption><p>How PPAR<italic>γ</italic>\ndownregulation of CXCR4 may act to decrease metastasis. Tumor cells typically have high levels of\nCXCR4 at their cell surface. During metastasis, cancer cells that find their\nway into the bloodstream lodge in tissues that have high concentrations of CXCL12\n(e.g., lungs, liver, and bone marrow). CXCL12 both encourages the entry of\ncells into the tissue and promotes growth of the cell population. Downregulation\nof CXCR4 by PPAR<italic>γ</italic> activation (endogenous 15dPGJ<sub>2</sub> or\nthiazolidinedione drugs, TZDs) will interfere with this process and may impede\nmetastasis.</p></caption></fig>" ]
[ "<table-wrap id=\"tab1\" position=\"float\"><label>Table 1</label><caption><p>Involvement of CXCL12/CXCR4 in different\ncancers.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Cancer</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Comments</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">References</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Acute lymphoblastic leukemia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Levels of CXCR4 are elevated on\nlymphoblasts. Elevated levels of CXCR4 are associated with increased\ninfiltration in liver and spleen</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##11736934##58##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Acute myelogenous leukemia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">High CXCR4 expression is associated with relapse and reduced survival</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##16888090##59##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Brain cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 expression is demonstrated\nin tissues and cell lines derived from glioblastoma, medulloblastoma, and\nastrocytoma. Cell lines respond to CXCL12 with increased proliferation,\nsurvival and migration. Gliomas expressing CXCR4 are associated with\nincreased tumor size and reduced survival</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##11591806##41##, ##REF##9808513##60##–##REF##17881971##64##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Breast cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">High CXCR4 expression is noted in breast cancer tissues compared to normal tissues and cell lines with invasive characteristics. CXCR4 expression is associated with more extensive lymph node metastasis and with liver metastasis, although CXCR4 expression in lymph node metastases may be lower than primary cancers. CXCR4 co-expression with HER2/neu is an indicator of more extensive lymph node involvement</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##11242036##25##, ##REF##12927045##28##, ##REF##16061624##65##–##REF##16756955##67##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cervical cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 expression is associated with increased tumor size, stromal invasion, lymph node metastasis, and reduced survival</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##17032700##68##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Chronic lymphocytic leukemia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Malignant B cells express 3- to 4-fold higher cell-surface CXCR4 levels than normal B cells. High CXCR4 expression on B cells is associated with reduced survival in patients with familial chronic lymphocytic leukemia</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##10602415##69##, ##REF##12150154##70##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Colorectal cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 is over-expressed in colorectal carcinoma tissues compared to normal tissues, and on certain established cell lines. In patients with liver metastasis, higher CXCR4 expression is found on liver metastases compared to the primary tumor. In patients with stage I/II disease, high CXCR4 mRNA expression in tumor samples is associated with increased disease recurrence. In patients with stage IV disease, patients with high CXCR4 have decreased overall survival. High CXCR4 expression is associated with increased lymph node involvement and distant metastasis, as well as reduced 3-year\nsurvival</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##15592929##40##, ##REF##12839981##71##–##REF##16675573##75##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Endometrial cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Endometrial adenocarcinoma\ntissues and human cell lines express CXCR4 protein. CXCL12 induces proliferation of endometrial\ncarcinoma cells</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##15146553##76##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Esophageal cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 expression is associated\nwith reduced survival and increased lymph node/bone marrow metastasis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##16368946##77##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gastric cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A majority of primary gastric tumors\nand many human gastric carcinoma cell lines express CXCR4. Primary tumors\nthat express CXCR4 protein are associated with peritoneal carcinomatosis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##16489019##78##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Head and neck squamous cell\ncancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 expression is found in\ntissues and cell lines. High CXCR4 expression is associated with increased\noccurrence of distant metastases and reduced survival</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##15837745##79##, ##REF##18357387##80##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hepatocellular carcinoma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 is correlated with tumor\nprogression, metastasis, and reduced survival</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##16819541##81##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Melanoma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 protein is expressed on\nhuman melanoma cell lines, as well as on cells isolated from melanoma surgical\nspecimens. CXCL12 enhances cell\nadhesion to fibronectin, the binding of murine melanoma cells to endothelial\ncells, and invasion of human melanoma cells across basement membranes. CXCR4\nexpression is associated with reduced disease-free survival and overall\nsurvival</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##15059909##35##, ##REF##14583470##43##, ##REF##11571298##82##, ##REF##15756007##83##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Multiple myeloma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Multiple myeloma cells isolated\nfrom bone marrow and multiple myeloma cell lines express cell-surface CXCR4\nprotein. CXCL12 enhances adhesion to fibronectin and stimulates cell\nmigration</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##11154207##84##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Nasopharyngeal cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Most primary human nasopharyngeal\ncarcinoma biopsy samples and metastatic lymph nodes stain positively for\nCXCR4 protein. Nasopharyngeal carcinoma cell lines also express CXCR4 mRNA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##16000558##85##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Non-Hodgkin's lymphoma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Most tissue samples and cell\nlines express high levels of CXCR4 mRNA and cell-surface protein. CXCR4 is\nimplicated in transendothelial migration and proliferation of non-Hodgkin's\nlymphoma cells</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##12036921##86##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Nonmelanoma skin cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 is expressed on invasive\nsquamous cell carcinoma and basal cell carcinoma tissues. Expression on invasive squamous cell\ncarcinoma is increased compared to normal skin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##18312436##87##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Non-small cell lung cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 mRNA is upregulated in\nNSCLC tissues compared to normal tissues, and levels are higher in tissue\nsamples taken from patients with metastasis than from those without\nmetastasis. Overexpression of CXCR4 in\nNSCLC cells leads to enhanced migratory, invasive, and adhesive responses to\nCXCL12. Nuclear CXCR4 staining is\nassociated with longer survival and reduced incidence of metastasis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##16322285##88##, ##REF##15033669##89##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Osteosarcoma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 mRNA is expressed in most\nhuman osteosarcoma samples, and two of three osteosarcoma cell lines. CXCR4\nexpression is higher at metastatic sites than in the primary tumor</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##15814634##90##, ##REF##16528367##91##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ovarian cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 mRNA is expressed in\novarian cancer cell lines, as well as in biopsies from primary tumors and\novarian cancer ascites. High levels of CXCL12 are present in ascitic fluid\ntaken from patients with ovarian cancer. CXCL12 stimulates the growth of\novarian cancer cells. CXCR4 expression is associated with increased\nrecurrence and reduced survival</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##11431324##26##, ##REF##12384559##33##, ##REF##16631235##92##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pancreatic cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Most human pancreatic cancer\ntissues stain positively for CXCR4 expression, and more than half of\npancreatic cancer cell lines express CXCR4 mRNA and cell-surface protein.\nCXCL12 induces chemotaxis of human pancreatic carcinoma cells, as well as\nstimulates proliferation and promoted survival</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##15548713##42##, ##REF##10999740##93##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer cell lines\nexpress CXCR4 mRNA and protein, and approximately half of prostate cancer\ntissues stain positively for CXCR4. Treatment of cells with CXCL12 increases\ntheir adherence to osteosarcoma cells and bone marrow endothelial cells,\ntransendothelial migration, and invasion into Matrigel. CXCR4 expression is a\npositive predictor of bone metastasis, particularly in patients with elevated\nprostate specific antigen (PSA) levels. High CXCR4 expression is associated\nwith increased cancer-specific mortality</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##11912162##29##, ##REF##15467730##36##, ##REF##15240098##94##, ##REF##18201276##95##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Renal cell cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">One of four human renal cell\ncancer lines express CXCR4 mRNA, which is upregulated in renal cell cancer tumor\nsamples compared to normal tissue. High CXCR4 expression is associated with\npoor tumor-specific survival, independent of tumour stage and differentiation\ngrade</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##11953881##96##, ##REF##13679920##97##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rhabdomyo sarcoma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Several rhabdomyosarcoma cell\nlines express cell-surface CXCR4 protein. CXCL12 increases cell motility,\ninduces chemotaxis, increases adhesion to extracellular matrix, and\nstimulates secretion of MMP-2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##12239174##37##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Small cell lung cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4 mRNA and cell-surface protein\nare detected in cell lines. CXCL12 induces proliferation, increases adherence\nand motility, and induces morphological changes such as filopodia formation</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##12414661##98##]</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Thyroid cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Human thyroid carcinoma cell\nlines express CXCR4 protein, and CXCR4 is upregulated in primary papillary\nthyroid carcinomas compared to normal thyroid tissue. CXCL12 increases\nproliferation, inhibits apoptosis, and increases migration and invasion of\nhuman thyroid cancer cells</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##12519884##99##, ##REF##15184868##100##]</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tab2\" position=\"float\"><label>Table 2</label><caption><p>Rosiglitazone\ndownregulation of CXCR4 on HT-29 cells and suppression by PPAR<italic>γ</italic> antagonists. HT-29 cells were treated with\nthe PPAR<italic>γ</italic> antagonists (I) GW9662 at 1 <italic>μ</italic>M or (II) T0070907 at 100 nM for 30 minutes before exposure to\nrosiglitazone (10 nM). Cell-surface CXCR4 protein expression was measured after\n48 hours. The data are mean values ± SE\n(<italic>n</italic> = 4). The table is taken from [##REF##17390024##101##] with permission.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\" colspan=\"1\">Experiment </th><th align=\"center\" rowspan=\"2\" colspan=\"1\">PPAR<italic>γ</italic> antagonist</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">Treatment </th><th align=\"center\" rowspan=\"2\" colspan=\"1\">Decrease due to rosiglitazone (%)</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Control</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Rosiglitazone</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">I</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Control</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.53 ± 0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.95 ± 0.09***\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">63</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">GW9662</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.47 ± 0.22</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.43 ± 0.27 n.s.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">II</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Control</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.90 ± 0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.81 ± 0.11**\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">57</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">T0070907</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.74 ± 0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.07 ± 0.18 n.s.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><fn id=\"TF1\"><p>Significant change due to\nrosiglitazone, ***<italic>P</italic> &lt; .001; **<italic>P</italic> &lt; .01; n.s.: not significant.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"PPAR2008-769413.001\"/>", "<graphic xlink:href=\"PPAR2008-769413.002\"/>", "<graphic xlink:href=\"PPAR2008-769413.003\"/>" ]
[]
[{"label": ["3"], "surname": ["Mellado", "Rodr\u00edguez-Frade", "Ma\u00f1es", "Mart\u00ednez-A"], "given-names": ["M", "JM", "S", "C"], "article-title": ["Chemokine signaling and functional responses: the role of receptor dimerization \nand TK pathway activation"], "italic": ["Annual Review of Immunology"], "year": ["2001"], "volume": ["19"], "fpage": ["397"], "lpage": ["421"]}, {"label": ["4"], "surname": ["Rossi", "Zlotnik"], "given-names": ["D", "A"], "article-title": ["The biology of chemokines and their receptors"], "italic": ["Annual Review of Immunology"], "year": ["2000"], "volume": ["18"], "fpage": ["217"], "lpage": ["243"]}, {"label": ["30"], "surname": ["Tang", "Chuang", "Fong", "Maa", "Way", "Hung"], "given-names": ["CH", "JY", "YC", "MC", "TD", "CH"], "article-title": ["Bone-derived SDF-1 stimulates IL-6 Release via CXCR4, ERK and NF-"], "italic": ["\u03ba", "Carcinogenesis"]}, {"label": ["107"], "surname": ["Chen", "Bai", "Yang", "Cong", "Chen"], "given-names": ["J-D", "X", "A-G", "Y", "S-Y"], "article-title": ["Inactivation of HIV-1 chemokine co-receptor CXCR-4 by a novel intrakine strategy"], "italic": ["Nature Medicine"], "year": ["1997"], "volume": ["3"], "issue": ["10"], "fpage": ["1110"], "lpage": ["1116"]}, {"label": ["135"], "surname": ["Fredholm", "Arslan", "Halldner", "Kull", "Schulte", "Wasserman"], "given-names": ["BB", "G", "L", "B", "G", "W"], "article-title": ["Structure and function of adenosine receptors and their genes"], "italic": ["Naunyn-Schmiedeberg's Archives of Pharmacology"], "year": ["2000"], "volume": ["362"], "issue": ["4-5"], "fpage": ["364"], "lpage": ["374"]}, {"label": ["168"], "surname": ["Marnett", "DuBois"], "given-names": ["LJ", "RN"], "article-title": ["COX-2: a target for colon cancer prevention"], "italic": ["Annual Review of Pharmacology and Toxicology"], "year": ["2002"], "volume": ["42"], "fpage": ["55"], "lpage": ["80"]}, {"label": ["175"], "surname": ["Soydan", "Tavares", "Weech", "Tremblay", "Bennett"], "given-names": ["AS", "IA", "PK", "NM", "A"], "article-title": ["High molecular weight phospholipase A"], "sub": ["2"], "italic": ["European Journal of Cancer"], "year": ["1996"], "volume": ["32"], "issue": ["10"], "fpage": ["1781"], "lpage": ["1787"]}, {"label": ["181"], "surname": ["Pai", "Soreghan", "Szabo", "Pavelka", "Baatar", "Tarnawski"], "given-names": ["R", "B", "IL", "M", "D", "AS"], "article-title": ["Prostaglandin E"], "sub": ["2"], "italic": ["Nature Medicine"], "year": ["2002"], "volume": ["8"], "issue": ["3"], "fpage": ["289"], "lpage": ["293"]}]
{ "acronym": [ "CAY10410:", "CXCL12:", "CXCR4:", "DCIS:", "GW9662:", "HIF-1:", "HRE:", "LESTR:", "NF-κB:", "NSAIDs:", "NSCLC:", "PPARγ:", "PPRE:", "pVHL:", "RCC:", "RXR:", "SDF-1:", "T0070907:", "TAM:", "VEGF:", "15dPGJ2:", "15-PGDH:", "Δ12‐PGJ2:" ], "definition": [ "9,10-dihydro-15-deoxy-Δ12,14-prostaglandin J2\n", "CXC chemokine ligand 12", "CXC chemokine receptor 4", "Ductal carcinoma in situ", "2-chloro-5-nitro-N-phenylbenzamide", "Hypoxia-inducible factor-1", "Hypoxia response element", "Leukocyte-expressed seven-transmembrane domain receptor", "Nuclear factor-κB", "Nonsteroidal anti-inflammatory drugs", "Non-small cell lung cancer", "Peroxisome proliferator-activated\nreceptor γ\n", "Peroxisome proliferator response element", "Von Hippel-Lindau tumor suppressor protein", "Renal cell cancer", "Retinoid X receptor", "Stromal cell-derived factor\n1", "2-chloro-5-nitro-N-(4-pyridyl)benzamide", "Tumor-associated macrophages", "Vascular endothelial growth\nfactor", "15-deoxy-Δ12,14‐PGJ2\n", "15-hydroxyprostaglandin dehydrogenase", "9-deoxy-Δ9,Δ12‐13,14-dihydro-PGD2." ] }
218
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no
2022-01-13 03:12:57
PPAR Res. 2008 Sep 2; 2008:769413
oa_package/fb/c3/PMC2528256.tar.gz
PMC2528299
18769604
[ "<title>Introduction</title>", "<p>There is substantial evidence for a humoral immune response to cancer in humans, demonstrated by the identification of autoantibodies to a number of intracellular and surface antigens in serum from patients with different tumor types (##REF##9547346##1##–##REF##9547328##3##). A tumor-specific humoral immune response directed against oncoproteins (##REF##2142141##4##–##REF##7903196##5##), or mutated proteins such as p53 (##REF##1322237##6##) or other aberrantly expressed proteins have been previously described. It is currently largely unknown whether the occurrence of such antibodies is beneficial. However, knowledge of potential tumor antigens that may evoke tumor-specific immune responses may have relevance to the development of effective strategies for cancer screening and diagnosis.</p>", "<p>Pancreatic cancer has the worst prognosis among cancers, with a 5-year survival rate of &lt;3%, accounting for the fourth largest number of cancer deaths in the United States (##REF##12568441##7##). The poor prognosis for pancreatic cancer is, in part, due to lack of early detection methods. Currently, there is a paucity of pancreatic cancer markers for early detection and for the diagnosis of pancreatic cancer.</p>", "<p>Autoimmunity in pancreatic cancer has been demonstrated against several proteins, including MUC1 (##REF##12455059##8##–##REF##8884844##9##), p53 (##REF##8884844##9##), calreticulin (##REF##15289361##13##) and Rad51 (##REF##11935313##10##) proteins. MUC1 is a transmembrane glycoprotein involved in cell-cell and cell-extracellular matrix interactions, and MUC1 autoantibodies have been observed in sera from patients with a variety of different tumors (##REF##7514493##11##). The presence of MUC1 IgG autoantibodies has been shown to be associated with a favorable prognosis (##REF##12455059##8##–##REF##8884844##9##). The recombination factor Rad51 is highly expressed in pancreatic adenocarcinoma (##REF##11935313##10##), and Rad51 autoantibodies have been observed in 7% of patients with pancreatic cancer.</p>", "<p>A large number of autoantibodies have been identified in various tumor types, but in most cases they occur in a small percentage of patient’s sera. Therefore, they are not effective individually for the early detection of cancer. Thus, the development of panels of such autoantibodies directed against a variety of tumor antigens may be effective (##REF##11302861##12##).</p>", "<p>We have implemented a proteomic approach for the identification of tumor antigens that elicit a humoral response in pancreatic cancer (##REF##15289361##13##). In this study we have utilized the pancreatic cancer cell line Panc-1 as the source of tumor cell proteins for antigen identification. We have utilized 2-D PAGE to separate protein constituents, followed by their transfer onto PVDF membranes. Sera from cancer patients and from controls were screened individually by Western blot analysis for antibodies that reacted against the resolved proteins. Mass spectrometric analysis was used for protein identification. Within, we report the identification of a vimentin isoform as an antigen that elicits a humoral immune response in pancreatic cancer.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Materials</title>", "<p>All cell culture reagents, including Dulbecco’s modified Eagle’s medium (DMEM, containing L-glutamine, sodium pyruvate and pyridoxine hydrochloride), Dulbecco’s phosphate buffered saline (PBS), fetal calf serum and penicillin/streptomycin were obtained from Invitrogen (Carlsbad, CA). The mouse monoclonal anti-vimentin antibody (Clone V9) was purchased from Lab Vision Corp. (Fremont, CA). The horseradish peroxidase-conjugated sheep anti-human IgG and the ECL (Enhanced Chemiluminescence) kits were obtained from Amersham (Piscataway, NJ). The Immobilon-P PVDF (polyvinylidene fluoride) membranes were purchased from Millipore Corp. (Bedford, MA). The acrylamide used in the first dimension electrophoresis, urea, ammonium persulfate and PDA (piperazine diacrylamide) were all purchased from BioRad (Rockville Center, NY). The acrylamide used in the second dimension electrophoresis was purchased from Serva (Crescent Chemical, Hauppauge, NY) and the carrier ampholytes (pH 4 to 8) and NP-40 were both purchased from Gallard/Schlessinger (Carle Place, NY). All other reagents and chemicals were obtained from either Fisher or Sigma and were of the highest purity available.</p>", "<title>Sera, tumor tissues and cell lines</title>", "<p>Serum and tumor tissue was obtained at the time of diagnosis following informed consent using IRB-approved guidelines. A total of 36 serum samples were obtained from patients with a confirmed diagnosis of pancreatic adenocarcinoma who were seen in the Multidisciplinary Pancreatic Tumor Clinic at the University of Michigan Comprehensive Cancer Center. Sera from the pancreatic cancer patients were randomly selected from a clinic population that sees, on average, at the time of initial diagnosis, 15% of pancreatic adenocarcinoma patients presenting with early stage (i.e. stage 1/2) disease and 85% presenting with advanced stage (i.e. stage 3/4). Inclusion criteria for the study included patients with a confirmed diagnosis of pancreatic cancer, the ability to provide written, informed consent, and the ability to provide 40 ml of blood. Exclusion criteria included inability to provide informed consent, patients actively undergoing chemotherapy or radiation therapy for pancreatic cancer, and patients with other malignancies diagnosed or treated within the last 5 years. Sera were also obtained from 18 patients with chronic pancreatitis who were seen in the Gastroenterology Clinic at University of Michigan Medical Center and at the Catholic University of Daegu, in Daegu, South Korea, and from 15 control healthy individuals collected at University of Michigan under the auspices of the Early Detection Research Network (EDRN). The mean age of the tumor group was 65.4 years (range 54–74 years) and from the chronic pancreatitis group was 54 years (range 45–65). The sera from the normal subject group was age and sex-matched to the tumor group. All of the chronic pancreatitis sera were collected in an elective setting in the clinic in the absence of an acute flare. All sera were processed using identical procedures. The samples were permitted to sit at room temperature for a minimum of 30 minutes (and a maximum of 60 minutes) to allow the clot to form in the red top tubes, and then centrifuged at 1,300 × g at 4°C for 20 minutes. The serum was removed, transferred to a polypropylene, capped tube in 1 ml aliquots, and frozen. The frozen samples were stored at −70°C until assayed. All serum samples were labeled with a unique identifier to protect the confidentiality of the patient. None of the samples were thawed more than twice before analysis. The human cancer cell lines used in this study were all individually cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 units/ml streptomycin (Invitrogen, Carlsbad, CA).</p>", "<title>2-D PAGE and Western blot analysis</title>", "<p>After excision, the tumor tissue was immediately frozen at −80°C. An aliquot was lysed in solubilization buffer (8 M urea (Bio-Rad), 2% Nonidet P-40, 2% carrier ampholytes, pH 4–8 (Gallard/Schlessinger), 2% β-mercaptoethanol, and 10 mM PMSF), then stored at −80°C until use. Cultured Panc-1 pancreatic adenocarcinoma cells were harvested in 300 μl of solubilization buffer by using a cell scraper and stored at −80°C until use. Proteins derived from the extracts of either cultured cells or solid tumors were separated into two dimensions as described previously (##REF##2915694##14##). Briefly, solubilized proteins were applied onto isoelectric focusing gels. Isoelectric focusing was performed using pH 4 to 8 carrier ampholytes at 700 V for 16 h, followed by 1000V for an additional 2 h. The first-dimension gel was loaded onto the second-dimension gel, after equilibration in 125 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 1% dithiothreitol, and bromophenol blue. For the second-dimension separation, a gradient of 11 to 14 % acrylamide (Crescent Chemical) was used. Proteins were transferred to an Immobilon-P PVDF membrane (Millipore) or visualized by silver staining of the gels.</p>", "<title>Western blotting</title>", "<p>After transfer, membranes were incubated with a blocking buffer consisting of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1.8% nonfat dry milk, and 0.01% Tween 20 for 2 h. The membranes were incubated for 1 h at room temperature with serum obtained from either patients or healthy individuals as a source of primary antibody at a 1:100 dilution. Following three washes with washing buffer (Tris-buffered saline containing 0.01% Tween 20), the membranes were incubated with horseradish peroxidase-conjugated anti-human (Amersham) IgG antibodies at a dilution of 1:1000 for 1 h at room temperature. Immunodetection was accomplished by ECL (Enhanced Chemiluminescence (Amersham)) followed by autoradiography on Hyperfilm MP (Amersham).</p>", "<title>Vimentin detection by western blotting</title>", "<p>A mouse anti-vimentin (Clone V9) monoclonal antibody (Lab Vision Corporation, Fremont, CA) was used at 1:100 dilution for western blotting and was processed as for incubations with patient sera, with a horseradish peroxidase-conjugated anti-mouse IgG (Amersham) as the secondary antibody.</p>", "<title>In-gel enzyme digestion and mass spectrometry</title>", "<p>For protein identification by mass spectrometry, 2-D gels were stained by a modified silver staining method, and excised proteins were destained for 5 min in 15mM potassium ferricyanide and 50 mM sodium thiosulfate as described (##REF##10217175##15##). Following three washes with water, the gel pieces were dehydrated in 100% acetonitrile for 5 min, and then dried. Digestion was performed by addition of 100 ng of trypsin (Promega, Madison, WI) in 200 nM ammonium bicarbonate. Following enzymatic digestion overnight at 37°C, the peptides were extracted twice with 50 μl of 60% acetonitrile/1% trifluoroacetic acid. Following removal of acetonitrile by centrifugation in a vacuum centrifuge, the peptides were concentrated by using pipette tips C18 (Millipore) and identified by nanoflow capillary liquid chromatography coupled with electrospray quadrupole-time of flight tandem mass spectrometry (ESI Q-TOF MS/MS) in the Q-TOF <italic>micro</italic> (MicroMass, Manchester, U.K.). The acquired spectra were processed and searched against a non-redundant Swiss-Prot protein sequence database using protein-Lynx Global Server (<ext-link ext-link-type=\"uri\" xlink:href=\"www.micromass.co.uk\">www.micromass.co.uk</ext-link>).</p>" ]
[ "<title>Results</title>", "<title>Autoantibodies to pancreatic tumor proteins in sera from patients with pancreatic cancer</title>", "<p>Panc-1 pancreatic tumor cell line proteins were resolved by 2-D PAGE, then transferred onto Immobilon-P PVDF membranes. Sera obtained from 36 newly diagnosed patients with pancreatic cancer, from 18 patients with chronic pancreatitis and from 15 healthy donors were screened individually for the presence of antibodies to Panc-1 pancreatic tumor cell line proteins (##TAB##0##Table 1##). Each membrane was treated with one serum sample as the primary antibody and with sheep anti-human IgG as the secondary antibody. In general, most pancreatic patient sera reacted against multiple proteins (##FIG##0##Fig. 1##). Some of the reactive proteins also reacted with control sera and thus were considered to represent nonspecific reactivity. The reactive proteins most commonly observed with pancreatic cancer patient sera, but not with controls included one protein with an estimated molecular mass of 55 kDa and a pI of 5. This protein showed reactivity with sera from 16 of 36 patients with pancreatic cancer (44.4%), with 1/18 sera (5.6%, p = 0.003 (one-sided Fisher’s exact test)) from chronic pancreatitis patients and none of 15 (0%, p = 0.001 (one-sided Fisher’s exact test)) sera from healthy donors (##TAB##0##Table 1##). Combining the chronic pancreatitis and healthy controls into a single group gave p = 4 × 10 − 5 (one-sided Fisher’s exact test) in comparison to cancer.</p>", "<title>Identification of the reactive protein as vimentin</title>", "<p>The protein spot of interest was extracted from the gels following 2-D PAGE and silver staining, digested with trypsin and the resulting peptides were analyzed by ESI Q-TOF Tandem MS spectrometry. The acquired spectra were processed and searched against a non-redundant SwissProt protein sequence database using proteinLynx Global Server (<ext-link ext-link-type=\"uri\" xlink:href=\"www.micromass.co.uk\">www.micromass.co.uk</ext-link>). The sequence of 12 tryptic peptides matched the predicted sequence of vimentin (MW 53.5 kDa. pI 5.1) (##FIG##1##Fig. 2##). The identity of this protein was confirmed with 2-D Western blotting using Panc-1 whole-cell extracts and anti-vimentin mouse monoclonal antibody (##FIG##2##Fig. 3##). Interestingly, although a number of vimentin isoforms were detected by monoclonal anti-vimentin antibody from Panc-1 whole cell lysates, only one vimentin isoform showed reactivity specifically with pancreatic cancer patient sera. We hypothesized that perhaps specific protease cleavage of intact vimentin resulted in the observed antigenicity. To this end, each of the three lowest vimentin isoforms which were reactive with patients serum (##FIG##2##Fig. 3##) were excised from modified silver-stained gels (the uppermost isoform was not visualized in the silver-stained gels), then subjected to analysis by tandem mass spectrometry in order to search for vimentin-specific tryptic peptides. We found multiple peptide coverage (7–13 peptides for each isoform), spanning residues 78–400 for each of the three isoforms (data not shown). Thus, as the same peptide coverage was identified for each of the isoforms, we were unable to find evidence of protease cleavage leading to antigenicity of the specific vimentin isoform that was specifically reactive with pancreatic cancer patient’s serum. Moreover, although some peptide modifications were found, such as deamination, methylation, phosphorylation and hydroxylation, none were specific to the vimentin isoform that showed reactivity specifically with pancreatic cancer patient’s serum (data not shown).</p>", "<title>Role of glycosylation in vimentin antigenicity</title>", "<p>We sought to determine whether aberrant vimentin glycosylation contributed to immunogenicity. Solubilized proteins from the Panc-1 cell line were subjected to N-deglycosylation by a combination of Endoglycosidase F, Endo-α-N-acetylgalactosaminidase, and α-2–3, 6, 8, 9-Neuraminidase. The resulting products were separated by SDS electrophoresis and analyzed by Western blotting. Although the deglycosylated positive control revealed a demonstrable mobility shift by SDS-PAGE, the deglycosylating enzyme treatment did not result in any mobility shifts of vimentin. Thus, Endoglycosidase F- sensitive glycosylation does not appear to be playing a role in the observed immunogenicity of the vimentin isoform (data not shown).</p>", "<title>Analysis of vimentin expression by 2-D PAGE</title>", "<p>We hypothesized that there might be changes in the expression level of the single minor antigenic isoform of vimentin that could lead to antigenicity in pancreatic cancer. Using 2-D PAGE, we examined the expression of the different vimentin isoforms in a variety of tissues and tumor types. The major vimentin isoforms were present in the different cell lines examined, including 6 pancreatic tumor cell lines, 4 lung tumor cell lines, 9 colon tumor cell lines and 33 ovarian tumor cell lines, at similar expression levels. A similar pattern of expression was also observed in 6 pancreatic tumors, 38 lung tumors, 7 colon tumors and 25 ovarian tumors (##FIG##3##Fig. 4##), suggesting that the major vimentin isoforms were ubiquitously expressed. However, the minor isoform that was found to be antigenic was specific to just a subset of the tumor cell lines. We analyzed (by Western blot) different tumor cell lines for the specific isoform of vimentin that was immunogenic in pancreatic tumor patients. These cell lines were derived from 4 lung tumors (3 adenocarcinoma and 1 small cell), 2 pancreatic tumors, 2 breast tumors, 7 colon tumors, 1 neuroblastoma and 1 testes. Interestingly, the isoform of vimentin was only found in 4 of the cell lines analyzed (in the Panc-1 pancreatic, in the H23 and A549 lung adenocarcinoma, and in the Hs1 testes tumor cell lines), thus suggesting that expression of the antigenic isoform was cell line-specific. Additionally, we explored whether the antigenic isoform was overexpressed in a variety of different adenocarcinomas, including 18 colon, 16 ovarian, 14 lung, 10 esophageal and pancreatic (4 tumor and 4 normal tissue). The integrated intensity measurement of the antigenic isoform in each tumor type was directly compared to that of a neighboring non-antigenic vimentin isoform that is ubiquitously expressed in all tumor types examined. Interestingly, the antigenic isoform of vimentin was expressed at 5–10 fold higher levels relative to the ubiquitous isoform in pancreatic tumors compared to other tumor types, and was expressed at approximately 50% higher levels than that found in normal pancreas (##TAB##1##Table 2##).</p>" ]
[ "<title>Discussion</title>", "<p>We have implemented a proteomics-based approach to identify proteins that elicit a humoral response in pancreatic cancer patients. This approach allows screening by Western blot analysis of patient sera for antibodies that react against separated tumor cell proteins. This study was focused on a search for autoantibodies to pancreatic tumor proteins present in the Panc-1 cancer cell line. We have shown that a humoral response directed against a single isoform of vimentin occurred in 44.4% patients with pancreatic cancer. One out of 18 (5.6%) chronic pancreatitis patients and none of the noncancer controls exhibited reactivity against the antigenic vimentin isoform.</p>", "<p>Intermediate filaments are one of the three major cytoskeleton networks in higher eukaryotic cells. These filaments consist of a number of different members, including vimentin and the cytokeratin proteins. Different types of intermediate filament protein genes are expressed depending on the tissue type. Little is known about the function of intermediate filaments in normal cells, although it is believed that they provide cellular integrity and resistance against mechanical stresses (##REF##10998598##16##). Their tissue-specific expression in normal cells and differential expression/assembly in cancer is of great pathologic value in tumor diagnostics. Expression of vimentin has been postulated to play a role in invasiveness and metastasis in cervical carcinoma (##REF##8976877##17##). However, Heatley et al. have shown that vimentin expression could not differentiate between benign and invasive breast lesions, although its expression was correlated with tumor grade and decreased survival in ductal carcinoma (##REF##7686566##18##).</p>", "<p>To date, autoantibodies to different classes of intermediate filaments, including vimentin, have been detected in human sera (##REF##7018769##19##–##REF##4040759##22##). Although the mechanism of induction of autoantibodies against vimentin still remains obscure, proteolysis of the intact, native protein may play a role in development of autoimmunity. In a previous study we have demonstrated that a particular form of calreticulin elicits a humoral response in hepatocellular carcinoma (##UREF##0##23##), with the reactive epitope occurring in a truncated form (CRT32, which includes the C-terminal portion), whereas the intact protein did not elicit reactivity. Importantly, <italic>in vitro</italic> studies have demonstrated that vimentin is subject to caspase-mediated proteolysis in an apoptosis-related manner (##REF##11423904##24##). Prasad et al. (##REF##9712696##25##) have reported apoptosis-associated proteolysis of vimentin in human prostate epithelial tumor cells and demonstrated that vimentin undergoes limited proteolysis in the apoptotic cells. Additionally, intact vimentin and some of its proteolytic fragments correspond to ubiquitinated polypeptides that are specific to the apoptotic process (##REF##10200476##26##–##REF##3899428##27##). Alcover et al. (##REF##3899428##27##) have demonstrated that anti-vimentin antibodies in patients with autoimmune diseases interact preferentially with a specific domain of the protein, a peptide with a molecular weight of 30 kDa that is close to the amino-terminal of intact vimentin. It should be noted, however, that although we have identified 12 peptides from vimentin by MS/MS analysis, the most N-terminal of these started at residue 78 of the intact protein. Although the epitope that reacts with autoantibodies in pancreatic cancer patient’s sera remains undefined, it is plausible that the antigenic epitope was exposed following proteolytic cleavage of the intact protein near the N-terminus.</p>", "<p>Most antigens recognized by autoantibodies are molecules that exist within cells under normal conditions. However the structure, processing and/or subcellular localization of some molecules, change with cell death. These processed forms are recognized by the immune system, leading to the development of autoimmunity particularly if the dead cells fail to be effectively cleared by phagocytosis (##REF##10200542##28##). For example, some autoantigens such as the nuclear autoantigen La, or ribonucleoprotein Ro move to a region near the cell periphery during apoptosis (##REF##2543764##29##–##REF##9794444##30##). Thus, it may be proposed that the processing and translocation of cellular antigens to a new sub-cellular compartment could have a causative role in autoimmunity. Interestingly, it has been recently demonstrated that vimentin is secreted by macrophages in response to pro-inflammatory signaling pathways (##REF##12483219##31##). In particular, whereas the anti-inflammatory cytokine interleukin-10 will inhibit vimentin secretion from macrophages, the pro-inflammatory cytokine tumor necrosis factor will trigger secretion (##REF##12483219##31##). These findings may also contribute to the development of autoantibodies against vimentin in pancreatic cancer.</p>", "<p>A prerequisite for an immune response against a cellular protein is its presentation as an antigen. It is not clear why only a subset of patients with a specific tumor type develop a humoral response to a particular antigen. Immunogenicity may depend on the level of expression, post-translational modification, or other types of processing of a protein, the extent of which may be variable among tumors of a similar type. Although vimentin expression is approximately three fold higher in pancreatic tumors at the protein level, as compared to other tumors analyzed in our study, only a single isoform of vimentin had demonstrable immunogenicity to autoantibodies in pancreatic cancer patient’s sera. Thus, the immunoreactivity of vimentin is unlikely to be related to the total level of vimentin protein expression. Further, we were unable to demonstrate aberrant N- or O-linked glycosylation of vimentin in the pancreatic tumor cell lines (data not shown).</p>", "<p>Although the vimentin autoantibodies were largely restricted to patients with pancreatic cancer among the subject groups we investigated, further studies are needed to determine the specificity of the vimentin antibodies to pancreatic cancer. For example, although increased levels of vimentin antibodies were found in pancreatic cancer, as compared to chronic pancreatitis and healthy individuals, the relationship between tumor burden, tumor staging and antibody levels needs further clarification. Assessment of the utility of vimentin autoantibodies as diagnostic markers in pancreatic cancer also needs to be addressed in further studies. It is clear, however, that the proteomic approach we have implemented, which allows for the screening of natural forms of proteins as expressed in tumor cells, has the potential to identify novel antigens that may have utility for cancer screening and diagnosis.</p>" ]
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[ "<p>Pancreatic cancer has a poor prognosis, in part due to lack of early detection. The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Proteins from a pancreatic adenocarcinoma cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for autoantibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis and 15 healthy subjects were analyzed. Autoantibodies were detected against a protein identified by mass spectrometry as vimentin, in sera from 16/36 patients with pancreatic cancer (44.4%). Only one of 18 chronic pancreatitis patients and none of the healthy controls exhibited reactivity against this vimentin isoform. Interestingly, none of several other isoforms of vimentin detectable in 2-D gels exhibited reactivity with patient sera. Vimentin protein expression levels were investigated by comparing the integrated intensity of spots visualized in 2-D PAGE gels of various cancers. Pancreatic tumor tissues showed greater than a 3-fold higher expression of total vimentin protein than did the lung, colon, and ovarian tumors that were analyzed. The specific antigenic isoform was found at 5–10 fold higher levels. The detection of autoantibodies to this specific isoform of vimentin may have utility for the early diagnosis of pancreatic cancer.</p>" ]
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[ "<title>Acknowledgements</title>", "<p>This project was supported, in part, by NCI grant U01 CA-84982 (to SMH) and the Tissue Core of the University of Michigan Comprehensive Cancer Center grant CA46952.</p>" ]
[ "<fig id=\"f1-bmi-2006-175\" position=\"float\"><label>Figure 1</label><caption><p>(<bold>A</bold>) A silver-stained image of the Panc-1 pancreatic tumor cell line, (<bold>B</bold>) as compared with a Western blot of the Panc-1 cell line with serum from a patient with pancreatic cancer, and (<bold>C</bold>) normal serum.</p></caption></fig>", "<fig id=\"f2-bmi-2006-175\" position=\"float\"><label>Figure 2</label><caption><p>Tandem mass spectrometry identification of vimentin. The MS/MS spectrum of vimentin (obtained after trypsin digestion) is shown by analysis with ESI-Q-TOF, coupled with nanoflow capillary high-performance liquid chromatography. The precursor ion shown in the figure is m/z 648.3487, and resultant peaks were searched against the non-redundant SwissProt protein sequence database using the ProteinLynx global server. A total of twelve tryptic peptides, as shown, matched the vimentin protein.</p></caption></fig>", "<fig id=\"f3-bmi-2006-175\" position=\"float\"><label>Figure 3</label><caption><p>Western blot analysis of vimentin with (<bold>A</bold>) sera from a pancreatic cancer patient, (<bold>B</bold>) from healthy individual, and (<bold>C</bold>) a monoclonal anti-vimentin antibody.</p></caption></fig>", "<fig id=\"f4-bmi-2006-175\" position=\"float\"><label>Figure 4</label><caption><p>Vimentin protein levels measured in human tumors and tumor cell lines. 2-D gels were prepared using solubilized proteins from a variety of human tumors and tumor cell lines, as described in “Materials and Methods”. Background corrected integrated intensity (volume) was measured (Visage software, Genomic Solutions, Ann Arbor, MI) for total vimentin. Bars represents the average intensities for 38 lung tumors, 6 pancreatic tumors, 7 colon tumors, 25 ovarian tumors, 4 lung cell lines, 6 pancreatic cell lines, 9 colon cell lines and 33 ovarian cell lines.</p></caption></fig>" ]
[ "<table-wrap id=\"t1-bmi-2006-175\" position=\"float\"><label>Table 1</label><caption><p>Identification of vimentin autoantibodies in patient’s sera. P value is in comparison to pancreatic cancer *, one-sided Fisher’s exact test.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Serum</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Number of subjects</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Vimentin-positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">P value*</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pancreatic cancer</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (44.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Chronic pancreatitis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 (5.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">P = 0.003</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Healthy Individuals</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">P = 0.001</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"t2-bmi-2006-175\" position=\"float\"><label>Table 2</label><caption><p>Expression levels of the antigenic vimentin isoform in human tumors. The numbers shown are averaged integrated intensity measurements (± SD) for two vimentin spots, with “A” being the ubiquitous spot and “B” being the identified antigenic spot.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Tumor Type</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Gels Analyzed</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Spot A measurement</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Spot B measurement</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">B/A</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Colon</bold></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.668 ± 0.255</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.574 ± 0.21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.859</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Ovarian</bold></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.284 ± 0.173</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.176 ± 0.086</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.620</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Lung</bold></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.359 ± 0.317</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.450 ± 0.205</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.253</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Esophageal Pancreas</bold></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.816 ± 0.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.861 ± 0.277</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.055</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Normal Pancreas</bold></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.471 ± 0.264</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.911 ± 0.263</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.057</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Tumor</bold></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.215 ± 0.119</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.369 ± 1.334</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.367</td></tr></tbody></table></table-wrap>" ]
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[ "<graphic xlink:href=\"bmi-2006-175f1\"/>", "<graphic xlink:href=\"bmi-2006-175f2\"/>", "<graphic xlink:href=\"bmi-2006-175f3\"/>", "<graphic xlink:href=\"bmi-2006-175f4\"/>" ]
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[{"label": ["23"], "surname": ["Le Naour", "Brichory", "Misek", "Brechot"], "given-names": ["F", "F", "DE", "C"], "year": ["2002"], "article-title": ["A distinct repertoire of autoantibodies in hepatocellular carcinoma identified by proteomic analysis"], "source": ["Mol Cell Prot"], "volume": ["1"], "fpage": ["197"], "lpage": ["203"]}]
{ "acronym": [], "definition": [] }
31
CC BY
no
2022-01-12 14:58:27
Biomark Insights. 2007 Feb 7; 1:175-183
oa_package/22/be/PMC2528299.tar.gz
PMC2528303
18769605
[ "<title>1. INTRODUCTION</title>", "<p>In recent decades, a paradigm shift has occurred in oncology, from treatment of disease to treatment of the whole person. In his influential <italic>The Structure of Scientific Revolutions</italic> ##UREF##0##1##, Thomas Kuhn argued that scientific theories do not evolve from the straightforward accumulation of facts, but from a set of changing historical and contextual circumstances. Medical practice is closely aligned with this concept, because medicine is a combination of science, art, and the humanities ##REF##470449##2##.</p>" ]
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[ "<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>Literature demonstrating the importance of social relationships for cancer survivorship is accumulating. Building on that literature, the term “Healing Ties” refers to the scientific and popular factors supporting the idea that relationships and community are essential for healing. However, difficulties arise in assessing the effect of social support for survivorship.</p>", "<p>The current paper reviews the role in survivorship of social support, with respect to the explanatory model provided by neuro-oncology and psycho-neuro-immunology. Taking cognizance of the importance of social relationships, the model of cancer rehabilitation aims, through its interdisciplinary framework, to restore a sense of well-being and to facilitate healing by optimizing the capability for full social relationships and engagement with the world.</p>" ]
[ "<title>2. HEALING TIES</title>", "<p>In her recent cultural history of mind–body medicine, the Harvard-based historian Anne Harrington refers to the constellation of scientific and popular factors informing particular theories as “narratives.” Narratives are templates, she writes ##UREF##1##3##:</p>", "<p>One particular narrative that Harrington describes as “Healing Ties” refers to the simple but revolutionary idea that social relationships and community are essential for the prevention of illness and the promotion of healing and well-being.</p>", "<p>The concept of social stress as a causal determinant for (ill) health was advanced through the pioneering studies of notable figures in social epidemiology such as John Cassell, Len Symes, and Michael Marmot. Marmot’s work—made famous through his Whitehall studies looking at British civil servants—emphasizes the importance of the social inequality gradient as an independent indicator of health and well-being ##UREF##2##4##.</p>", "<p>Another related field, psychobiology, examines the pathways through which psychosocial factors stimulate biologic systems by central nervous system activation of autonomic, neuroendocrine, and immunologic responses ##REF##11741358##5##. In a classic study that applied those ideas to cancer, Vernon Riley showed that rates of tumour growth and quickened mortality were associated with a stressful environment ##REF##168638##6##. In mice with the Bittner tumour virus, a group raised in harsh surroundings showed an accelerated tumour onset and course as compared with a group maintained, apparently, as mice like to be maintained. This basic science model accords with studies examining the effects of social relationships for human cancer survivorship.</p>", "<p>In his 1978 review of research in psycho-oncology, Bernard Fox hypothesized about the interconnected biologic and psychological pathways that should be studied to determine the influence of states of mind on the promotion of cancer and its subsequent progress ##REF##556113##7##:</p>", "<p>In the decades since Fox’s review was published, a number of studies (but not all) have demonstrated the association between social support and increased survival for patients with various cancers <xref ref-type=\"fn\" rid=\"fn1-co15_4p185\">a</xref>.</p>", "<p>The conflicting evidence about the importance of psychological interventions for cancer survivorship and healing is also exemplified by three seemingly identical randomized controlled trials concerning the effects of group therapy on life expectancy for women suffering with late-stage (metastatic) breast cancer. The initial study in 1989 by the psychiatrist David Spiegel and colleagues reported that a cohort of women in support groups appeared to live, on average, twice as long as a control cohort not in such groups ##REF##2571815##15##; more recent studies seem convincingly to controvert that finding ##REF##11742045##16##,##REF##17647221##17##. Admittedly, these studies deal with the very narrow context of group therapy and not the broader one of social support as provided by family and community, but Harrington uses Spiegel’s example to question the validity of the template of Healing Ties.</p>", "<p>The difficulty in assessing the effect of social support for survivorship exists in defining the terms of research in narrow methodologic language, in evaluating quantitatively that which is essentially numinous, and then in determining the causative role of the defined idea in a particular disease. Yet despite the complexity of the design process, sophisticated techniques are currently being developed, particularly in relation to the collateral health effects of social networks ##REF##15271805##18##.</p>", "<p>Although the weight of evidence suggests that social support increases survival for people living with cancer, it is certain that social support can help to reduce distress and suffering. The emphasis on assessing longevity, important as it is, might actually diminish the perceived importance of social relationships for experiential aspects of being ill and caring for ill people, an essential element of relationship-centered medicine ##REF##17397699##19##.</p>", "<title>3. WHOLE-PERSON CARE</title>", "<p>If the shift toward whole-person care is a recent phenomenon in modern Western medicine, the insights upon which it draws are ancient. Plato is often justifiably invoked as the father of medical holism. Thus, in a well-known dialogue in the <italic>Phaedrus</italic> (270c), Plato compares the art of medicine with rhetoric. Here, Socrates says that whereas the task of medicine is to define the nature of the body, the task of rhetoric is to define the nature of the soul. Moreover, one cannot know the nature of the soul without knowing the nature of the whole. To which Phaedrus responds that Hippocrates the Asclepiad says that even the nature of the body can be understood only as a whole. Medicine, like hermeneutic philosophy <xref ref-type=\"fn\" rid=\"fn2-co15_4p185\">b</xref> relates part to whole, and whole to part.</p>", "<p>Commenting on this passage, the philosopher Hans-Georg Gadamer in his essays on the enigma of health notes that “the nature of the whole includes and involves the entire life situation of the patient, and even of the physician” ##UREF##3##20##. It is in the manner that medicine relates the disease to the person and to the larger community that the ultimate validity for the importance of social relationships in the health care context needs to be sought, rather than in the results of any one particular study. As noted by Plato, there is a parallel between the structure of the body—the subject of medicine—and the structure of the psyche, the self (or in Platonic terms, the soul).</p>", "<title>4. CANCER REHABILITATION</title>", "<p>To elucidate the foregoing idea in contemporary terms, consider the example of cancer rehabilitation, a movement that exemplifies the shifting emphasis from diagnosis of disease to symptom specificity or whole-patient care. Perhaps the key concept for cancer rehabilitation is that of interdisciplinarity. Rehabilitation requires an interdisciplinary team approach because of the variety of potential problems that patients may face during the course of their illness ##REF##18596892##21##. For this reason, the cancer rehabilitation team that we are involved with includes people trained in the disciplines of medicine, nursing, dietetics, physiotherapy, psychology, social work, occupational therapy, and medical ethics. One consequence of this interdisciplinarity is the acknowledgment that rehabilitation and healing can occur only when treatment incorporates as many relevant approaches and stories as have a bearing on the well-being of the person—including, of course, those of the patient and the patient’s family. The emphasis on social relationships is an important aspect of the approach espoused in cancer rehabilitation. Not only are social relationships important for particular interventions, but the aim of cancer rehabilitation is to restore a sense of well-being of self by optimizing the capability for full social relationships and engagement with the world.</p>", "<title>5. SUMMARY: STORIES AS THE HEALING MATRIX OF SOCIAL RELATIONSHIPS</title>", "<p>As indicated earlier, the effects of social relationships are not, however, simply social, but biological. Consider cachexia. There are both biologic reasons (the association of chronic inflammation with tumour progress and symptoms) and social imperatives to support a comprehensive care model from the time of diagnosis for cancer patients ##REF##17500503##22##. The effects of interdisciplinary care directly affect the constellation of physiologic factors that are associated with cancer. At a conceptual level, strengthening social relationships allows healing and survivorship to occur because it provides support for an ailing organism to self-organize. The constellation of pathologic factors is matched by the constellation of care that crystallizes in the narrative template of Healing Ties.</p>", "<p>Just as social stress provokes biologic weakening of an organism, social relations provide the opportunity for the whole person to flourish through the optimization of the physiologic functions upon which the human self is based. It is therefore not surprising that there is also evidence to suggest that the act of storytelling itself has positive effects on health and well-being. The social psychologist James Pennebaker has demonstrated in well-replicated studies the immunologic benefits that follow disclosure of traumatic events ##UREF##4##23##. Stories are not just stories, but an essential part of the feedback loop between the body and the self, the biologic and cultural matrix for the healing effects of social relationships. These stories may not come forward in the usual sequential trajectories of cancer care—a situation that isolates biochemotherapy from other components of cancer rehabilitation. They do emerge in the course of including patients and their families as partners in a community of care—a central tenet of cancer rehabilitation.</p>" ]
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[ "<disp-quote><p>They provide us with tropes and plotlines that help us understand that larger import of specific stories we hear, read, or see in action. They also help us construct specific stories of our own—including ones about our own experience—that others can recognize and affirm. We learn these narrative templates from our own culture, not in the way we might formally learn the rules of grammar in school, but in the way we might unconsciously learn the rules of grammar at home—by being exposed to multiple examples of living stories that rely on them.</p></disp-quote>", "<disp-quote><p>Cancer is a multi-step process; whatever the random mutation and other biological initiators of the cancer process, its further production and spread will depend in part on homeostatic controls that can be influenced by psychological factors through neurohormonal and immunological pathways. This hypothesis is valuable in focusing the attention of researchers on the patient, stimulating studies on the following: (1) the influence of specific states of mind on etiology (promotion) and prognosis of certain cancers, (2) the effect of psychological therapy on duration of survival of patients, and (3) the biological pathways that medicate the posited effects of states of mind.</p></disp-quote>" ]
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[ "<fn-group><fn id=\"fn1-co15_4p185\"><label>a</label><p>See, for example, Barraclough <italic>et al.,</italic> 1992 ##REF##1586819##8##; Goodkin <italic>et al.,</italic> 1986 ##REF##3701669##9##; Goodwin <italic>et al.</italic>, 1987 ##REF##3669259##10##; Graham <italic>et al.,</italic> 2002 ##REF##12065263##11##; Neale <italic>et al.,</italic> 1986 ##REF##3764489##12##; Priestman <italic>et al.,</italic> 1985 ##REF##3978029##13##; and Waxler–Morrison <italic>et al.,</italic> 1991 ##REF##1887281##14##.</p></fn><fn id=\"fn2-co15_4p185\"><label>b</label><p>“Hermeneutics” refers to the methodologic principles of textual interpretation. Hermeneutic philosophy analyses the philosophical foundations of interpretative methodologies. Hans–Georg Gadamer, one of whose essays is quoted in the next paragraph, is the most notable contemporary exponent of this philosophical method. </p></fn></fn-group>" ]
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[{"label": ["1"], "surname": ["Kuhn"], "given-names": ["TS"], "source": ["The Structure of Scientific Revolutions"], "publisher-loc": ["Chicago"], "publisher-name": ["University of Chicago Press"], "year": ["1970"]}, {"label": ["3"], "surname": ["Harrington"], "given-names": ["A"], "source": ["The Cure Within: A History of Mind\u2013Body Medicine"], "publisher-loc": ["New York"], "publisher-name": ["W.W. Norton and Company"], "year": ["2008"], "fpage": ["24"], "lpage": ["5"]}, {"label": ["4"], "surname": ["Marmot"], "given-names": ["M"], "source": ["Status Syndrome"], "publisher-loc": ["London"], "publisher-name": ["Bloomsbury"], "year": ["2004"]}, {"label": ["20"], "surname": ["Gadamer"], "given-names": ["HG"], "source": ["The Enigma of Health: The Art of Healing in a Scientific Age"], "publisher-loc": ["Stanford"], "publisher-name": ["Stanford University Press"], "year": ["1996"], "fpage": ["41"]}, {"label": ["23"], "surname": ["Pennebaker", "Wilce"], "given-names": ["JW", "JM"], "article-title": ["Telling stories: the health benefits of disclosure"], "source": ["Social and Cultural Lives of Immune Systems"], "publisher-loc": ["London"], "publisher-name": ["Rutledge"], "year": ["2003"], "fpage": ["19"], "lpage": ["35"]}]
{ "acronym": [], "definition": [] }
23
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2022-01-12 14:47:25
Curr Oncol. 2008 Aug; 15(4):185-187
oa_package/4f/98/PMC2528303.tar.gz
PMC2528304
18769606
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[ "<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>" ]
[ "<p>For having stolen fire and given it to mankind, Prometheus was punished by Zeus by being chained to the side of Mount Caucasus, where, every day, an eagle would eat away at his liver. Prometheus’s liver would regenerate itself overnight, ready to be eaten again with the coming dawn. Despite significant advances in supportive care, modern day chemotherapy can still be a Promethean experience for many cancer patients.</p>", "<p>The use of cytotoxic chemotherapy to treat cancer dates back to the 1940s, and although progress in the ability to treat various types of cancer has been significant, much of that progress has relied on the introduction of new cytotoxic agents with novel, but non-selective mechanisms of action ##REF##15630416##1##. The availability of supportive care agents such as 5-hydroxytryptamine<sub>3</sub> (5-HT<sub>3</sub>) receptor antagonists, neurokinin 1 (NK<sub>1</sub>) receptor in- hibitors, and growth factors such as filgrastim and eryth-ropoietin have also allowed treatment advances to be made by providing tools to better manage the side effects of chemotherapy, facilitating our ability to optimize the delivery of traditional cytotoxic agents, to push the boundaries of their steep dose–response curve, and to widen the narrow therapeutic index.</p>", "<p>However, with the exception of anti-hormonal therapy for breast and prostate cancer, the ability to truly exploit the differences between cancer cells and normal cells was realized only in 2001, with the introduction of imatinib, the first molecularly targeted agent for the treatment of chronic myeloid leukemia positive for the Philadelphia chromosome ##REF##15630416##1##. What makes today’s molecularly targeted therapies different from the more traditional cytotoxic agents is that they have been developed with a predefined extracellular or intracellular target or pathway in mind. These pathways have been identified as functioning in an aberrant manner in cancer cells relative to normal cells. To date, agents have been developed that disrupt pathways controlling cancer cell growth, differentiation, transcription, or angiogenesis. These agents also tend to have a reversible pharmacologic effect, to be cytostatic rather than cytotoxic, and to be most often given on a regular ongoing daily schedule rather than in cycles ##REF##12401902##2##,##REF##16982470##3##.</p>", "<p>The currently available molecularly targeted therapies fall into two broad categories: monoclonal antibodies that target cell surface proteins, and small-molecule kinase inhibitors that inhibit intracellular signalling pathways. From the perspective of mechanism of action, the first generation of agents either interact with epidermal growth factor pathways or inhibit angiogenesis. The newer multi-targeted agents (some currently available, and many more in development) affect multiple intracellular kinase targets ##REF##16929325##4##–##REF##18378537##7##. A discussion comparing modes of action and clinical efficacies of currently available molecularly targeted therapies (rituximab, trastuzumab, bevacizumab, imatinib, erlotinib, sunitinib, and sorafenib to name a few) is beyond the scope of this editorial, but recent reviews are readily available ##REF##16929325##4##,##REF##18043264##8##.</p>", "<p>In the rush to bring molecularly targeted therapies into day-to-day clinical practice, the side effects associated with these agents—used either alone or in combination with traditional cytotoxic agents—have received little attention. It had been postulated that, because of their increased selectivity for cancer cells, these agents would be less toxic than the traditional cytotoxic agents. However, it has been learned that these agents can indeed cause toxicities in patients—perhaps not surprisingly, in retrospect, because they target key signalling pathways for cellular growth and development. These toxicities are, for the most part, different from the toxicities of traditional cytotoxic agents, but they can nevertheless lead to dose reductions and delays and reduced quality of life for oncology patients.</p>", "<p>In addition to familiar side effects such as diarrhea, mucosal membrane toxicity, palmer–plantar erythrodysesthesia, and infusion reactions (for the monoclonal antibodies), the targeted agents cause relatively unique side effects, including proteinuria, hypertension, and skin reactions (acneiform rash, dry skin, nail changes, hair depigmentation) ##REF##16929325##4##,##UREF##0##9##,##REF##18390464##10##. Relative to the body of literature supporting the clinical efficacy of molecularly targeted therapies, information regarding their side effects is lacking. These unique side effects are no less distressing to patients, and they affect quality of life as much as the side affects associated with traditional cytotoxic therapy. Indeed, when targeted therapies are used in combination with traditional cytotoxic treatment, practitioners are adding to the range of toxicities experienced by patients.</p>", "<p>The advent of new molecularly targeted therapies brought with it the belief that the oncology community, like Heracles (“Hercules” in Roman mythology), who freed Prometheus, would free patients from the cyclical experience of the side effects associated with traditional cytotoxic chemotherapeutic agents. To a certain extent, this expectation has been realized, but these agents pose other challenges that require vigilance with respect to treatment-related side effects.</p>", "<p>The name Prometheus means “forethought”; as advocates for patients, we must act with foresight and learn to anticipate treatment-related side effects, implementing strategies to prevent their occurrence and acting to mitigate the severity of these side effects when they do occur.</p>", "<p>For the molecularly targeted therapies, the challenges that lie ahead include characterization of their toxicity profile (onset, severity, duration) in the broader cancer patient population, development of instruments that can be used in day-to-day practice by patients or by health care practitioners to assess the occurrence of side effects, systematic evaluation of strategies to prevent or manage treatment-related side effects, and development of tools that can help to identify patients at risk for development of side effects. Only then can the true potential of individualized anticancer therapy with molecularly targeted therapies be realized—and the chains of Prometheus broken once and for all for the sake of our patients.</p>" ]
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[ "<fn-group><fn><p>Educational service provided to physicians by an unrestricted grant from Sanofi-Aventis</p></fn><fn><p>Introducing our new series of expert guest editorials reporting upon different aspects of practice. These are independently reviewed and supported by unrestricted educational grants from our colleagues in industry.</p></fn></fn-group>" ]
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[{"label": ["9"], "surname": ["Mulder", "Punt"], "given-names": ["SF", "CJA"], "article-title": ["Managing toxicities of targeted therapies"], "source": ["Eur J Cancer Suppl"], "year": ["2007"], "volume": ["15"], "fpage": ["394"], "lpage": ["397"]}]
{ "acronym": [], "definition": [] }
10
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2022-01-12 14:47:25
Curr Oncol. 2008 Aug; 15(4):198-199
oa_package/a1/6c/PMC2528304.tar.gz
PMC2528305
18769607
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[ "<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>" ]
[ "<title>FOREWORD</title>", "<p>The editorial that follows departs from our usual expository style in favour of an introduction from Dr. Stephen Sagar of a very special supplement to <italic>Current Oncology.</italic> We trust that you will enjoy reading the upcoming supplement on integrative oncology, whose plethora of contributions from talented practitioners is being published in a combination of hard copy and online articles at the <italic>Current Oncology</italic> Web site, <ext-link ext-link-type=\"uri\" xlink:href=\"www.current-oncology.com\">www.current-oncology.com</ext-link>. Furthermore, I hope you can find the time to respond to Dr. Sagar’s appeal in the supplement to add your voice to the unique follow-up discussion that will be hosted at the Web site.</p>", "<p>M. McLean <sc>md</sc></p>", "<p>The concept “integrative” is defined as “to join with something else,” “unite,” “make part of a larger unit,” and be “open to people with various cultural values as equals” ##UREF##0##1##. One of the fundamental tenets of integrative oncology ##UREF##1##2##,##REF##16737670##3## is to consider the cultural values of patients and to incorporate those values into the decision plan, using various self-empowerment tools that are safe, that improve outcome and that are, preferably, cost-effective. Conceptually, the same is true for education and learning. The term “integrative learning” was coined by Jerry Perez de Tagle ##UREF##2##4## and comes in many varieties: connecting skills and knowledge from multiple sources and experiences, applying skills and practices in various settings, making use of diverse and even contradictory points of view, and understanding issues and positions contextually.</p>", "<p>Thanks to a generous grant from the Lotte and John Hecht Memorial Foundation ##UREF##3##5##, a special supplement of <italic>Current Oncology</italic> on integrative oncology is being printed concurrently with the regular issue. The Foundation continues the principles of its founders to integrate diverse societal values and has taken a particular interest in the investigation and evaluation of complementary and alternative medicine (<sc>cam</sc>), particularly as part of a cancer treatment program. In line with the principles of integration, the <italic>Current Oncology</italic> supplement will incorporate peer-reviewed manuscripts that focus on <sc>cam</sc>, plus educational articles on integrative oncology services and research. This material will be presented in a hybrid “integrative” format that uses the resources both of print and of the Internet, including appropriate links, colour photographs, video streams, and lectures accompanied by slides.</p>", "<p>In his publication “The New Alchemy: Transmuting Information into Knowledge in an Electronic Age,” Professor Alejandro Jadad eloquently discusses the transition into the new age of media communications and the integration of the new media into health care ##REF##10906918##6##. In that article, he concludes, “[T]his is meant to be an interactive feature.... [T]he new media and tools to which we are being exposed will undoubtedly change the way in which we communicate, learn and think.” Those media are currently the ones in which our patients are exploring information—in a virtual world with no boundaries. With structure, guidance, and the separation of fact from fiction, the Internet is a powerful tool for education and decision-making. However, communication and integration of virtual information requires guidance based on knowledge derived from evidence. In the integrative oncology supplement of <italic>Current Oncology,</italic> my co-editor (Anne Leis of the University of Saskatchewan) and I have integrated these concepts, especially as they relate to cancer patients utilizing <sc>cam</sc>.</p>", "<p>Part of the impetus for the supplement was the conference titled Integrating Wellness into Cancer Care, held at the University of Toronto, October 4–5, 2007. The conference was led by Dr. Paul Fortin, in memory of his wife, Dr. Veronique Benk. Veronique was a radiation oncologist, clinician, and researcher who specialized in breast cancer, and she was devoted to her patients. Her personal experience of breast cancer and myeloid leukemia was transformative, and she embraced a wider approach to cancer treatment. Her approach integrated state-of-the-art medical care with a new emphasis on spirituality, wellness, and quality of life.</p>", "<p>From that point, a host of distinguished authors contributed their knowledge and research to the supplement—Lynda Balneaves, Alison Brazier, Alastair Cunningham, Gary Deng, Meghan Duncan, Jane Maher, Doreen Oneschuk, Dugald Seeley, Simon Sutcliffe, Mary Vachon, Marja Verhoef, Raimond Wong, and Anne Leis and I. We hope that you will find the issue stimulating and thought-provoking.</p>" ]
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[ "<disp-quote><p>Integrative oncology is both a science and a philosophy that focuses on the complexity of the health of cancer patients and proposes a multitude of approaches to accompany the conventional therapies of surgery, chemotherapy, molecular therapeutics, and radiotherapy to facilitate health.</p><p>—<italic>Stephen Sagar</italic></p></disp-quote>" ]
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[{"label": ["1"], "collab": ["Farlex"], "article-title": ["The Free Dictionary. Dictionary/Thesaurus > Integrative [Web definition]"], "publisher-loc": ["Huntington Valley, PA"], "publisher-name": ["Farlex"], "comment": ["n.d. [Available at: "], "ext-link": ["www.thefreedictionary.com/integrative"]}, {"label": ["2"], "surname": ["Sagar", "Cassileth"], "given-names": ["SM", "BR"], "article-title": ["Integrative oncology for comprehensive cancer centres: definitions, scope and policy"], "source": ["Curr Oncol"], "year": ["2005"], "volume": ["12"], "fpage": ["103"], "lpage": ["17"]}, {"label": ["4"], "collab": ["Wikipedia"], "article-title": ["Jerry Perez de Tagle [Web article]"], "publisher-loc": ["San Francisco"], "publisher-name": ["Wikimedia"], "comment": ["n.d. [Available at: "], "ext-link": ["en.wikipedia.org/wiki/Jerry_Perez_de_Tagle"]}, {"label": ["5"], "collab": ["Lotte and John Hecht Memorial Foundation"], "article-title": ["Welcome to the Lotte & John Hecht Memorial Foundation [Web home page]"], "publisher-loc": ["Vancouver"], "publisher-name": ["Lotte and John Hecht Memorial Foundation"], "comment": ["n.d. [Available at: "], "ext-link": ["www.hecht.org"]}]
{ "acronym": [], "definition": [] }
6
CC BY
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2022-01-12 14:47:25
Curr Oncol. 2008 Aug; 15(4):166-167
oa_package/83/70/PMC2528305.tar.gz
PMC2528306
18769608
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[ "<title>2. DISCUSSION</title>", "<p>In patients with breast cancer, the presence of either sternal involvement or an isolated sternal metastasis is relatively uncommon, with reported incidences of 5.2% and 1.9%–2.4% respectively ##REF##3346742##1##,##REF##8016400##2##. Sternal involvement may occur either from direct invasion by enlarged internal mammary lymph nodes or from hematogenous spread. However, in contrast with vertebral lesions, which tend to result in multicentric bony disease from spread through the paravertebral plexus ##REF##4966305##3##, some sternal lesions have been observed to remain solitary with time and may be amenable to surgical resection with curative intent ##REF##2607606##4##,##REF##6313272##5##.</p>", "<p>Sternectomy for isolated breast cancer recurrence remains a controversial issue, and the literature consists predominantly of retrospective case series. Noguchi <italic>et al.</italic> ##REF##3416279##6## performed sternal resections with parasternal and mediastinal lymph node dissection on 9 patients before chemo-endocrine therapy. Eventual relapse in 8 patients revealed that lymph node dissection had no effect on locoregional control. Nevertheless, the dissection provided prognostic information, because all patients with involved parasternal and mediastinal lymph nodes relapsed and died within 30 months, but 3 patients without lymph node involvement survived for more than 6 years.</p>", "<p>Lequaglie <italic>et al.</italic> ##REF##12052759##7## performed radical, curative-intent sternectomies in a subgroup of 28 patients with isolated breast cancer recurrence and found that the 10-year overall survival in the group was 41.8%. These authors suggested that sternectomy could be curative in carefully selected patients.</p>", "<p>An isolated sternal metastasis should, however, be regarded with caution, because it is more likely to herald systemic disease than to be truly solitary. Kwai <italic>et al.</italic> ##REF##3346742##1## demonstrated that 54% of patients with breast cancer and solitary sternal metastasis developed other foci of distant disease within 20 months. The predominance of pulmonary metastasis and distant skeletal disease found is their study was attributed to drainage of the internal mammary nodes into the subclavian vein.</p>", "<p>Consequently, patients with breast cancer presenting with a solitary sternal metastasis require thorough restaging evaluation to rule out other foci of metastatic disease. Treatment should be based on a multimodality approach. With an isolated recurrence, local therapy with radiation would be appropriate. In the presence of distant disease, systemic options should be offered. Surgical resection should be reserved for palliation or for instances in which the other treatment modalities are not possible.</p>", "<p>Furthermore, the natural history of the initial breast cancer and the prognosis following recurrence should be considered. An important aspect of the present case is that our patient had a triple-negative breast cancer with axillary node involvement. Compared with other breast cancers, this disease subtype is highly aggressive, with an increased rate of early development of distant recurrence that peaks at 3 years after diagnosis ##REF##17671126##8##. Progression is rapid, with a median survival of only 9 months following recurrence. In addition, the extensive literature on relapsed breast cancer demonstrates that a shorter disease-free interval from time of initial treatment is associated with worse prognosis ##REF##11319846##9##.</p>" ]
[ "<title>3. CONCLUSIONS</title>", "<p>Although resection of a so-called isolated sternal metastasis is surgically feasible, it should be assessed and discussed with patients on a case-by-case basis. In the case of our patient, her history of a treated lymph node–positive tumour with triple-negative phenotype and recurrence within 3 years following mastectomy, chemotherapy, and radiotherapy should realistically have predicted this tragic outcome of widespread relapse within weeks following her extensive palliative surgery.</p>" ]
[ "<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>The occurrence of a solitary sternal metastasis from breast cancer is relatively uncommon, and its treatment is controversial. Most case reports on the role of sternal resection in what is termed a “solitary sternal metastasis” tend to present a rather optimistic outcome.</p>", "<p>Here, we report the case of a premenopausal woman with axillary lymph node–positive, triple-negative breast cancer treated with mastectomy followed by adjuvant chemotherapy and radiation therapy. She developed a radiologically isolated sternal recurrence 3 years later, which was treated with partial sternectomy. The present case report reviews the use of sternectomy for breast cancer recurrence and highlights the need for thorough clinical and radiologic evaluation to ensure the absence of other systemic disease before extensive surgery is undertaken.</p>" ]
[ "<title>1. CASE REPORT</title>", "<p>A 35-year-old woman with a history of breast cancer diagnosed 3 years earlier was referred to us for discussion of bisphosphonate use after surgical excision of an isolated sternal metastasis. Her initial breast cancer was treated with left mastectomy and axillary node dissection. Pathology revealed multifocal, poorly-differentiated invasive ductal carcinoma with three tumour masses (2.3 cm, 2.0 cm, and 1.1 cm). Of 14 lymph nodes, 5 were involved. Margins were negative. Immunohistochemistry was negative for estrogen, progesterone, and human epidermal growth factor (<sc>her</sc>2/<italic>neu</italic>) receptors. This patient received adjuvant epirubicin/5-fluorouracil/cyclophosphamide (<sc>fec</sc>-100) chemotherapy for 6 cycles and locoregional radiation treatment. Subsequently, she also had a prophylactic contralateral mastectomy and bilateral transrectus abdominal muscle flap reconstructions. Genetic testing was negative for <italic>BRCA1</italic> and <italic>BRCA2</italic> mutations.</p>", "<p>Three years after completion of adjuvant systemic therapy, this woman presented to her family physician with new-onset chest and neck tenderness during pregnancy. Investigations were deferred for 6 months until she was postpartum. A computed tomography scan of the neck and chest revealed a 5-cm soft-tissue mass with manubrial destruction consistent with recurrent disease (##FIG##0##Figure 1##). Restaging revealed no other foci of metastatic disease, and a bone scan showed uptake only in the manubrium. Given the clinical and radiologic impression of an isolated focus of recurrence, the patient elected to have a partial sternectomy.</p>", "<p>Pathology revealed a 5.4-cm, poorly differentiated invasive ductal carcinoma infiltrating the manubrium with involvement of the left sternocleidomastoid muscle and left clavicular head. Three lymph nodes were positive (one sternocleidomastoid and two thymic). Margins were clear. Once again, immunohistochemistry was triple-negative.</p>", "<p>There were no postoperative complications, and after a 6-week recovery, the woman was referred to our team for a second opinion regarding bisphosphonate use following surgical excision of the sternal metastasis. At presentation, she was asymptomatic. On physical examination, the scars from her breast reconstructions and sternal surgery were evident. She also had two palpable 0.5-cm right supraclavicular nodes.</p>", "<p>Restaging radiologic imaging performed 6 weeks after her surgery confirmed sub-centimetre right supraclavicular lymph nodes and also an enlarged 1.6-cm left supraclavicular lymph node (##FIG##1##Figure 2##). Fine-needle aspiration of the latter node was positive for triple-negative, poorly differentiated invasive ductal carcinoma.</p>" ]
[]
[ "<fig id=\"f1-co15_4p193\" position=\"float\"><label>FIGURE 1</label><caption><p>Computed tomography of the thorax, performed preoperatively, shows a soft-tissue mass with manubrial destruction (arrows). (A) Soft-tissue window. (B) Bone window.</p></caption></fig>", "<fig id=\"f2-co15_4p193\" position=\"float\"><label>FIGURE 2</label><caption><p>Computed tomography of the thorax, performed postoperatively, shows a 1.6-cm left supraclavicular lymph node deep to the sternocleidomastoid muscle.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"co15_4p193f1\"/>", "<graphic xlink:href=\"co15_4p193f2\"/>" ]
[]
[]
{ "acronym": [], "definition": [] }
9
CC BY
no
2022-01-12 14:47:25
Curr Oncol. 2008 Aug; 15(4):193-195
oa_package/06/74/PMC2528306.tar.gz
PMC2528307
18769609
[]
[]
[]
[ "<title>2. DISCUSSION AND CONCLUSIONS</title>", "<p>We believe that this is the first case report of the safety of cetuximab monotherapy in a patient with metastatic and psoriasis. Trivin <italic>et al.</italic> ##REF##15370619##1## <sc>crc</sc> reported on a patient who had both a tumour response and durable remission of psoriasis after treatment with cetuximab in combination with <sc>folfiri</sc>. However, that case did not differentiate between a potential beneficial effect of the chemotherapy and the effect of the cetuximab.</p>", "<p>Durable remissions of psoriasis have also been reported in patients undergoing high-dose chemotherapy for hematologic malignancies ##REF##15156169##2##–##REF##12221673##4##.</p>", "<p>We conclude that a previous history of psoriasis or active lesions should not be regarded as a contraindication for cetuximab treatment in patients with advanced <sc>crc</sc>. Psoriasis is associated with abnormal expression of <sc>egfr</sc> in the involved skin ##UREF##0##5##. It is possible that cetuximab may induce remission of psoriasis through regulation of this abnormal <sc>egfr</sc> metabolism.</p>" ]
[ "<title>2. DISCUSSION AND CONCLUSIONS</title>", "<p>We believe that this is the first case report of the safety of cetuximab monotherapy in a patient with metastatic and psoriasis. Trivin <italic>et al.</italic> ##REF##15370619##1## <sc>crc</sc> reported on a patient who had both a tumour response and durable remission of psoriasis after treatment with cetuximab in combination with <sc>folfiri</sc>. However, that case did not differentiate between a potential beneficial effect of the chemotherapy and the effect of the cetuximab.</p>", "<p>Durable remissions of psoriasis have also been reported in patients undergoing high-dose chemotherapy for hematologic malignancies ##REF##15156169##2##–##REF##12221673##4##.</p>", "<p>We conclude that a previous history of psoriasis or active lesions should not be regarded as a contraindication for cetuximab treatment in patients with advanced <sc>crc</sc>. Psoriasis is associated with abnormal expression of <sc>egfr</sc> in the involved skin ##UREF##0##5##. It is possible that cetuximab may induce remission of psoriasis through regulation of this abnormal <sc>egfr</sc> metabolism.</p>" ]
[ "<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>Cetuximab, a monoclonal antibody directed against the epidermal growth factor receptor, has activity against colorectal cancer. Treatment is associated with skin toxicity, and the safety of cetuximab in patients with psoriasis is unknown. We report the case of a male patient with stage <sc>iv</sc> colorectal cancer (<sc>crc</sc>) and a life-long history of extensive psoriasis. This patient experienced a durable remission of his <sc>crc</sc> and major improvement of his psoriasis during single-agent treatment with cetuximab. We conclude that, despite its known skin toxicity, cetuximab treatment can be offered to colorectal patients suffering from psoriasis.</p>" ]
[ "<title>1. CASE REPORT</title>", "<p>Cetuximab, an immunoglobulin G1 monoclonal antibody directed against the human epidermal growth factor receptor (<sc>egfr</sc>), is effective for the treatment of metastatic colorectal cancer (<sc>crc</sc>). Few data on the safety of cetuximab in patients with pre-existing chronic skin diseases are available, but here, we report on an 80-year-old man, successfully treated with cetuximab, who had suffered from extensive treatment-resistant psoriasis since adolescence. Diverse lifelong treatments had provided only partial relief. Recently, he had used calcipotriol ointment up to 4 times weekly.</p>", "<p>Our patient underwent resection of a well-differentiated adenocarcinoma (sigmoid, stage <sc>iii</sc>, pT4N1M0) in June 2003, followed by 8 months of adjuvant chemotherapy [bolus 5-fluorouracil (5-<sc>fu</sc>) and leucovorin (<sc>lv</sc>)]. In September 2004, bilateral inoperable liver metastases were treated with capecitabine until April 2005; best response was stable disease. At progression, second-line treatment was 5-<sc>fu</sc>/<sc>lv</sc>/irinotecan (<sc>folfiri</sc>) from September 2005 until May 2006, achieving a partial response. Disease progressed in August 2006, and <sc>lv</sc>/5-<sc>fu</sc>/oxaliplatin (<sc>folfox</sc>7) was administered until December 2007. Disease stabilized before progressing again.</p>", "<p>After a short treatment-free interval, cetuximab monotherapy (400 mg/m<sup>2</sup>, intravenously over 2 hours on day 1, and 250 mg/m<sup>2</sup>, intravenously over 1 hour on day 8 and once weekly thereafter) was initiated in February 2007. Immunohistochemistry for <sc>egfr</sc> on the primary tumour was negative. Over the next 6 weeks, the psoriasis lesions lessened considerably (##FIG##0##Figure 1##), and the patient reduced calcipotriol ointment applications to once weekly.</p>", "<p>The main adverse effects of cetuximab are dermatologic. Grade 2 dermatitis and folliculitis developed in this patient, but were controlled by topical clindamycin and then lessened in intensity. On re-evaluation in May 2007, almost complete regression of the liver metastases and a normalization of blood concentrations of carcinogenic embryonic antigen were observed. Throughout this period, no local or systemic corticosteroids were administered. In September 2007, irinotecan was added to cetuximab treatment because of disease progression. Disease control was regained, and the patient is still receiving cetuximab (July 1, 2008). He also continues to experience a marked improvement of his psoriasis.</p>" ]
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[ "<fig id=\"f1-co15_4p196\" position=\"float\"><label>FIGURE 1</label><caption><p>Tumour response and resolution of psoriasis in a patient with metastatic colorectal cancer (<sc>crc</sc>) treated with cetuximab monotherapy. An 80-year-old man was treated with cetuximab third-line for metastatic <sc>crc</sc>. His psoriasis on lower limbs, elbows, forearms, trunk, and face diminished in intensity after 6 weeks of treatment. This improvement was accompanied by almost complete regression of liver metastases. (A,C) February 2007. (B,D) May 2007.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"co15_4p196f1\"/>" ]
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[{"label": ["5"], "surname": ["King", "Gates", "Stoscheck", "Nanney"], "given-names": ["LE", "RE", "CM", "LB"], "suffix": ["Jr"], "article-title": ["Epidermal growth factor/transforming growth factor alpha receptors and psoriasis"], "source": ["J Invest Dermatol"], "year": ["1990"], "volume": ["95"], "fpage": ["10S"], "lpage": ["12S"]}]
{ "acronym": [], "definition": [] }
5
CC BY
no
2022-01-12 14:47:25
Curr Oncol. 2008 Aug; 15(4):196-197
oa_package/a0/94/PMC2528307.tar.gz
PMC2528308
18769610
[ "<title>1. INTRODUCTION</title>", "<p>In Atlantic Canada, approximately 90 children between birth and 14 years of age are diagnosed with cancer annually ##UREF##0##1##. Although childhood cancer is rare, it is nevertheless the most common disease-related cause of death among children. Fortunately, the survival rate for children with cancer has increased dramatically since the late 1990s. Understandably, the psychological, sociologic, and financial effects of the disease can be extremely stressful for families ##UREF##1##2##. Few studies have been conducted to document these issues, particularly from the perspective of the families who care for a child with cancer.</p>" ]
[ "<title>3. METHODS</title>", "<p>A qualitative research method was chosen for this project. Using qualitative methods, researchers can study social and cultural phenomena in the context of people’s day-to-day lives and from the viewpoint of the participants. This approach permits researchers to address the uniqueness of the particular situation and to generate a hypothesis or theory ##UREF##6##17##.</p>", "<title>3.1 Research Ethics Board Certification</title>", "<p>The research protocol used in our study was reviewed and approved by the River Valley Health Research Ethics Committee and the research ethics boards of Memorial University of Newfoundland and Université de Moncton. All participants signed an informed consent form before commencing their interview.</p>", "<title>3.2 Inclusion Criteria</title>", "<p>For inclusion in the study, we recruited parents or caregivers whose children were 19 years or younger when diagnosed with cancer. The child’s diagnosis had to have occurred no more than 10 years before recruitment.</p>", "<title>3.3 Recruitment Process</title>", "<p>In New Brunswick, staff members at the Canadian Cancer Society–New Brunswick Division mailed a letter to the parents of children who had participated in summer camps for children with cancer. The parents, if interested, could directly contact the study team for more information and to set up an interview. Participants were also recruited through newspaper articles and other French and English media.</p>", "<p>In Newfoundland and Labrador, participants were recruited with the assistance of Candlelighters Canada–Newfoundland and Labrador Division, a childhood cancer support foundation. A notice of invitation was posted in the Candlelighters newsletter, followed by a letter sent to 25 specific families that fit the inclusion criteria.</p>", "<p>The recruitment phase was concluded when the research team agreed that demographic and linguistic diversity was achieved in the sample and that no new themes were emerging from the interviews. With a total response rate to the individual mailings of approximately 30%, 9 anglophone and 12 francophone parents in New Brunswick and 7 anglophone parents in Newfoundland and Labrador participated in the study. Participants included one or both parents or caregivers.</p>", "<title>3.4 The Interview</title>", "<p>A semi-structured format was used to guide the interviews with the participants. The interview questions were all open-ended. The interview schedule started with general questions and then moved to questions about social supports, the effect of cancer on the child and the family, and specific questions about the economic effects of cancer. Most interviews took place in the participants’ homes, although a few took place in one of the research offices. In general, the interviews lasted between 1 and 2 hours. All participants agreed to have their interviews audiotaped on a digital recorder. After the interview was completed, the participants were asked to complete a sociodemographic form.</p>", "<title>3.5 Data Analysis</title>", "<p>All interviews were transcribed verbatim, and a rigorous constant comparative thematic analysis was applied ##REF##12518472##18##. In a thematic analysis, researchers identify themes and common patterns among the experiences of the participants ##REF##9168386##19##,##UREF##7##20##.</p>", "<p>The three researchers and their three assistants all read 6 selected transcripts representing the three distinct interview groups: English New Brunswick, French New Brunswick (translated transcripts), and English Newfoundland and Labrador. All six researchers coded the transcripts independently. The researchers then convened for a 2-day team meeting to discuss the codes. Most codes were easily agreed on; in cases where disagreement arose, team discussions ensued to reach a consensus about the code. An English coding scheme was developed, and this coding scheme was used to analyze the rest of the transcripts from all three interview groups. The coding scheme was slightly revised and updated during two subsequent teleconferences.</p>", "<p>Of the French interviews, 8 were translated into English by an official translation agency. Four interviews were coded in French by a bilingual researcher using the English coding scheme.</p>", "<title>3.6 Confidentiality</title>", "<p>Because of the relatively small number of pediatric cancer cases and the small population in the two provinces, as little detail as possible regarding the identities of the study participants is revealed here. Quotes may have been slightly modified to ensure that no identifying information is disclosed.</p>" ]
[ "<title>4. RESULTS</title>", "<title>4.1 Profile of the Study Population</title>", "<p>Of the 28 families, 5 had children still in active treatment at the time of the interview, 3 had experienced the death of their child, and the rest had children in the follow-up care stage of the cancer care continuum. Just over half (57%) of the families lived in rural areas, and almost three quarters considered themselves to be religious (68%) at the time of the interview. Most of the parents were married, with an average of 2 children, and they had postsecondary educations and good incomes (see ##TAB##0##Table I##).</p>", "<p>Among the parents, 17 mothers (61%) were working full- or part-time and 11 (39%) were not working (1 retired, 7 not in the labour force, 3 on sick leave or stress leave) at the time of the interview; 24 fathers (86%) were working full- or part-time, 2 (7%) were seasonal workers, and 2 (7%) were not in the workforce.</p>", "<p>The age of the affected children at the time of diagnosis in New Brunswick ranged from 6 months to 17 years. In Newfoundland and Labrador, the age of the affected children at diagnosis ranged from 3 to 16 years. The most common diagnosis was acute lymphocytic leukemia. All children had received chemotherapy, a few had received radiation therapy, and a few had undergone bone marrow transplantation.</p>", "<title>4.2 Themes Related to Economic Effects</title>", "<p>The four major themes that emerged as contributing factors to the severe economic effects on the families were</p>", "<p>travel expenses for treatment and follow-up care,</p>", "<p>loss of income because of a reduction or termination of parental employment,</p>", "<p>out-of-pocket expenses for treatment, and</p>", "<p>an inability to draw on programs for assistance or income supplementation.</p>", "<title>4.2.1 Travel Costs</title>", "<p>Of the 28 families interviewed, all but 3 were required to travel to other cities, frequently out-of-province, for treatments. In Newfoundland and Labrador, all families traveled to the Janeway Children’s Hospital in St. John’s. Most of the New Brunswick families traveled to the <sc>iwk</sc> Health Centre in Halifax, Nova Scotia. Some families living in western and northern New Brunswick traveled to children’s hospitals in Quebec. A few parents whose children underwent very specialized treatments such as bone marrow transplantation, traveled to Ontario hospitals.</p>", "<p>It was not uncommon for parents to report having to travel immediately after a medical consultation to a large children’s hospital without returning home for weeks or sometimes months. Parents were then forced to make alternative arrangements for other children at home and (if they were working for pay) with their employers. As one parent said, “We just had to jump in the van and drive to [city] with literally the clothes on our backs and a few dollars. It was all we had. When we got there, we did not think about where we [were] going to stay” (interview 7).</p>", "<p>The costs associated with travel and accommodations were substantial for many parents. One father said, “Financially, it set us back 10 years because of the loss of my salary and the wretched trips” (interview 9). Many families were able to take advantage of reduced-rate accommodations such as the Ronald McDonald House or hospital rates at local hotels; however, because of prolonged stays, the costs still added up for these families. As one mother described it, “even though you had Ronald MacDonald house to stay at for $11 a night, of course the phone bill[....] Every day, every report, we phoned home. Parking, meals at the hospital, and we tried to get groceries and eat when we could at Ronald MacDonald house, but that was ... you know, if I was going to take a guess, I would say it was couple thousand dollars for those three weeks” (interview 14).</p>", "<title>4.2.2 Work-Related Issues</title>", "<p>In this study, caring for a child with cancer greatly affected the work patterns of the parents in general, but particularly the work patterns of the mothers. The work status of the parents at the time of their child’s cancer diagnosis and during treatment shows a considerable gendered change (see ##FIG##0##Figures 1## and ##FIG##1##2##). At the time of diagnosis, 24 fathers and 22 mothers were working for pay either full-time or part-time. Of the fathers, 61% reported that their work hours changed during the child’s treatment; among the mothers, the proportion was 86%. Five mothers (18%) simply stopped working altogether.</p>", "<p>One father described why his wife stopped working and looked after their sick child: “[She] is a nurse, and our salaries are pretty much equal. She earns a little bit more than I do[....] If I left for a year, six months or two years, I would have to transfer a lot of the projects to somebody else. It would be hard for me to start again, but it seems to be a lot easier for her [...] plus, she’s the mother.” During treatment, this mother was unable to continue to work. The father said, “[She] tried to work through [the child’s treatment] for a few months, and then she had to go on sick leave[....] The company denied her benefits. They said, ‘You’re not sick, your son is.’” (interview 13).</p>", "<p>Self-employed parents often experienced an immediate loss of income: “There were times, you know, when we weren’t able to work the hours that we normally work, so there was much less money coming in[....] If you do not go to work, you don’t get paid” (interview 2).</p>", "<title>4.2.3 Out-of-Pocket Medical Costs</title>", "<p>Not only do travel, lodging, and meals away from home add to costs, but so too do medications and medical supplies not covered by the <sc>php</sc>. Private supplemental health insurances covers some medication and supply costs, but parents without insurance must pay for out-of-hospital drugs and medical supplies themselves. Many of the interviewees spoke of spending many thousands of dollars for treatment equipment such as feeding tubes, needles, and medication. One mother was so overwhelmed by these costs for medical supplies for her child that she felt “the last thread snapped” when she was negotiating with the supplemental private health insurer on what they would cover and what they would not cover. This mother was ultimately forced to cancel her daughter’s Registered Education Savings Plan and to withdraw the funds to pay for medical expenses.</p>", "<title>4.2.4 Inability to Draw On Income Support Programs</title>", "<p>Few parents were able to draw on formal programs for financial support. Only 1 parent remarked that supplemental private health insurance provided a per diem when the child was hospitalized; in most cases, however, the parents were unable to find any respite from the demands on their finances. Many parents reported that programs were contingent on the parent being able to “look for work” and that financial assistance was based on “previous earnings.”</p>", "<p>One mother described her struggle with the federal Employment Insurance program: “We [husband and wife] tried to get on unemployment, but we couldn’t get that. We tried to get social assistance to help us, and they did a little bit, but it wouldn’t be enough to butter your bread, because they had said something about ‘You made too much money the month before.’” She noted that her husband had been laid off just before their daughter’s diagnosis of cancer: “They gave us the weekend off to pack up and to drive to the <sc>iwk</sc>. We went to the unemployment office to see if they would help us because he was laid off, but they refused. We did not lie about anything. I told him that my daughter had just found out that she has cancer, and we are leaving for Halifax. But they said there is nothing we can do[....] So you go with the credit cards you have” (interview 3).</p>" ]
[ "<title>5. DISCUSSION AND CONCLUSIONS</title>", "<p>Our research clearly demonstrates the severity of the negative financial effects on families in New Brunswick and Newfoundland and Labrador when they care for a child with cancer. The emerging theory from this research is that government programs are inadequate to support families who care for children with catastrophic illnesses such as cancer. Although none of the interviewed families suggested that they had to withdraw care from their child because of financial constraints, we demonstrated that many parents struggled with financial hardship and that these concerns imposed additional stresses on the families.</p>", "<p>Debts accrued over the course of the treatment, and the follow-up phase of the disease could have long-term effects on the financial stability of the family. Many families discussed how they were still paying off debts years after the treatment. A few parents even discussed having to re-mortgage their homes or to take money out of registered retirement or education savings plans to pay for the medical and out-of-pocket expenses.</p>", "<p>Care for a child with cancer was, not surprisingly, gendered. It was most often the mother who reduced or terminated employment to care for the sick child, regardless of prior earning power in the family. Her reduced income not only was responsible for the immediate drop in family income, but also potentially jeopardized her future and retirement earnings. Inability to contribute to a registered retirement savings plan or a company pension plan could also affect her income in old age ##UREF##8##21##.</p>", "<p>Caregivers in the formal health care system need to be aware that parents of pediatric cancer patients not only have to deal with the stress of the illness, but also with stressors in their immediate environment, which can be very severe ##REF##11118467##6##,##REF##11352405##11##.</p>", "<p>Canada lacks support programs for parents caring for a child with a catastrophic illness. The federal government has introduced a “compassionate leave” program, but that program is geared toward caring for a spouse or parent with a terminal illness ##UREF##9##22##. Parents who care for a child with cancer are particularly vulnerable to financial ruin at the time of cancer treatments and, in most cases, for many years afterward, because cancer survivorship for children increases with technological and medical advances. Canada needs to develop programs for parents who care for children with catastrophic illnesses so that parents do not have to struggle financially to properly care for the child with cancer and that child’s siblings.</p>" ]
[ "<title>5. DISCUSSION AND CONCLUSIONS</title>", "<p>Our research clearly demonstrates the severity of the negative financial effects on families in New Brunswick and Newfoundland and Labrador when they care for a child with cancer. The emerging theory from this research is that government programs are inadequate to support families who care for children with catastrophic illnesses such as cancer. Although none of the interviewed families suggested that they had to withdraw care from their child because of financial constraints, we demonstrated that many parents struggled with financial hardship and that these concerns imposed additional stresses on the families.</p>", "<p>Debts accrued over the course of the treatment, and the follow-up phase of the disease could have long-term effects on the financial stability of the family. Many families discussed how they were still paying off debts years after the treatment. A few parents even discussed having to re-mortgage their homes or to take money out of registered retirement or education savings plans to pay for the medical and out-of-pocket expenses.</p>", "<p>Care for a child with cancer was, not surprisingly, gendered. It was most often the mother who reduced or terminated employment to care for the sick child, regardless of prior earning power in the family. Her reduced income not only was responsible for the immediate drop in family income, but also potentially jeopardized her future and retirement earnings. Inability to contribute to a registered retirement savings plan or a company pension plan could also affect her income in old age ##UREF##8##21##.</p>", "<p>Caregivers in the formal health care system need to be aware that parents of pediatric cancer patients not only have to deal with the stress of the illness, but also with stressors in their immediate environment, which can be very severe ##REF##11118467##6##,##REF##11352405##11##.</p>", "<p>Canada lacks support programs for parents caring for a child with a catastrophic illness. The federal government has introduced a “compassionate leave” program, but that program is geared toward caring for a spouse or parent with a terminal illness ##UREF##9##22##. Parents who care for a child with cancer are particularly vulnerable to financial ruin at the time of cancer treatments and, in most cases, for many years afterward, because cancer survivorship for children increases with technological and medical advances. Canada needs to develop programs for parents who care for children with catastrophic illnesses so that parents do not have to struggle financially to properly care for the child with cancer and that child’s siblings.</p>" ]
[ "<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Objective</title>", "<p>In a study conducted in New Brunswick and Newfoundland and Labrador, we examined the economic impact on families caring for a child with cancer.</p>", "<title>Methods</title>", "<p>We undertook semi-structured interviews with 28 French and English families with a child diagnosed with cancer in the last 10 years.</p>", "<title>Results</title>", "<p>Families who care for a child with cancer incur considerable costs during the diagnostic, treatment, and follow-up care phases of the disease. Four major themes emerged from this qualitative study as contributing factors for these expenses: necessary travel; loss of income because of a reduction or termination of parental employment; out-of-pocket treatment expenses; and inability to draw on assistance programs to supplement or replace lost income. In addition, many of the decisions with regard to the primary caregiver were gendered. Typically, the mother is the one who terminated or reduced work hours, which affected the entire family’s financial well-being.</p>", "<title>Conclusions</title>", "<p>For families with children diagnosed with cancer, financial issues emerged as a significant concern at a time when these families were already consumed with other challenges. This economic burden can have long-term effects on the financial security, quality of life, and future well-being of the entire family, including the siblings of the affected child, but in particular the mother. Financial assistance programs for families of seriously ill children need to be revisited and expanded.</p>" ]
[ "<title>2. BACKGROUND</title>", "<p>The experience of pediatric cancer patients is different from that of adults with cancer, because the whole family—particularly the parents, and in some cases, the grandparents—are usually completely involved in the child’s illness ##REF##15188446##3##,##REF##17847987##4##. It has been reported that parents can develop posttraumatic stress disorder from dealing with a child’s illness ##REF##12509953##5##,##REF##11118467##6##. Siblings have reported feeling lost and ignored by parents who are preoccupied with the sick child and who may be absent from home for extended periods of time accompanying a child receiving treatment out of town. These feelings can lead to behavioural challenges in the siblings left at home ##REF##15188446##3##.</p>", "<p>In addition to the disruptions of family dynamics, families describe financial hardship associated with caring for a child with cancer. In one study, 37% of families reported that they were forced to borrow money to cover the extra cost of treatment related to the child’s illness ##REF##12755929##7##. Another study reported that parents of children with cancer suffered greater financial hardship than did parents of children with other serious illness such as diabetes ##REF##16728365##8##.</p>", "<p>Barr and Sala ##REF##14608192##9## reported that few studies have specifically examined the out-of-pocket expenses incurred by families dealing with childhood cancer and other chronic diseases. A small qualitative Canadian study by Scott–Findlay and Chalmers ##REF##11588761##10## reported that, among other hardships, families with children who had cancer were required to travel 400 km on average (round trip) to receive treatment. Yantzi <italic>et al.</italic> ##REF##11352405##11## reported a relationship between the distance a family has to travel to the hospital for children with chronic illness and the quality of family relationships, because of the travel time and time spent away from home.</p>", "<p>In the present study, we were interested in learning about the experiences of families in New Brunswick and Newfoundland and Labrador who had cared or were caring for a child with cancer, and the effect of this experience on the family’s financial well-being.</p>", "<title>2.1 Study Setting</title>", "<p>New Brunswick and Newfoundland and Labrador are unique in that, in both provinces, half the population lives in rural areas or small towns. As a result, many people must travel to receive specialized treatments. For example, in New Brunswick, pediatric cancer patients are usually treated out-of-province in either Nova Scotia or Quebec. In Newfoundland and Labrador, patients can attend the Janeway Hospital for Children in St. John’s, but the province is large, and travel from remote areas can involve journeys of hundreds of kilometres.</p>", "<title>2.2 Health Care System</title>", "<p>All Canadian residents are entitled to enrol in a provincial health plan (<sc>php</sc>). The <sc>php</sc> covers all medically necessary physician and hospital costs and the cost of drugs provided in hospital.</p>", "<p>Prescription drugs provided outside a hospital setting are not covered by the <sc>phps</sc>. For cancer patients, these drugs may include oral chemotherapy agents that can be administered at home ##REF##17695439##12## or supportive drugs (such as antiemetics or pain medications) given to combat the side effects of treatment. Provincial drug insurance programs may offset the costs of some of these drugs for low-income families, and both New Brunswick and Newfoundland and Labrador have such programs ##UREF##2##13##. Out-of-pocket drug expenses may also be cost-shared through private supplemental health insurance programs (Blue Cross or Medavie, for example). Private supplemental medical insurance (including a prescription drug plan) is often offered as an employment benefit, and individuals can also purchase supplemental health insurance on their own (but usually at much higher premiums). Costs covered through private supplemental insurance policies vary and may require a 20%–30% co-payment ##UREF##3##14##. Overall, 20% of Canadians lack private supplemental health insurance, and the proportion is higher in the Atlantic Provinces ##UREF##4##15##. In 2001, it was reported that 30% of Newfoundland and Labrador residents and 32% of New Brunswick residents did not have private supplemental health insurance ##UREF##5##16##.</p>" ]
[ "<title>Acknowledgments</title>", "<p>6. ACKNOWLEDGMENTS</p>", "<p>We thank Dr. Sue Tatemichi for reviewing this manuscript. Financial support for the study was provided by the Canadian Cancer Society–New Brunswick Division and from research funding provided as part of a Canadian Institutes of Health Research/<sc>rpp</sc> New Investigator Award held by Maria Mathews.</p>" ]
[ "<fig id=\"f1-co15_4p173\" position=\"float\"><label>FIGURE 1</label><caption><p>Employment of parents of children with cancer at the time of diagnosis.</p></caption></fig>", "<fig id=\"f2-co15_4p173\" position=\"float\"><label>FIGURE 2</label><caption><p>Employment status change of parents of children with cancer during the treatment phase.</p></caption></fig>" ]
[ "<table-wrap id=\"tI-co15_4p173\" position=\"float\"><label>TABLE I</label><caption><p>Income and education of parents of children with cancer at time of interview</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" rowspan=\"1\" colspan=\"1\">NB</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">NL</th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Total [</italic>n <italic>(%)]</italic></th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Education</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Mother</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  High school or less</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (14)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  Professional diploma</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (25)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  Undergraduate degree</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (25)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  Graduate/professional degree</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 (32)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  Did not wish to answer</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (04)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Father</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  High school or less</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 (32)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  Professional diploma</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (21)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  Undergraduate degree</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (25)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  Graduate/professional degree</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (18)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  Did not wish to answer</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (04)</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\"> Annual family income</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  $20,000 or less</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (04)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  $20,001 to $30,000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (15)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  $30,001 to $40,000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  $40,001 to $50,000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (15)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  $50,001 to $60,000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (11)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  $60,001 to $70,000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (08)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  $70,001 to $80,000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (08)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  $80,001 or more</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 (32)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  Did not wish to answer</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (11)</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><fn id=\"tfn1-co15_4p173\"><p>NB = New Brunswick; NL = Newfoundland and Labrador.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"co15_4p173f1\"/>", "<graphic xlink:href=\"co15_4p173f2\"/>" ]
[]
[{"label": ["1"], "collab": ["Public Health Agency of Canada ("], "sc": ["phac"], "source": ["The Canadian Childhood Cancer Surveillance and Control Program: Facts and Figures"], "publisher-loc": ["Ottawa"], "publisher-name": ["PHAC"], "year": ["2007"]}, {"label": ["2"], "surname": ["Lightfoot", "Streggles", "Gauthier\u2013Frohlick"], "given-names": ["N", "S", "D"], "article-title": ["Psychological, physical, social, and economic impact of travelling great distances for cancer treatment"], "source": ["Curr Oncol"], "year": ["2005"], "volume": ["12"], "fpage": ["1"], "lpage": ["7"]}, {"label": ["13"], "collab": ["New Brunswick, Department of Health"], "article-title": ["Prescription drug program [Web resource]"], "publisher-loc": ["Fredericton"], "publisher-name": ["Department of Health"], "comment": ["n.d. [Available at: "], "ext-link": ["www.gnb.ca/0051/0212/index-e.asp"]}, {"label": ["14"], "collab": ["Canadian Life and Health Insurance Association ("], "sc": ["clhia", "clhia", "clhia"], "comment": ["n.d. [Available at: "], "ext-link": ["www.clhia.ca/e2_g.htm"]}, {"label": ["15"], "collab": ["Canada, Statistics Canada"], "article-title": ["Population urban and rural, by province and territory (Newfoundland and Labrador) [Web page]"], "publisher-loc": ["Ottawa"], "publisher-name": ["Statistics Canada"], "year": ["2005"], "comment": ["[Available at: "], "ext-link": ["www40.stat-can.ca/l01/cst01/demo62b.htm"]}, {"label": ["16"], "collab": ["Canadian Life and Health Insurance Association ("], "sc": ["clhia"], "source": ["The Role of Supplementary Health Insurance in Canada\u2019s Health System"], "comment": ["Submission to the Commission on the Future of Health Care in Canada"], "publisher-loc": ["Toronto"], "publisher-name": ["CLHIA"], "year": ["2001"]}, {"label": ["17"], "surname": ["Creswell"], "given-names": ["JW"], "source": ["Qualitative Inquiry and Research Design: Choosing Among Five Traditions"], "publisher-loc": ["Thousand Oaks, CA"], "publisher-name": ["Sage Publications"], "year": ["1997"]}, {"label": ["20"], "surname": ["Thorne"], "given-names": ["S"], "article-title": ["Data analysis in qualitative research"], "source": ["Evid Based Nurs"], "year": ["2000"], "volume": ["3"], "fpage": ["68"], "lpage": ["70"]}, {"label": ["21"], "surname": ["Miedema"], "given-names": ["B"], "source": ["Mothering for the State: The Paradox of Fostering"], "publisher-loc": ["Halifax"], "publisher-name": ["Fernwood"], "year": ["1999"]}, {"label": ["22"], "collab": ["Canada, Human Resources and Social Development Canada ("], "sc": ["hrsdc"], "source": ["Information on Labour Standards: 5A Compassionate Care Leave"], "publisher-loc": ["Gatineau, QC"], "publisher-name": ["HRSDC"], "year": ["2006"]}]
{ "acronym": [], "definition": [] }
22
CC BY
no
2022-01-12 14:47:25
Curr Oncol. 2008 Aug; 15(4):173-178
oa_package/19/fb/PMC2528308.tar.gz
PMC2528309
18769611
[ "<title>1. INTRODUCTION</title>", "<p>Prostate cancer is the most common malignancy in Canadian men and ranks third behind lung and colon cancer in terms of cancer-related mortality. However, from 1994 to 2003, mortality from prostate cancer declined at a rate of 2.7% annually. That decline is attributed both to the widespread use of testing for prostate-specific antigen (<sc>psa</sc>), which has led to a shift in stage and grade at diagnosis, and to the existence of effective therapies for clinically localized disease.</p>", "<p>In 2007, estimates placed new cases of prostate cancer at 22,300 and deaths from the disease at 4300 ##UREF##0##1##. Those statistics highlight some important facts about prostate cancer:</p>", "<p>In most cases, prostate cancer is not a fatal condition.</p>", "<p>Current treatment options still fail to cure or control disease in a significant proportion of cases, and approximately 20% of patients die from their prostate cancer.</p>", "<p>Not surprisingly, the treatment strategies under evaluation in ongoing clinical trials in early prostate cancer reflect the biologic heterogeneity of the disease. They include such diverse therapies as active surveillance for good-prognosis disease and the addition of cytotoxic chemotherapy to radical radiation or prostatectomy for disease with high risk of relapse. The present article reviews ongoing studies in localized prostate cancer conducted by the National Cancer Institute of Canada (<sc>ncic</sc>) Clinical Trials Group (<sc>ctg</sc>).</p>" ]
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[ "<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>The designation “clinically localized prostate cancer” comprises a group of biologically heterogeneous tumours with different growth rates and risks of relapse. Because prostate cancer is primarily a disease of older men, treatment selection must take into account the prognosis of the tumour, patient age, comorbidities, side effects of treatment, and patient preferences. Clinical trials must identify the various prognostic groups and test the appropriate treatment strategies within these subgroups.</p>" ]
[ "<title>2. NCIC CTG PR.11: A PHASE III STUDY OF ACTIVE SURVEILLANCE THERAPY AGAINST RADICAL TREATMENT IN PATIENTS DIAGNOSED WITH FAVOURABLE-RISK PROSTATE CANCER (START)</title>", "<title>2.1 Background</title>", "<p>Tumours detected because of <sc>psa</sc> testing comprise most of the localized prostate cancer cases diagnosed today. Although testing may allow for diagnosis and the use of curative therapy at an earlier stage in a potentially life-threatening disease, it also clearly identifies a group of patients with biologically indolent tumours in whom radical therapy may be unnecessary and detrimental because of its associated morbidity and costs ##REF##12813170##2##,##REF##12096083##3##. Previous nonrandomized studies have identified the prognostic significance of stage and grade in patients treated with conservative therapy or observation, thus identifying a patient population for whom curative therapy can potentially be withheld without compromise to long-term outcome ##REF##8272085##4##–##REF##15870412##6##. An extension of the concept of observation is that of “active surveillance,” which entails close follow-up of disease and intervention with curative intent triggered by early signs of disease progression.</p>", "<p>The strategy of active surveillance for patients with favourable-risk prostate cancer was evaluated in a large phase <sc>ii</sc> study by Klotz. Active surveillance was applied in 331 patients with favourable-risk disease (defined as <sc>psa</sc> below 15 ng/mL, Gleason score of 7 or less, and tumour stage less than T2B), following them until criteria of early disease progression [defined by biochemical, histologic (grade), or clinical progression] were met. Of those patients, 80% had a Gleason score of 6 or less, and the same proportion had a <sc>psa</sc> below 10 ng/mL. With a median follow-up of 72 months, 34% of patients discontinued active surveillance. Biochemical progression led to discontinuation in 15%; clinical progression, in 3%; histologic progression, in 4%; and patient preference, in 12%. With a median follow-up of 7 years, overall survival was 85%, and disease-specific survival was 99%. The median <sc>psa</sc> doubling time for the entire cohort was 7.0 years; a <sc>psa</sc> doubling time of less than 2 years was associated with a high risk of local progression for patients who underwent radical prostatectomy. At January 2007, 134 patients remained on active surveillance ##REF##18047961##7##.</p>", "<title>2.2 Study Design</title>", "<p>The PR.11 trial is an Intergroup study led by the <sc>ncic ctg</sc> (study chair, Dr. Laurence Klotz) that compares active surveillance with radical therapy (prostatectomy or radiotherapy depending on physician and patient choice) at the time of diagnosis in a randomized phase <sc>iii</sc> setting. Eligible patients are those with a life expectancy of more than 10 years and favourable-risk prostate cancer [defined as clinical stage T1B, T1C, T2A, or T2B at the time of diagnosis; clinical (diagnostic biopsy) Gleason score of 6 or less; <sc>psa</sc> 10.0 ng/mL or lower]. Patients randomized to the active surveillance arm will undergo radical intervention (again, prostatectomy or radiotherapy depending on physician and patient choice) at the time one or more of the following pre-specified criteria are met:</p>", "<p>Biochemical progression—<sc>psa</sc> doubling time less than 3 years, based on at least 5 separate consecutive measurements over a minimum of 12 months from the date of the baseline measurement or from the date that the <sc>psa</sc> reached a value greater than or equal to the <sc>psa</sc> before initiation of androgen deprivation therapy (if applicable), as assessed by the local investigator.</p>", "<p>Histologic or grade progression—Gleason pattern predominant 4 or greater (that is, a Gleason score of 7 (4+3) or higher) in re-biopsy of the prostate.</p>", "<p>Clinical progression—”local progression” defined as local progression of prostate cancer resulting in urinary retention, gross hematuria, or hydronephrosis; or “distant metastasis” defined by radiology, cytology, or histology (or a combination) at sites remote from the prostate and regional lymph nodes.</p>", "<p>Using a non-inferiority design to rule out a greater than 5% difference in 15-year survival between the radical treatment and active surveillance groups, 2130 patients will be accrued over a 5-year period. The primary endpoint is disease-specific survival. That endpoint, rather than overall survival, was selected because of the need to determine the effect of the active surveillance strategy specifically on prostate cancer mortality. Secondary endpoints include overall survival, quality of life, distant disease-free survival, <sc>psa</sc> relapse or progression after radical intervention, initiation of androgen deprivation therapy, proportion of patients on the active surveillance arm receiving radical intervention, prognostic significance of <sc>psa</sc> doubling time before diagnosis, and prognostic significance of molecular biomarkers. Quality of life is an important part of the study, and the Expanded Prostate Cancer Index, <sc>rand</sc> SF-12, and State–Trait Anxiety Inventory will be used to provide a comprehensive examination of the various components of patient-reported outcomes on study. The feasibility phase has commenced in designated centres of participating cooperative groups. If the results of the feasibility phase indicate sufficient patient and physician willingness to participate in the randomization process, then accrual will be opened widely.</p>", "<title>3. NCIC CTG PR.12: A PHASE III STUDY OF NEOADJUVANT DOCETAXEL AND ANDROGEN SUPPRESSION PLUS RADIATION THERAPY VERSUS ANDROGEN SUPPRESSION ALONE PLUS RADIATION THERAPY FOR HIGH-RISK LOCALIZED ADENOCARCINOMA OF THE PROSTATE (DART)</title>", "<title>3.1 Background</title>", "<p>Radical radiotherapy and long-term androgen suppression constitute an accepted treatment option for localized but high-risk disease as defined by clinical stage (T3) and high Gleason score (8 or higher) or high <sc>psa</sc> (20 ng/mL or more), or both. Results from previously conducted randomized studies are consistent with 5-year disease-free survival rates of 46%–74% with combined therapy ##REF##12126818##8##–##REF##15817329##10##, thus providing the rationale for continued evaluation of therapies to improve outcome by control of micrometastatic disease.</p>", "<p>Docetaxel is a good candidate drug. The mechanism of action of this agent involves disruption of the microtubular network critical for mitotic and inter-phase cellular functions. Doses of 75–100 mg/m##REF##12813170##2## intravenously (IV) administered are well tolerated, with neutropenia, alopecia, cutaneous reactions, gastro-intestinal effects (nausea, diarrhea), neurotoxicity, and edema being among the most frequently reported adverse events. Severe hypersensitivity reactions characterized by respiratory or circulatory instability or generalized rash or erythema occur in fewer that 5% of patients, although lesser grades are more common ##UREF##1##11##. Using overall survival as the primary endpoint, two pivotal studies have demonstrated the efficacy of docetaxel in advanced hormone-refractory prostate cancer ##REF##15470214##12##,##REF##15470213##13##. Efficacy and adverse event data support the use of the every-three-weeks docetaxel schedule, and that schedule has been widely adopted for use in this patient population.</p>", "<p>Using changes in <sc>psa</sc> as a marker of antitumour effect, studies have shown that docetaxel is also active against hormone-sensitive prostate cancer ##REF##15183967##14##–##REF##16033841##17##. Furthermore, based on preclinical data that suggest that docetaxel may result in phosphorylation and inactivation of the anti-apoptotic protein Bcl-2 (which is upregulated with androgen suppression), combination therapy with docetaxel and androgen suppression may lead to greater antitumour effect ##REF##9000560##18##–##REF##7671257##21##.</p>", "<p>Timing of therapy appears to be important. Eigl <italic>et al.</italic> implanted LNCaP human prostate cancer and Shionogi mouse mammary carcinoma cell lines into mice, and followed up with treatment using one of these three regimens: castration with paclitaxel on progression, paclitaxel with castration on progression, or concurrent castration and paclitaxel ##REF##16000589##22##. As compared with sequential castration followed by paclitaxel, concurrent therapy resulted in significantly longer time-to-progression and time-to-sacrifice in the mice. Notably, a marked lack of response to castration in the mice treated initially with paclitaxel was seen.</p>", "<p>Clinical studies in patients with locally advanced prostate cancer have demonstrated the feasibility and tolerability of combined therapy with docetaxel and androgen suppression in the neoadjuvant setting before prostatectomy ##UREF##3##23## or radiotherapy ##UREF##4##24##. In 54 men with high-risk prostate cancer, McKenzie <italic>et al.</italic> used one of two neoadjuvant treatment schedules before radical radiotherapy: 6 months of androgen suppression, plus 2 cycles of docetaxel 35 mg/m##REF##12813170##2## IV weekly for 6 weeks out of 8; or 5 months of androgen suppression, plus 4 cycles of docetaxel 75mg/m##REF##12813170##2## IV every 3 weeks. Androgen suppression was continued after completion of radiotherapy for a total duration of 3 years. The primary endpoint was unacceptable toxicity. Eight patients (14.8%) developed unacceptable toxicity: 5 in the weekly docetaxel regimen [grade 3 acute genitourinary radiotherapy-related adverse events (<italic>n</italic> = 3), grade 3 docetaxel hypersensitivity (<italic>n</italic> = 1), grade 3 fatigue lasting more than 2 weeks (<italic>n</italic> = 1)] and 3 in the every-three-weeks arm [febrile neutropenia (<italic>n</italic> = 1), grade 4 neutropenia lasting more than 7 days, grade 3 acute genitourinary radiotherapy-related adverse event (<italic>n</italic> = 1)]. Compliance with the radiotherapy was excellent, and all patients completed planned treatment. Long-term follow-up continues. The neoadjuvant regimen containing androgen suppression and every-three-weeks docetaxel was chosen for further study based on the promising results of this pilot and the proven efficacy and tolerability of every-three-weeks docetaxel dosing in the advanced-disease setting.</p>", "<title>3.2 Study Design</title>", "<p>The <sc>ncic ctg</sc> PR.12 trial is a phase <sc>iii</sc> study comparing the every-three-weeks docetaxel and neoadjuvant androgen suppression regimen piloted by McKenzie <italic>et al.</italic> to androgen suppression alone in addition to radical radiotherapy (three-dimensional conformal radiotherapy, 46 Gy in 23 fractions, with 24–28 Gy in 12–14 fractions). Study chairs are Drs. Michael McKenzie and Kim Chi. In both treatment arms, androgen suppression will be given for a total duration of 3 years. Patients with high-risk disease (defined as at least clinical stage T3 or T4, Gleason score of 8 or higher, or <sc>psa</sc> above 20 ng/mL) are eligible for the study. The primary endpoint is disease-free survival. The sample size for this study is estimated based on detecting an estimated 33.3% risk reduction in disease progression favouring the experimental arm [hazard ratio (<sc>hr</sc>): 0.667], using a 1-sided log-rank test at the 2.5% significance level and 90% power. An estimated 530 patients (assuming a 14.8% loss to follow-up) will be accrued over 4.5 years, with an additional 5 years of follow-up. Secondary endpoints include overall survival, time to biochemical disease progression, time to local disease progression, time to distant disease progression, time to next anticancer therapy, progression-free survival, degree of <sc>psa</sc> suppression before radiotherapy, quality of life, and adverse events. Centres will be credentialed by <sc>ncic ctg</sc> for delivery of radiotherapy before randomization of the first patient. Tumour and biologic specimens will be collected during the study to determine the prognostic role of cytokines and insulin-like growth factor axis markers. In addition, cytokine levels and changes in levels over time will be correlated with fatigue (as measured by the Common Terminology Criteria, version 3.0) and quality of life.</p>", "<title>4. NCIC CTG PR.13: RADIOTHERAPY AND ANDROGEN DEPRIVATION IN COMBINATION AFTER LOCAL SURGERY (RADICALS)</title>", "<title>4.1 Background</title>", "<p>The PR.13 study represents a collaborative effort between the Medical Research Council Clinical Trials Unit (United Kingdom) and the <sc>ncic ctg</sc> (Canadian study chairs: Drs. Charles Catton and Fred Saad). This large pragmatic study is addressing two fundamental issues in the postoperative management of patients with resectable prostate cancer: What is the optimal timing of radiotherapy in these patients? And what role, if any, does androgen suppression play in determining outcome? The relevance of the study to current practice is underscored by the fact that prostatectomy is a standard of care in men presenting with operable prostate cancer. In Ontario alone, the number of radical prostatectomies between 1993–1994 and 2003–2004 rose by 171% ##UREF##5##25##.</p>", "<p>The role of postoperative radiotherapy has been addressed in three randomized studies:</p>", "<p>In <sc>eortc</sc> 22911, 1005 patients with pT3 disease post radical prostatectomy were randomized to either observation or adjuvant radiotherapy ##REF##16099293##26##. The primary endpoint, local control, was modified to clinical progression-free survival and later to biochemical progression-free survival. After a median follow-up of 5 years, biochemical progression-free survival was significantly improved in the irradiated group [74.0%; 98% confidence interval (<sc>ci</sc>): 68.7 to 79.3] as compared with the observation group (52.6%; 98% <sc>ci</sc>: 46.6 to 58.5; <italic>p</italic> &lt; 0.0001). Clinical progression-free survival was significantly better with adjuvant radiation (<sc>hr</sc>: 0.61; 98% <sc>ci</sc>: 0.43 to 0.87; <italic>p</italic> = 0.0009). No difference in overall survival was detected. The rate of 5-year grade 3 or higher toxic effects was 2.6% in the no-further-treatment group and 4.2% in the postoperative irradiation group (<italic>p</italic> = 0.0726). The incidence of grade 3 urethral stricture and incontinence was 1.4% (6 patients) in each group.</p>", "<p>A similar design was used in <sc>swog</sc> 8794 (<sc>ncic ctg</sc> PR.2), which randomized 425 men with pT3 disease to observation or to radiotherapy to the prostate bed ##REF##17105795##27##. The primary endpoint was metastasis-free survival, defined as the time from randomization to first evidence of metastatic disease or death from any cause. With a median follow-up of 10.6 years, the metastases-free survival was not significantly different between the two arms (<sc>hr</sc>: 0.75; 95% <sc>ci</sc>: 0.55 to 1.02; <italic>p</italic> = 0.06). Overall survival favoured the adjuvant radiotherapy arm, but did not reach statistical significance (<sc>hr</sc>: 0.80; 95% <sc>ci</sc>: 0.58 to 1.09; <italic>p</italic> = 0.16). The rate of biochemical relapse was significantly lower in men with an undetectable <sc>psa</sc> level post prostatectomy (<italic>n</italic> = 249) treated with adjuvant radiotherapy (<sc>hr</sc>: 0.43; 95% <sc>ci</sc>: 0.31 to 0.58; <italic>p</italic> &lt; 0.001), as was recurrence-free survival [defined as survival without evidence of measurable or evaluable disease, excluding <sc>psa</sc> relapse (<sc>hr</sc>: 0.62; 95% <sc>ci</sc>: 0.46 to 0.82; <italic>p</italic> = 0.001)]. Approximately one third of patients randomized to the observation arm ultimately received pelvic radiotherapy. Rectal complications (3.3% vs. 0%, <italic>p</italic> = 0.02), urethral stricture [17.8% VS. 9.5%; risk ratio (<sc>rr</sc>): 1.9; 95% <sc>ci</sc>: 1.1 to 3.1; <italic>p</italic> = 0.02), and urinary incontinence (6.5% vs. 2.8%; <sc>rr</sc>: 2.3; 95% <sc>ci</sc>: 0.9 to 5.9; <italic>p</italic> = 0.11) were more frequent in the adjuvant radiotherapy arm.</p>", "<p>Results from the <sc>aro</sc> 96–02 study were reported at the 2007 meeting of the American Society of Clinical Oncology ##UREF##6##28##. That study randomized patients with pT3 disease to adjuvant radiotherapy or a “wait-and-see policy.” Those who failed to achieve an undetectable <sc>psa</sc> level postoperatively on either arm were given a designation of progressive disease and offered radiotherapy. The primary endpoint, biochemical control, was significantly improved in the adjuvant radiotherapy arm (<sc>hr</sc>: 0.53, <italic>p</italic> = 0.0015).</p>", "<p>Taken together, the results of the foregoing trials fail to fully inform physicians and patients about the role of post-prostatectomy radiotherapy in current practice because of differences in outcome definitions used in the trials, lack of consistent effect of adjuvant radiotherapy on clinical (non-<sc>psa</sc>) endpoints, variable use of late radiotherapy in patients randomized to the observation arm, and current use of assays for <sc>psa</sc> testing that are more sensitive than those used during the studies.</p>", "<p>The situation regarding the use of hormone therapy in this group of patients is even less clear. No randomized controlled trials have reported addressing the role and optimal duration of hormone therapy in men receiving post-prostatectomy radiotherapy. The uncertainty among clinicians regarding the role of adjuvant radiotherapy and hormone therapy is reflected in recent surveys of urologists and oncologists, indicating a wide variation in use of these therapies in the post-prostatectomy patient population ##REF##15214652##29##,##REF##15999120##30##.</p>", "<title>4.1 Study Design</title>", "<p>The <sc>radicals</sc> trial is designed to address the issues of radiotherapy timing (immediate vs. early salvage) and of hormone therapy duration (none vs. short-term vs. long-term). The primary endpoint is disease-specific survival. It is estimated that the radiotherapy timing randomization will have to recruit 2600 patients and the hormone-duration randomization, 3500 patients. Many patients will be in both randomizations. The trial is planned to address these questions over 12–13 years with 5.5 years of accrual and 7 years of further follow-up. Secondary endpoints include freedom from treatment failure, clinical progression-free survival, overall survival, non-protocol hormone therapy, treatment toxicity, and patient-reported outcomes.</p>", "<p>The radiotherapy timing randomization involves immediate radiotherapy to the prostate bed versus a salvage radiotherapy policy at the time of <sc>psa</sc> failure. The radiotherapy will use standard techniques and dose fractionation schedules: 66 Gy in 33 fractions over 6.5 weeks or 52.5 Gy in 20 fractions over 4 weeks. The hormone duration randomization involves no hormone therapy with radiotherapy, compared with short-term (6 months) hormone therapy beginning shortly before radiotherapy, compared with long-term (24 months) hormone therapy beginning shortly before radiotherapy. Patients who decide not to enter the three-way randomization will be able to choose randomization between two of the three arms: 0 as compared with 6 months of hormone therapy if they do not want to be randomized to a long duration of treatment, or 6 as compared with 24 months of hormone therapy if they do not want to be randomized to the no-hormonetherapy treatment arm.</p>", "<p>Key eligibility criteria for the radiotherapy timing randomization include a postoperative serum <sc>psa</sc> below 0.4 ng/mL within 3 months after radical prostatectomy, and uncertainty in the opinion of the clinician and patient regarding the need for immediate postoperative radiotherapy. For the hormone duration randomization, patients must be expected to receive radiotherapy (adjuvant or salvage) and must have a <sc>psa</sc> of 10 ng/mL or more at the time of randomization. In an 18-month feasibility stage, <sc>radicals</sc> will carefully assess randomization rates and the trial as a whole. Continuation of the trial beyond the feasibility stage will be conditional on satisfactory patient accrual.</p>", "<title>5. SUMMARY</title>", "<p>Ongoing studies at the <sc>ncic ctg</sc> are addressing fundamental questions regarding the management of localized prostate cancer.</p>", "<p>The randomized phase <sc>iii</sc> Intergroup study PR.11 led by <sc>ncic ctg</sc> is asking the single most important question regarding the management of favourable-risk prostate cancer: Is active surveillance with a radical intervention based on signs of disease progression as good as radical intervention at diagnosis? The results of this study, whether positive or negative, have the potential to define the management of low-risk prostate cancer globally and to clarify the role of <sc>psa</sc> doubling time in decision-making.</p>", "<p>The hypothesis being tested in PR.12 is whether the addition of docetaxel to standard treatment with androgen suppression combined with radiotherapy improves outcome in a high-risk prostate cancer population. This study builds on preclinical data demonstrating the interaction between taxanes, androgen suppression, and development of androgen resistance, and also the extensive literature demonstrating activity of docetaxel in prostate cancer.</p>", "<p>Finally, PR.13 is a large study that seeks to clarify the roles of post-prostatectomy radiotherapy timing (adjuvant vs. relapse) and the optimal duration of hormone therapy (0 months vs. 6 months vs. 24 months) in patients already treated with prostatectomy.</p>" ]
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[{"label": ["1"], "collab": ["Canadian Cancer Society and the National Cancer Institute of Canada"], "source": ["Canadian Cancer Statistics 2007"], "publisher-loc": ["Toronto"], "publisher-name": ["Canadian Cancer Society"], "year": ["2007"]}, {"label": ["11"], "collab": ["Canadian Pharmacists Association ("], "sc": ["cp", "a"], "source": ["Compendium of Pharmaceuticals and Specialties 2007: The Canadian Drug Reference for Health Professionals"], "publisher-loc": ["Ottawa"], "publisher-name": ["CPHA"], "year": ["2007"]}, {"label": ["19"], "surname": ["Friedland", "Cohen", "Miller"], "given-names": ["D", "J", "R"], "article-title": ["A phase "], "sc": ["ii"], "source": ["Proc Am Soc Clin Oncol"], "year": ["1999"], "fpage": ["18"], "comment": ["[Available online at: "], "ext-link": ["www.asco.org/ASCO/Abstracts+%26+Virtual+Meeting/Abstracts?&vmview=abst_detail_view&confID=17&abstractID=15490"]}, {"label": ["23"], "surname": ["Winquist", "Chi", "Chin"], "given-names": ["E", "KN", "J"], "article-title": ["Multicenter phase II study of combined neoadjuvant docetaxel and androgen ablation ("], "sc": ["adt", "rp", "lc", "p"], "source": ["Proc Am Soc Clin Oncol"], "year": ["2007"], "fpage": ["25"], "comment": ["[Available online at: "], "ext-link": ["www.asco.org/ASCO/Abstracts+%26+Virtual+Meeting/Abstracts?&vmview=abst_detail_view&confID=47&abstractID=30613"]}, {"label": ["24"], "surname": ["McKenzie", "Chi", "Neville"], "given-names": ["MR", "KN", "A"], "article-title": ["Phase "], "sc": ["II", "as", "rt", "hrlc", "p"], "source": ["Proc Am Soc Clin Oncol"], "year": ["2006"], "fpage": ["24"], "comment": ["[Available online at: "], "ext-link": ["www.asco.org/ASCO/Abstracts+%26+Virtual+Meeting/Abstracts?&vmview=abst_detail_view&confID=40&abstractID=34492"]}, {"label": ["25"], "surname": ["Hodgson", "Urbach", "Przybysz", "Gong", "Sullivan", "Rabeneck", "Tu", "Pinfold", "McColgan", "Laupacis"], "given-names": ["D", "D", "R", "Y", "T", "L", "JV", "SP", "P", "A"], "article-title": ["Cancer surgery. Chapter 2"], "source": ["Access to Health Services in Ontario"], "publisher-loc": ["Toronto"], "publisher-name": ["Institute for Clinical Evaluative Sciences"], "year": ["April 2005"], "fpage": ["10"], "comment": ["[Available online at: "], "ext-link": ["www.ices.on.ca/WebBuild/site/ices-internet-upload/file_collection/Chp2_v5new.pdf"]}, {"label": ["28"], "surname": ["Wiegel", "Bottke", "Willich"], "given-names": ["T", "D", "N"], "article-title": ["Phase "], "sc": ["iii", "rt", "rp", "aro", "auo ap"], "source": ["Proc Am Soc Clin Oncol"], "year": ["2007"], "fpage": ["25"], "comment": ["[Available online at: "], "ext-link": ["www.asco.org/ASCO/Abstracts+%26+Virtual+Meeting/Abstracts?&vmview =abst_detail_view&confID=47&abstractID=35176"]}]
{ "acronym": [], "definition": [] }
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CC BY
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2022-01-12 14:47:25
Curr Oncol. 2008 Aug; 15(4):179-184
oa_package/4d/5e/PMC2528309.tar.gz
PMC2528310
18769612
[ "<title>INTRODUCTION</title>", "<p>After basal and squamous cell skin cancers, prostate cancer is the most frequent cancer in men in the United States, with 186,320 men estimated to be diagnosed with the disease and 28,660 expected to die from it in 2008 ##REF##18287387##1##. Approximately 90% of hematogenous metastases in prostate cancer patients occur in bone ##REF##10836297##2##, making skeletal complications the leading cause of morbidity and mortality in these patients.</p>", "<p>Although skeletal metastases from prostate cancer are usually considered osteoblastic by radiologic analysis, histologic and biochemical studies suggest that osteolytic and osteoblastic responses are sequentially linked ##REF##3102281##3##–##REF##14991763##5##. A vicious cycle in which prostate cancer cells secrete factors that stimulate bone matrix turnover, which in turn releases growth factors that enhance tumour proliferation, has been proposed ##REF##12548574##6##. In the normal bone microenvironment, endosteal cells (osteoblasts, osteoclasts) and bone marrow cells (fibroblasts, pre-osteoblasts, pre-osteoclasts, lymphoid and myeloid cells, endothelial cells, hematopoietic and mesenchymal stem cells) communicate with each other in a homeostatic process that involves transmission of various biologic signals. However, this delicate equilibrium is perturbed when prostate cancer cells colonize the bone, leading to an alteration of the physiologic balance between bone formation and bone resorption, and to a hospitable environment for expansion of the metastases ##REF##17826661##7##.</p>", "<p>A role for matrix metalloproteinases (<sc>mmps</sc>) in bone metastasis has long been suspected. Because <sc>mmps</sc> contribute to the process of normal bone remodelling, and because enhanced turnover of bone matrix occurs when tumour cells metastasize to bone, a role can be predicted for these enzymes in metastasis-associated bone modification.</p>" ]
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[ "<p>Richard J. Ablin, <sc>p</sc>h<sc>d</sc>, Research Professor of Immunobiology, University of Arizona College of Medicine and the Arizona Cancer Center, Tucson, Arizona, U.S.A., and Phil Gold, <sc>p</sc>h<sc>d md</sc>, Professor of Medicine, Physiology, and Oncology, McGill University, Montreal, Quebec, Canada, Section Editors.</p>", "<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>" ]
[ "<title>MMPs AT A GLANCE</title>", "<p>The <sc>mmp</sc> family of Zn<sup>2+</sup>-dependent endopeptidases comprises at least 24 members, which have the combined capacity to degrade virtually all the structural components of extracellular matrix [<sc>ecm</sc> (for review see ##REF##11990853##8##,##REF##12209155##9##)]. Most <sc>mmps</sc> are secreted, although 6 of them are membrane-tethered (<sc>mt</sc>) proteins that have the ability to mediate proteolytic events at both the cell surface and in the immediate pericellular milieu. The <sc>mmps</sc> consist of an N-terminal signal sequence, a propeptide domain, a catalytic domain, and a C-terminal domain known as the hemopexin-like domain. In the case of the <sc>mt-mmp</sc>s, a C-terminal transmembrane domain or a glycosylphosphatidylinisotol link domain is added.</p>", "<p>All <sc>mmps</sc> are synthesized as inactive zymogens and are maintained as such by coordination of a cysteine residue within the propeptide domain, with a Zn<sup>2+</sup> ion present in the catalytic domain, which prevents binding and cleavage of the substrate. The dissociation of cysteine from the Zn<sup>2+</sup> atom, known as “cysteine switch,” is a complex process, the details of which have emerged only recently##REF##17929919##10## The activity of <sc>mmps</sc> is specifically inhibited by tissue inhibitors of <sc>mmps</sc> (<sc>timps: timp</sc>-1, <sc>timp</sc>-2, <sc>timp</sc>-3, and <sc>timp</sc>-4), which usually bind to the active species, inhibiting catalysis. In some <sc>mmps</sc>, the <sc>timps</sc> can bind to the zymogens.</p>", "<p>For a long time, the significance of the binding of certain <sc>timps</sc> to the latent form of some <sc>mmps</sc> was unclear. It is now known that, for example, low concentrations of <sc>timp</sc>-2 can promote activation of pro-<sc>mmp</sc>-2 through a mechanism in which <sc>timp</sc>-2 acts as a linker between the pro-<sc>mmp</sc>-2 zymogen and <sc>mt</sc>1-<sc>mmp</sc> at the cell surface. In this way, the propeptide of the pro-<sc>mmp</sc>-2 present in this ternary complex formed on the cell surface is cleaved by an adjacent active <sc>mt</sc>1-<sc>mmp</sc>. Conversely, high levels of <sc>timp</sc>-2 relative to <sc>mt</sc>1-<sc>mmp</sc> would inhibit activation by blocking all free <sc>mt</sc>1-<sc>mmp</sc> molecules.</p>", "<p>Initially, <sc>mmps</sc> were recognized as proteinases exclusively committed to <sc>ecm</sc> remodelling during invasive, angiogenic, and metastatic processes. They are now known to have additional roles, such as activation of growth factors, cleavage of cell surface receptors, release of angiogenic factors, release of apoptotic ligands, and generation of angiostatic molecules, to mention just a few. These multiple roles demonstrate the complexity and multiplicity of the substrates of <sc>mmps</sc> and their ample range of biologic activity in normal and pathologic conditions alike.</p>", "<title>MMPs AND PROSTATE CANCER BONE METASTASES</title>", "<p>Cancer cells that disseminate to bone alter the normal skeletal remodelling process, upsetting the balance between bone formation and bone resorption. Because <sc>mmps</sc> are involved in the physiologic turnover of <sc>ecm</sc> and in bone remodelling, a contribution of <sc>mmps</sc> to prostate cancer bone metastasis seems to be reasonable. In fact, the participation of <sc>mmps</sc> in the pathogenesis of osteolytic prostate cancer bone metastasis was confirmed by several experimental studies.</p>", "<p>More than 10 years ago, it was shown that the combined administration of alendronate (a bisphosphonate compound) and of paclitaxel inhibits bone metastases produced in severe combined immunodeficient (<sc>scid</sc>) mice by intravenous inoculation of human PC-3 ML cancer cells. This effect was mainly the result of a complete abrogation of the production and secretion of <sc>mmps</sc> ##REF##9186547##11##. <italic>In vitro,</italic> it was found that PC-3 cells produce s when placed on bone surfaces ##UREF##0##12##, and secrete <sc>mmp</sc> and that <italic>MMP2</italic> and <italic>MMP9</italic> are among a set of genes altered when prostate cancer cells and bone marrow stromal cells interact ##REF##16596270##13##. Those findings were further supported by studies in prostate cancer patients with skeletal metastases who had elevated serum levels of and <sc>mmp</sc>-2 <sc>mmp</sc>-9.##REF##12168874##14##,##REF##16475674##15##</p>", "<p>Using the <sc>scid</sc>-hu model for bone metastasis, in which human prostate cancer cells are grown in human bone xenografts in <sc>scid</sc> mice ##REF##10213511##16##, we confirmed high expression of <sc>mmp</sc>-2 and <sc>mmp</sc>-9 in cancer cells and in neighbouring bone stromal cells. Those data were consistent with our observations in bone biopsy specimens obtained from prostate cancer patients ##REF##11773278##17##. Moreover, systemic administration to the mice of the broad-spectrum <sc>mmp</sc> inhibitor BB-94 (batimastat) inhibited the proliferation of prostate tumour cells growing within the human bone implants. That inhibition was accompanied by reduced degradation of bone marrow trabeculae and decreased osteoclast recruitment ##REF##11773278##17##.</p>", "<p>Recently, we showed an upregulation of net <sc>mmp</sc>-9 activity shortly after establishment of PC-3 cells in human bone xenografts—an increase that coincided with a wave of osteoclast recruitment and vigorous bone degradation ##REF##15793297##18##. Experimentally, the activation of <sc>mmp</sc>-9 that occurs during prostate cancer–bone interaction is species-specific, because active <sc>mmp</sc>-9 was found when human prostate cancer cells grew within human bone tissue, but not when they grew within mouse bones ##REF##17445684##19##.</p>", "<p>Tumour-associated osteoclasts are known initially to dissolve the mineralized bone matrix by acid secretion and then to disrupt the exposed non-mineralized <sc>ecm</sc> by using proteases. Although <sc>mmp</sc>-9 does not degrade type <sc>i</sc> collagen, the most abundant organic component in bone, we found that the protease is expressed mainly by osteoclasts. That finding suggests that active <sc>mmp</sc>-9 induced by prostate cancer–bone interaction most likely contributes to osteolysis by indirect mechanisms. For instance, <sc>mmp</sc>-9 may cause the release of <sc>ecm</sc>-bound vascular endothelial growth factor into a soluble and bioactive form ##REF##11025665##20##, thereby favouring the angiogenesis of intraosseous prostate tumours and the subsequent growth of those tumours. That effect ultimately stimulates new osteoclastic activity necessary to gain more space for expansion of the tumours.</p>", "<p>The foregoing hypothesis was confirmed by experimental therapy with SB-3CT, a novel mechanism-based <sc>mmp</sc> inhibitor with high selectivity for <sc>mmp</sc>-9 ##UREF##1##21##,##REF##16046398##22##. This inhibitor is primed, in a chemical step, for potent inhibition of gelatinases once bound to the active sites of these enzymes. Treatment with SB-3CT of <sc>scid</sc> mice bearing prostate cancer bone tumours resulted in significant inhibition of angiogenesis and intraosseous tumour growth, together with reduction in osteolysis ##REF##16381009##23##. Those results indicate an important contribution of <sc>mmp</sc>-9 to neovascularization of expanding bone metastases and to subsequent bone degradation. Moreover, pro-<sc>mmp</sc>-9 has been shown to play a crucial role in osteoclast recruitment ##REF##11076971##24##. The existence of abnormalities in developmental angiogenesis and ossification in mice with null mutation in <italic>MMP9</italic> ##REF##11076971##24##,##REF##9590175##25## further supports the importance of <sc>mmp</sc>-9 not only in normal but in pathologic processes occurring in the skeleton.</p>", "<p>Membrane-tethered 1 <sc>mmp</sc> knockout mice also present severe skeletal abnormalities, mostly as a result of their incapacity to degrade crosslinked fibrillar type <sc>i</sc> collagen prevalent in the bone matrix##REF##10520996##26##. In prostate cancer, <sc>mt</sc>1-<sc>mmp</sc> expression correlates with a more aggressive behaviour and has been shown to promote invasion and metastasis ##REF##15841326##27##–##REF##16619495##29##. Immunohistochemical studies of primary prostate adenocarcinomas revealed a heterogeneous pattern, with some malignant glands positive and others negative for <sc>mt</sc>1- <sc>mmp</sc> ##REF##10632347##30##. That finding, together with a uniform and strong <sc>mt</sc>1-<sc>mmp</sc> expression observed in all cases of prostate cancer bone metastases analyzed ##REF##17525276##31##, suggests the existence of a selective process in which <sc>mt</sc>1-<sc>mmp</sc>–expressing prostate cancer cells may have more tendency to metastasize to skeleton. Alternatively, the bone microenvironment might induce the expression of <sc>mt</sc>1-<sc>mmp</sc> in prostate cancer cells after their arrival in the marrow.</p>", "<p>We conducted a series of studies to assess the contribution of prostate cancer cell–derived <sc>mt</sc>1-<sc>mmp</sc> to bone metastasis. We overexpressed <sc>mt</sc>1-<sc>mmp</sc> in LNCaP human prostate cancer cells (which have baseline non-detectable expression of the protease), while its expression was attenuated in DU145 cells (which have baseline high <sc>mt</sc>1-<sc>mmp</sc> expression) using small interfering <sc>rna</sc>. We showed that intratibial injection of those cells resulted in completely opposite phenotypes in terms of intraosseous tumour growth and bone response. Compared with controls, tibiae injected with LNCaP cells overexpressing <sc>mt</sc>1-<sc>mmp</sc> showed increased osteolysis (as demonstrated by radiography and histomorphometry) and enhanced intraosseous tumour growth. In contrast, <sc>mt</sc>1-<sc>mmp</sc> downregulation in high-expressing <sc>mt</sc>1-<sc>mmp</sc> DU145 prostate cancer cells led to diminished intraosseous tumour growth and a mixed bone reaction, in which osteosclerotic responses predominated ##REF##17525276##31##. We further showed that <sc>mt</sc>1-<sc>mmp</sc> upregulation in cancer cells resulted in the release of one or more factors that promoted osteoclast differentiation <italic>in vitro.</italic> That effect was abrogated by pharmacologic inhibition of <sc>mt</sc>1-<sc>mmp</sc> activity and by osteoprotegerin, a soluble decoy receptor of the osteoclastogenic receptor activator of nuclear factor κB ligand (<sc>rankl</sc>). Our results strongly suggest the possibility that prostate cancer–associated <sc>mt</sc>1-<sc>mmp</sc> promotes an osteolytic response by shedding membrane-bound <sc>rankl</sc> (m<sc>rankl</sc>) from the cancer cell surface. Recently, <sc>mmp</sc>-7, produced mainly by osteoclasts at the prostate tumour–bone interface, has also been reported as a <sc>rankl</sc> “sheddase,” promoting osteolysis ##REF##15894268##32##. Together, these data suggest that specific inhibition of certain <sc>mmps</sc> in prostate cancer bone metastasis may be therapeutically beneficial.</p>", "<title>NEW THERAPEUTIC CHALLENGES</title>", "<p>Much of the initial excitement associated with the use of broad-spectrum <sc>mmp</sc> inhibitors in animal tumour models has been mitigated by a lack of therapeutic efficacy and undesired side effects observed in clinical trials with cancer patients ##REF##11923519##33##–##REF##12784996##35##. Some of the potential reasons for the failure of these agents include</p>", "<p>testing of patients with advanced high-volume disease refractory to other treatments;</p>", "<p>use of broad-spectrum inhibitors of <sc>mmps</sc> with unspecific and reversible effects;</p>", "<p>unknown repertoire of proteases expressed by the patients’ tumours (protease “degradome” ##REF##12094217##36##) before and during treatment;</p>", "<p>unintended inhibition of <sc>mmps</sc> with important physiologic roles (anti-targets), probably resulting in neutralization of the effects of the inhibitors on actual <sc>mmp</sc> “targets” that truly contribute to disease ##REF##16498445##37##;</p>", "<p>lack of studies to monitor <sc>mmp</sc> inhibition during treatment; and</p>", "<p>unknown effective doses of <sc>mmp</sc> inhibitors and diminished therapeutic index because of forced dose reduction to tolerable levels.</p>", "<p>The first drugs developed for <sc>mmp</sc> inhibition were peptidomimetic hydroxamate compounds with potent inhibitory effects, but no selectivity (for example, batimastat). The second-generation <sc>mmp</sc> inhibitors exhibited some marginal selectivity (for example, prinomastat). Because those compounds failed in clinical trials, a third generation of selective <sc>mmp</sc> inhibitors is now being developed and considered for cancer therapy ##REF##16538215##38##,##REF##17627541##39##, aiming to obtain the maximal effect on the disease in which the <sc>mmp</sc> target is involved with minimal adverse reactions. These selective inhibitors are hoped to have a ratio of at least 1000 between the <italic>K<sub>i</sub></italic> (the dissociation constant for binding of inhibitor) values for <sc>mmp</sc> anti-targets and those for <sc>mmp</sc> targets ##REF##16538215##38##,##REF##17627541##39##. These prospects should be explored and might prove efficacious, but even selectivity by a factor of 1000 might not solve the clinical riddle of compounds that serve as linear competitive inhibitors.</p>", "<p>For example, potent linear competitive inhibition of target <sc>mmps</sc> at low nanomolar or picomolar levels <italic>in vitro,</italic> despite the “factor of 1000” selectivity, might not prove selective <italic>in vivo</italic> because a low micromolar or high nanomolar level of activity against the anti-target <sc>mmps</sc> will still foster <italic>in vivo</italic> consequences. The challenge is not necessarily an issue of affinity for the target, but rather of the mechanism for discrimination other than mere recognition events between the drug and the target. In that vein, the mechanism-based gelatinase inhibitor SB-3CT and its new structural variants stand out. This inhibitor class takes advantage of the active site of the enzyme to undergo a specific chemical transformation facilitated by the target enzyme itself. Whether a given <sc>mmp</sc> might be able to perform this reaction, or whether it might not, a process that leads to potent enzyme inhibition is at the root of its ability to serve as a selective <sc>mmp</sc> inhibitor to the given target. The concepts pertinent to inhibition of <sc>mmp</sc> have been discussed in a recent review ##REF##16680577##40##.</p>", "<p>In the particular case of prostate cancer patients, no clinical trials have been performed to evaluate the therapeutic efficacy of <sc>mmp</sc> inhibitors on bone metastasis. We believe that the knowledge obtained in recent years using animal models has provided validated <sc>mmp</sc> targets that, together with the development of third-generation <sc>mmp</sc> inhibitors, would justify use of those inhibitors in the treatment of prostate cancer patients with skeletal metastasis. For instance, prostate cancer patients with locally advanced disease who have a high probability of developing bone metastasis and no current prospect of curative treatment could benefit from therapy with novel <sc>mmp</sc> inhibitors.</p>", "<p>For successful treatment, it is crucial that the protease degradome for tumours in each patient be assessed and that inhibition of the <sc>mmp</sc> being targeted be confirmed by appropriate methods during treatment. Alternatively, combination therapy using inhibitors for <sc>mmp</sc> targets and agents that can block osteoclastic action (such as bisphosphonates or anti-<sc>rankl</sc> drugs) could reasonably be employed in clinical trials involving prostate cancer patients with potential to develop bone metastases. The experimental evidence described herein showing a key role for <sc>mmps</sc> in bone metastasis suggests that targeting those <sc>mmps</sc> could have therapeutic value. New approaches must be explored, especially given that current approaches for treating bone metastasis in prostate cancer patients are still limited and only palliative.</p>" ]
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[{"label": ["12"], "surname": ["Sanchez\u2013Sweatman", "Orr", "Singh"], "given-names": ["OH", "FW", "G"], "article-title": ["Human metastatic prostate PC3 cell lines degrade bone using matrix metalloproteinases"], "source": ["Invasion Metastasis"], "year": ["1998\u20131999"], "volume": ["18"], "fpage": ["297"], "lpage": ["305"]}, {"label": ["21"], "surname": ["Brown", "Bernardo", "Zhi\u2013Hong"], "given-names": ["S", "M", "L"], "article-title": ["Potent and selective mechanism-based inhibition of gelatinases"], "source": ["J Am Chem Soc"], "year": ["2000"], "volume": ["122"], "fpage": ["6799"], "lpage": ["800"]}]
{ "acronym": [], "definition": [] }
40
CC BY
no
2022-01-12 14:47:25
Curr Oncol. 2008 Aug; 15(4):188-192
oa_package/04/12/PMC2528310.tar.gz
PMC2528311
18769613
[ "<title>1. INTRODUCTION</title>", "<p>Testicular cancers are the most common—and the most curable—malignancies among young men in North America ##REF##15069322##1##,##REF##14560141##2##. Seminomas account for approximately half of these cancers, and most patients (80%) present with stage <sc>i</sc> disease ##REF##14726503##3##,##REF##15124546##4##. Treatment is highly successful, with 5-year overall and disease-specific survivals approaching 100% for stage <sc>i</sc> seminoma ##REF##14560141##2##,##REF##15124547##5##.</p>", "<p>For decades, the standard treatment for this cancer has been radical inguinal orchiectomy, followed by adjuvant radiotherapy to the para-aortic and ipsilateral pelvic regions ##REF##14560141##2##,##REF##15124547##5##,##REF##9728212##6##. This approach led to a recurrence rate of less than 5%, with salvage chemotherapy being highly effective in the few patients that did relapse ##REF##14560141##2##,##REF##14726503##3##,##REF##15124547##5##,##REF##9728212##6##. However, long-term follow-up data (beyond 10–15 years) now indicate that treatment-related morbidity and mortality (especially from a second malignancy) are significant concerns ##REF##14726503##3##,##REF##16174857##7##,##REF##14770428##8##. Various approaches have therefore been investigated to minimize the toxicity associated with routine use of radiotherapy. One approach has been to minimize toxicity by reducing radiotherapy field sizes and doses ##REF##10561173##9##,##REF##15718317##10##; another approach has been to avoid radiation altogether ##REF##14726503##3##. This change has, in turn, resulted in the evolution of surveillance after orchiectomy as a viable option because of the general availability of computed tomography (ct) imaging for follow-up purposes ##REF##16414499##11##. In addition, the use of single-agent chemotherapy (most commonly 1–2 cycles of carboplatin) has also been recognized as a potential option in place of radiotherapy ##REF##12796024##12##–##REF##16039331##14##.</p>", "<p>As a result, management of stage <sc>i</sc> seminoma is currently focused not only on maintaining high rates of cure, but also minimizing both short- and long-term toxicity ##REF##14726503##3##,##REF##16414499##11##,##REF##17437287##15##. Previously, a survey of radiation oncologists conducted by Choo <italic>et al.</italic> in 2001 regarding management of stage <sc>i</sc> seminoma revealed considerable variation in practice, especially with regard to treatment ##REF##12010592##16##. Most radiation oncologists at that time routinely discussed surveillance as an option, but thought that only 20% of patients would choose that option. At that time, the authors found that adjuvant radiotherapy was usually the preferred choice. However, data have continued to accumulate regarding the viability of options that do not include the routine use of adjuvant radiotherapy.</p>", "<p>Review of seminoma management at our own institution ##REF##16197597##17## showed a steady decline in the use of adjuvant radiotherapy since the 1990s and the increasing use of surveillance for stage <sc>i</sc> seminoma patients—a trend that has also been reported elsewhere ##REF##16527585##18##,##REF##15708251##19##. Although recent studies suggest that the use of single-agent chemotherapy with carboplatin is very encouraging and the use of adjuvant chemotherapy is becoming increasingly popular in Europe ##REF##17100155##20##, this approach is still not routinely considered at our institution. Our experience has been that chemotherapy tends to be reserved for the salvage of radiotherapy failures or in patients wanting adjuvant treatment who are not deemed suitable for radiation. Based on the increasing options for management of stage <sc>i</sc> seminoma, we decided to survey Canadian radiation oncologists to see if their management approaches had changed since earlier in the decade.</p>" ]
[ "<title>2. METHODS</title>", "<p>We developed an electronic survey to assess management of stage <sc>i</sc> testicular seminoma patients. The survey was specifically designed for radiation oncologists, and the categories evaluated included staging investigations, treatment options, radiotherapy treatment planning details, surveillance protocols, and respondent demographic information. Intended for self-completion, the survey takes approximately 15 minutes to finish. After obtaining approval from the research ethics board of the Ottawa Hospital to proceed with this survey study, we sent the survey by e-mail in 2005 to Canadian radiation oncologists identified as treating genitourinary malignancies. The list of radiation oncologists was formulated from information obtained from the directory of the Canadian Association of Radiation Oncologists at that organization’s Web site and from correspondence with radiation oncology department heads at cancer centres across Canada. Initial non-responders were sent reminder notices, also by e-mail. Remuneration was not offered for completing the survey.</p>", "<p>The completed surveys were collated and analyzed for this study. The chi-square statistic was used to assess associations between the respondent’s practice history and that person’s responses to survey questions. To compare differences in choice of treatment attributable to age and years of practice, a test of mean differences was applied using analysis of variance. The SPSS software package (SPSS, Chicago, IL, U.S.A.) was used to perform the analyses.</p>" ]
[ "<title>3. RESULTS</title>", "<p>Electronic surveys were sent to 119 Canadian radiation oncologists, and 93 responses were received (78% response rate). Of the respondents, 14 indicated that they did not treat seminoma patients, and 1 declined to complete the survey. The survey completion rate among eligible responders was therefore 74% (78/105). Among respondents completing the survey, 89% were men and 11% were women. Median age was 43 years. Mean length of practice was 13 years, and 80% of the respondents worked in academic centres.</p>", "<p>##FIG##0##Figure 1## shows that the staging investigations most commonly used are <sc>ct</sc> scans of the abdomen and pelvis (100%) and chest radiographs (84%). Serum tumour markers are also commonly assessed, with alpha-fetoprotein (<sc>afp</sc>) being measured 95% of the time, and beta–human chorionic gonadotropin (<sc>bhcg</sc>) levels 100% of the time.</p>", "<p>Adjuvant radiation and surveillance were considered the most common standard treatment options by 90% and 81% of respondents respectively. However, 30% of respondents also listed adjuvant chemotherapy as a standard treatment option (##FIG##1##Figure 2##). However, when asked to indicate the management that they felt was most appropriate for most patients, surveillance was chosen by 56%; radiotherapy, by 31%; and chemotherapy, by 1%. The remaining 12% were unsure of the most appropriate management (##FIG##2##Figure 3##). The most common concerns related to the use of adjuvant radiotherapy were second cancers (84%), infertility (77%), late gastrointestinal toxicity (67%), acute nausea and vomiting (61%), and late renal toxicity (60%).</p>", "<p>Most respondents (91%) said that they routinely discuss surveillance with patients, but that tumour-related risk factors (size, local extension, and lymphovascular invasion), together with patient age and compliance, influence their recommendations. Nearly all respondents (&lt;95%) started offering surveillance during the last 10 years. Most respondents felt that at least 50% of patients are now choosing surveillance. The most commonly listed reasons, in order of importance, for not offering surveillance were patient fears and anxieties, patient reluctance, increased costs, and the belief that survival was actually better with the use of adjuvant radiotherapy.</p>", "<p>For patients on surveillance protocols, the investigations commonly used are <sc>ct</sc> scans of the abdomen and pelvis (93%), chest radiographs (81%), <sc>bhcg</sc> levels (92%), and <sc>afp</sc> levels (84%). Surveillance investigations are usually carried out every 3–4 months by 84% of respondents, every 6 months by 15%, and every 12 months by 1%.</p>", "<p>When treating with radiotherapy, 50% use para-aortic fields only, and 50% use para-aortic and ipsilateral pelvic fields. Planning <sc>ct</sc> is used for simulation by all respondents (100%); intravenous pyelograms and lymph angiograms are used by only 1%. A dose of 2500 cGy in 15–20 fractions over 3–4 weeks was recommended by more than 90% of respondents, and all respondents use linear accelerators (≥ 6 MV photons) for treatment delivery. Scrotal shielding is routinely used by 49% of respondents to reduce dose to the contralateral testicle, and thermoluminescent dosimeters are used by 47% for verifying dose to the contralateral testicle. Almost all respondents (96%) discussed fertility issues and sperm banking with patients before the start of adjuvant radiotherapy.</p>", "<p>Prophylactic antiemetics are ordered by 58% of the respondents, with ondansetron, prochlorperazine, dimenhydrinate, and metoclopramide being prescribed by 48%, 25%, 18%, and 8% respectively. Following treatment, 67% recommend that patients take contraceptive measures for at least 3 months (14%), 6 months (38%), 12 months (40%), or 24 months (8%).</p>", "<p>We observed a trend, although not statistically significant, for older radiation oncologists (&gt; 45 years vs. ≤ 45 years) to choose radiation for their patients (<italic>p</italic> = 0.07). However, years in practice, type of practice (academic vs. community), and provincial location did not appear to influence management choices.</p>" ]
[ "<title>4. DISCUSSION</title>", "<p>Cancer treatment approaches evolve with time and accumulation of research findings—first to improve and maximize cure rates, and then to minimize toxicity. Stage <sc>i</sc> seminoma management certainly confirms this paradigm. The use, since the 1960s, of adjuvant radiotherapy after orchiectomy reduced relapse rates to less than 5% and established adjuvant radiation as the standard practice until the 1990s ##REF##14560141##2##,##REF##9728212##6##. In fact, overall 5-year survival rates were in the 98%–99% range, because chemotherapy was highly successful in salvaging the few patients that did relapse post radiotherapy ##REF##17100155##20##. However, with the accumulation of long-term follow-up data over 25 years or more, it became obvious that second malignancies are a significant problem following radiation ##REF##14726503##3##,##REF##16174857##7##. The prevailing focus therefore moved to reduction of toxicity, because salvage therapies were so effective ##REF##14726503##3##,##REF##9728212##6##.</p>", "<p>This change in focus led to numerous studies evaluating alternatives to standard radiotherapy, and reductions in radiotherapy treatment volumes and lower radiation doses were both shown to be possible without significantly reducing efficacy ##REF##10561173##9##,##REF##15718317##10##. In prospective and retrospective studies, surveillance approaches have also been shown to be an excellent alternative that do not compromise cure rates, although they require more frequent imaging investigations ##REF##16414499##11##,##REF##16197597##17##–##REF##15708251##19##. More recently, the use of adjuvant single-agent chemotherapy (most commonly with single-agent carboplatin) has been shown to yield results, in terms of relapse rates and overall survival, similar to those seen with the use of adjuvant radiation ##REF##16039331##14##. Therefore, currently, there is evidence that all of the above approaches are effective and reasonable options that have their own unique advantages and limitations. However, long-term follow-up data for the newer approaches are limited, and questions still exist regarding long-term efficacy and toxicity.</p>", "<p>The effect of recent studies and their influence on actual current practice patterns across North America and Europe has not been fully evaluated. However, it is well known that medical practice patterns often change gradually after research findings are published, as physicians reflect on the available evidence and perhaps discuss them with their colleagues ##REF##12383461##21##–##REF##11033714##23##. Therefore, we believe that it is important to determine whether the accumulating evidence regarding management of stage <sc>i</sc> testicular seminoma has had an effect at the level of clinical practice in Canada, and that is what we attempted to do in the present study.</p>", "<p>With a very favourable survey completion rate of 74% ##UREF##0##24##, we believe that our results generally reflect current opinion among Canadian radiation oncologists regarding stage <sc>i</sc> seminoma management. Our findings show that staging is fairly consistent and reflects the emergence of <sc>ct</sc> scans of the abdomen and pelvis as standard, together with chest radiographs ##REF##16414499##11## and serum tumour markers (<sc>afp</sc>, b<sc>hcg</sc>). Radiation treatment planning with <sc>ct</sc> scans and delivery approaches using linear accelerators, as indicated by the respondents, are also consistent with the published literature, as are the dose and fractionation regimens commonly used ##REF##15124547##5##. However, opinion continues to vary regarding the necessity of ipsilateral pelvic lymph node irradiation.</p>", "<p>The surveillance protocols among respondents are very similar to published recommendations ##REF##17437287##15##. Although variations in practice still occur, a preponderance of respondents indicate that they routinely discuss both surveillance and adjuvant radiotherapy with their patients, and most believe that surveillance is the preferred option, with at least 50% of patients making the latter choice.</p>", "<p>Adjuvant chemotherapy is also starting to be recognized as a treatment option by almost one third of radiation oncologists even though our survey was conducted before the results of the Medical Research Council E19 study, published by Oliver <italic>et al.</italic> ##REF##16039331##14##, indicated that at a median follow-up of 4 years, adjuvant single-agent carboplatin chemotherapy was essentially equivalent in terms of relapse rates and overall survival to adjuvant radiotherapy. Our finding in this regard represents a major shift from the survey results reported in 2002 by Choo <italic>et al.</italic> ##REF##12010592##16##, when most radiation oncologists thought that adjuvant radiotherapy should be standard, although surveillance was considered an option. Also, chemotherapy was not even considered an option by any respondents at that time, likely reflecting the limited published data to that point.</p>", "<p>The biggest issue remains the attempt to achieve a balance between minimizing relapse rates and patient fears and anxiety related to recurrences, and avoiding unnecessary treatment for the preponderance of patients that will not relapse and the potential for toxicity that can occur decades later. This balance will likely remain controversial for some time to come, and it is uncertain whether consensus can be achieved in the near future. Individual patient factors, including personal choice, will also be essential in determining the management option that is chosen. However, it is reassuring to confirm that many radiation oncologists have been re-evaluating their approach to management of stage <sc>i</sc> testicular seminomas over the last 5 years, in parallel with growing evidence of newer management approaches. This re-evaluation may in part be attributable to the growing sub-specialization among many practicing radiation oncologists in terms of site-specific treatment and the resulting familiarity with recently published studies, and also to the Canadian sources of much of the published literature regarding surveillance for management of stage <sc>i</sc> seminoma ##REF##16414499##11##,##REF##16197597##17##–##REF##15708251##19##. We expect that management approaches will continue to evolve as ongoing studies mature, especially those evaluating the use of single-agent carboplatin chemotherapy.</p>" ]
[ "<title>5. CONCLUSIONS</title>", "<p>Canadian radiation oncologists are routinely discussing surveillance in addition to adjuvant radiotherapy as treatment options for patients with stage <sc>i</sc> testicular seminoma, but surveillance is usually considered the preferred option. Chemotherapy also appears to be emerging as a viable option among a growing number of radiation oncologists. As a result, the use of radiation is declining.</p>" ]
[ "<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>Recently published studies clearly indicate that there are now several acceptable options for managing stage <sc>i</sc> testicular seminoma patients after orchiectomy. We therefore decided to survey Canadian radiation oncologists to determine how they currently manage such patients and to compare the results with previous surveys.</p>", "<p>Our results demonstrate that adjuvant single-agent chemotherapy is being considered as an option by an increasing proportion of radiation oncologists (although it is not considered the preferred option), the routine use of radiotherapy is declining, and surveillance is becoming increasingly popular and is recommended most often.</p>" ]
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[ "<title>Acknowledgments</title>", "<p>6. ACKNOWLEDGMENTS</p>", "<p>This study received financial support from the Ottawa Radiation Oncology Partners, and preliminary results were presented at the June 2006 meeting of the Canadian Urological Association in Halifax, Nova Scotia. The authors thank Mr. Victor Gallant for providing statistical analyses, and Ms. Betty McAndrew for secretarial support.</p>" ]
[ "<fig id=\"f1-co15_4p168\" position=\"float\"><label>FIGURE 1</label><caption><p>The frequency of specific staging investigations used for stage <sc>i</sc> seminoma patients, from a sample of Canadian radiation oncologists. <sc>ct</sc> = computed tomography; <sc>afp</sc> = alpha-fetoprotein; β-<sc>hcg</sc> = beta–human chorionic gonadotropin.</p></caption></fig>", "<fig id=\"f2-co15_4p168\" position=\"float\"><label>FIGURE 2</label><caption><p>Standard management options used for stage <sc>i</sc> seminoma patients, from a sample of Canadian radiation oncologists.</p></caption></fig>", "<fig id=\"f3-co15_4p168\" position=\"float\"><label>FIGURE 3</label><caption><p>The “most appropriate option” for most stage <sc>i</sc> seminoma patients, from a sample of Canadian radiation oncologists.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"co15_4p168f1\"/>", "<graphic xlink:href=\"co15_4p168f2\"/>", "<graphic xlink:href=\"co15_4p168f3\"/>" ]
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[{"label": ["24"], "surname": ["Guadagnoli", "Cunningham"], "given-names": ["E", "S"], "article-title": ["The effects of nonresponse and late response on a survey of physician attitudes"], "source": ["Eval Health Prof"], "year": ["1989"], "volume": ["12"], "fpage": ["318"], "lpage": ["28"]}]
{ "acronym": [], "definition": [] }
24
CC BY
no
2022-01-12 14:47:25
Curr Oncol. 2008 Aug; 15(4):168-172
oa_package/b8/f8/PMC2528311.tar.gz
PMC2528551
18769614
[]
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[]
[ "<p>Cancer can lead to spiritual transformation, which can be seen as a form of alchemy. During this process, patients, family members, and even professional caregivers can find themselves having spiritual experiences that go beyond any they had previously encountered. This paper provides qualitative descriptions of the “Field” or “Soul Wisdom” experienced by patients and caregivers.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/272/237\">http://www.current-oncology.com/index.php/oncology/article/view/272/237</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s107.es48-s107.es52
oa_package/f3/0b/PMC2528551.tar.gz
PMC2528552
18769615
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[ "<p>The use of complementary and alternative medicine (<sc>cam</sc>), including the ingestion of natural health products (<sc>nhp</sc>s), is common among cancer patients. Of concern to clinicians and patients alike is the possibility that <sc>cam</sc>, used concurrently with biomedical therapy, may interact poorly with that therapy, especially chemotherapy and radiotherapy. Proponents of <sc>nhp</sc>s argue that taking such products can help to reduce the side effects of conventional therapy and can provide an additional anticancer effect. However, opponents insist that the potential for harm is too great to warrant the risk of concurrent administration. There are promising examples of specific <sc>nhp</sc>s that may provide patient benefit even when given in close proximity both to chemotherapy and to radiotherapy, but unfortunately, in part because of a rather limited evidence base, caution is warranted when considering the issue of therapeutic interactions. Strategic application of <sc>nhp</sc>s before or after conventional therapy may be considered; however, concurrent application should be avoided as a general principle until further evidence is available regarding specific interactions.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/282/247\">http://www.current-oncology.com/index.php/oncology/article/view/282/247</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s109.es81-s110.es86
oa_package/22/77/PMC2528552.tar.gz
PMC2528553
18769571
[ "<title>1. INTRODUCTION</title>", "<p>Since the early 1980s, reports have emerged of cancer patients declining conventional cancer treatment and using complementary and alternative medicine (<sc>cam</sc>) instead (for example, Cassileth <italic>et al.</italic>\n##REF##6732073##1##). Although some patients decline all conventional treatments and use <sc>cam</sc> as an alternative, others decline only some conventional treatments and complement the treatment they accept with <sc>cam</sc>. For physicians, this choice by a patient is often difficult and troublesome, because it involves risks such as delays in conventional oncology treatments, side effects of <sc>cam</sc>, and decreased survival time. Many terms have been used to describe this decision—”abandoning,” “non-compliance,” “refusing,” and “rejecting”—most of which carry a negative or pejorative connotation.</p>", "<p>How many patients make this decision is not very well known, but the number appears substantial enough to warrant close attention. For example, Cassileth <italic>et al.</italic>\n##REF##6732073##1## examined cancer patients attending a university cancer centre (<italic>n</italic> = 304) and patients recruited through various U.S. media sources who were receiving treatments from <sc>cam</sc> practitioners or <sc>cam</sc> clinics (<italic>n</italic> = 356). Of the 378 patients who used <sc>cam</sc>, 53 (14%) declined conventional treatment of any kind. Another survey ##REF##11448703##2## revealed that 13% of patients being referred for postsurgical cancer treatment (<italic>n</italic> = 158) rejected all further treatment, and 19% declined some treatments. A third study ##REF##11800343##3## found that about 3% of women under age 65 with breast cancer (<italic>n</italic> = 302) had refused conventional treatment.</p>", "<p>Valid estimates of the prevalence of the choice to decline conventional treatments are not available, but a number of relatively small-scale qualitative studies have focused on understanding why cancer patients make this decision.</p>", "<p>In general terms, qualitative research consists of the investigation of phenomena, typically in a detailed and holistic fashion, through the collection of rich narrative materials using a flexible research design ##UREF##0##4##. Drawing on the literature and on our own ongoing research, we here describe why cancer patients decide to decline conventional cancer treatments, who those patients are, and the response of physicians to patients who make such decisions.</p>" ]
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[ "<title>2. DISCUSSION</title>", "<title>2.1 Factors Contributing to the Decision to Decline Conventional Cancer Treatment</title>", "<p>In 1999–2000, we conducted a qualitative study with 31 cancer patients who had declined all conventional cancer treatments and were using <sc>cam</sc>\n##REF##12100104##5##. Numerous motivations for this decision were reported, including a negative experience with mainstream medicine, loss of family members or friends to cancer while on conventional treatment, <sc>cam</sc> use before diagnosis, and a strong belief system in favour of whole-person (holistic) healing. Because these factors existed before the cancer diagnosis, we considered them to be “predisposing.” Factors affecting a decision to decline treatment after diagnosis included poor doctor–patient communication, the emotional effect of the diagnosis, perceived severity of conventional treatment side effects, a high need for decision-making control, and strong beliefs in holistic healing and the mind–body–spirit connection.</p>", "<p>In Hawaii, Shumay and colleagues ##REF##11742609##6## conducted a similar study (<italic>n</italic> = 14) with comparable results. They also identified factors such as beliefs about conventional treatment, the relationship with treatment providers, and beliefs about <sc>cam</sc> as an alternative treatment option.</p>", "<p>In a small, ethnographic study of cancer patients who had declined conventional treatment (<italic>n</italic> = 8), Montbriand described factors similar to those in the previous studies and identified that emotional factors such as anger and fear were commonly expressed during the interviews ##REF##9494229##7##.</p>", "<p>Authors van Kleffens and van Leeuwen ##REF##15738431##8## concluded that medical and personal reasons both play a role in a patient’s decision to refuse treatment, but that personal values and experiences predominate. This study also revealed that patients find quality of life to be very important and seemed to believe that quality of life is incompatible with oncology treatment. They presented a list of 22 reasons why patients refuse recommended conventional cancer treatment, including some including general reasons similar to the ones we found (for example, “want to stay in control”) and more specific motivations, such as not wanting a stoma or loss of a breast, and not wanting to fight any more.</p>", "<p>We recently completed a mixed-methods study of 29 men with prostate cancer who declined all conventional treatments ##REF##21614154##9##. A similar study of women with breast cancer declining one or more conventional treatments is currently underway. To date, 32 women have enrolled in this 2-year observational case-controlled study. In-depth baseline interviews were conducted with all participants in both studies. ##TAB##0##Table I## shows the sociodemographic descriptions and treatment choices of the participants.</p>", "<p>Findings from the qualitative interviews highlight the extent to which the type of cancer, and possibly sex, influence these choices. In men, we studied the choice to decline all conventional treatments, and in women, the choice to decline at least one conventional treatment. Men and women differed in how they verbalized their treatment decision-making experiences, but both groups raised very similar issues. Foremost, participants described conducting an extensive search for information to evaluate cancer treatment (conventional and <sc>cam</sc>) options and to make informed choices. Sources of evidence cited by participants included personal experience, scientific evidence (medical literature), anecdotal information, and finding treatment consistent with their health beliefs. Men in particular acknowledged how much their decision was influenced by their perception of the negative experiences shared by other men with prostate cancer who depended on conventional treatments alone. Men and women both cited having control over decision-making and healing approaches as being essential during their cancer experience. They felt that being in control brought on feelings of well-being. Beliefs about conventional medicine (for example, “Western medicine treats the tumour, not the whole person”), <sc>cam</sc> (for example, “holistic medicine treats the whole person”), and causes of cancer also played a very important role in the decision by men and by women to decline treatment. Although men were found to emphasize the role of spirituality in their treatment decisions and cancer management in more depth than women did, the interplay between mind, body, and spirit was a vital part of the healing approach for men and women alike. Along the same line, physical, emotional, spiritual, and whole-person outcomes of treatment were all considered important indicators of treatment success. Last, in the search for informed treatment decisions, support by family and friends and <sc>cam</sc> practitioners was highly valued by participants. Cancer specialists were mentioned, but more often support came from family physicians. Men and women both mentioned the huge support received from integrative cancer clinics, which assist patients to make informed choices about the integration of conventional and <sc>cam</sc> cancer treatments. Many patients also indicated that they valued the ongoing follow-up care from their oncologists provided that they felt supported in their health beliefs. “Keeping the door open” was an important theme that emerged, because most patients wanted to keep their options open. Patients appreciated oncologists who were able to openly communicate that, although they did not agree with the patient’s decision to decline treatment, they would continue to support the patient and provide follow-up care. Conversely, patients who perceived that their cancer specialist was threatening them with a “death sentence,” pressuring them into accepting treatment, or making disparaging comments about <sc>cam</sc> were more likely to drop out of the conventional cancer system.</p>", "<p>Sex differences were also observed in the manner in which participants framed their recommendations for health professionals involved in cancer care. Men mentioned allowing patients sufficient time to adjust to the diagnosis and to make treatment decisions, considering how cancer treatment affects all aspects of well-being; encouraging patients to play an active role in treatment decisions and healing; and being open to assisting patients to find a physician who can support their philosophy of healing. Women identified reducing cancer-related stress at early diagnosis and supporting patients in making the best treatment choices for themselves. They also highlighted that health professionals should pay attention to both the individual woman and the whole person. Men and women both emphasized the need for health professionals to be aware of and to refer patients to integrative cancer care clinics or services.</p>", "<title>2.2 Psychosocial Characteristics of Patients Declining Some or All Conventional Treatments</title>", "<p>In the prostate and breast cancer studies, we used the Multiple Health Locus of Control (<sc>mhlc</sc>) scale ##REF##7844739##10##, the General Self-Efficacy (<sc>gse</sc>) scale ##UREF##1##11##, and the Control Preferences Scale (<sc>cps</sc>) ##REF##9505581##12## to assess psychosocial characteristics of participants. The <sc>mhlc</sc> scale measures the degree to which people believe that internal resources or external factors such as luck, chance, doctors, or powerful others affect their disease outcome. The <sc>gse</sc> scale assesses an individual’s perceived sense of general self-efficacy and is suitable for studies examining adaptation after a life change or a stressful event. The <sc>cps</sc> allows for the identification of the role (that is, active, collaborative, passive) that patients wish to play in disease management and treatment decisions.</p>", "<p>##TAB##1##Table II## presents the results from the <sc>mhlc</sc> scale, in which the means and 95% confidence intervals (<sc>cis</sc>) for the four subscales completed by the prostate cancer and breast cancer patients who declined conventional treatment are compared to published normative data from patients with a diagnosis of cancer ##REF##7844739##10##. Men and women who declined conventional cancer treatment both had scores on the internal scale that were higher than the published normative data from patients with a diagnosis of cancer, but scores on the chance, doctors, and powerful others scales that were lower.</p>", "<p>Scores on the <sc>gse</sc> scale were higher both for the prostate cancer group (mean: 34.8; 95% <sc>ci</sc>: 33.4 to 36.2) and for the breast cancer group (mean: 32.3; 95% <sc>ci</sc>: 30.9 to 33.7) than the normative scores for the American adult population (mean: 29.4; 95% <sc>ci</sc>: 9.2 to 29.7) ##UREF##1##11##.</p>", "<p>The two study groups were similar on the <sc>cps</sc>, with none of the participants in either group preferring to play a passive role and have the doctor make the final decision (Fisher exact test: <italic>p</italic> = 0.100). Men and women were both most likely to prefer an active role and to make the final decision after seriously considering the opinions of their doctors (72% for the breast cancer group, 46% for the prostate cancer group). Only 16% of the breast cancer group and 21% of the prostate cancer group indicated that they would prefer to make the final decision about their treatment. With regard to collaborative decision-making, 32% of the prostate cancer group indicated that they preferred decision-making to be shared between them and their doctors, but only 12% of the breast cancer group preferred that option.</p>", "<title>2.3 Response of Physicians to Patients Who Decline Conventional Cancer Treatment</title>", "<p>Little is known about how physicians regard the choices patients make, in particular when patients decide to decline potentially curative treatments. Again, work in this area is mostly qualitative. Authors van Kleffens and van Leeuwen ##REF##15738431##8## assessed how oncologists and general practitioners (<italic>n</italic> = 16) evaluated such a decision by a patient. They found that although patients base their decisions mostly on personal values or experience, physicians emphasize a goal-oriented medical perspective. From the point of view of the doctors, the decision to decline conventional treatment appears irrational, especially when the proposed treatment is curative. In the case of palliative treatment, physicians have less difficulty accepting the patient’s decision.</p>", "<p>Recently, Madjar <italic>et al.</italic>\n##REF##17416697##13## followed up on this notion in a qualitative study of medical and radiation oncologists (<italic>n</italic> = 12) and found that physicians tend to view (“construct”) patients and their decisions in terms of mutually exclusive categories. In addition to distinguishing between curable and non-curable diseases, and between rational and irrational treatment decisions, physicians also distinguished between patients who took a passive or an active role in decisions. Although most patients will go along with their physician’s recommendation and are fairly passive in decision-making, active patients are perceived to be different and possibly to seek alternative health options for which limited scientific evidence is available, and sometimes to decline conventional treatments. It is thus not surprising that some physicians see patients who decline conventional treatment in favour of <sc>cam</sc> as difficult, irrational patients who require extra time and challenge physicians’ authority. What physicians consider to be the salient features of the situation, such as the nature of the disease, the nature of the patient’s decision, and the personal characteristics of the patient, is in each of these studies characterized by a dichotomy.</p>", "<p>It is important to acknowledge the feelings, concerns, and reflections of physicians about their role when faced with patients who wish to take an active role in decision-making and to pursue alternative options to conventional care. The main themes arising from interviews with physicians were feelings of uncertainty, of failure (for example, failure to understand or to get to the bottom of the problem), of helplessness, and of concern (about the patient and the implications of the patient’s decision). According to Madjar <italic>et al.,</italic> the tendency of the physicians to perceive a patient’s decision to decline conventional treatment as either a rational or irrational decision may contribute to such feelings of uncertainty and concern, and may interfere with the ability of physicians to respond to such decisions with sensitivity and understanding.</p>" ]
[ "<title>3. CONCLUSIONS</title>", "<p>The picture that emerges from studying people who decline conventional treatments is not necessarily one of “problem patients,” but of a unique group of self-directed, confident, and active patients who have thought deeply about the meaning of cancer and about their cancer treatment options. It may not always be easy for clinicians to deal with these patients as they deviate from the norm and challenge current evidence, but in the end, relationships with these patients can be rewarding and insightful.</p>", "<p>Without exception, we found that these patients spend much time researching their treatment options. The sources of information they use reflect, to some degree, the definition of evidence set out by Sackett <italic>et al.</italic>\n##UREF##2##14##, which emphasizes the integration of best available evidence from systematic research, professional judgment, and patient values. In this context, patients ideally make treatment decisions that are informed by evidence; that meet their values, beliefs, and expectations; and that are supported by the clinical expertise of (conventional and <sc>cam</sc>) practitioners. For many patients, individual authority and the “lived experience” are also emerging as valuable information sources.</p>", "<p>The rationality of the decision by patients to decline some or all conventional cancer treatments has been discussed in the literature. For example, Huijer and van Leeuwen ##REF##11055039##15## concluded that what might appear to be an irrational decision in a medical context actually results from a balancing process in the patient’s personal context over time. This point has also been discussed by Kingston ##UREF##3##16##, who indicates that these patients are often erroneously labelled “difficult”: “Horses refuse at a jump, badly behaved dogs refuse to obey their masters. Our patients, I hope, make decisions.”</p>", "<p>We have also identified a desire on the part of patients to be in control of the treatment decision-making process and a belief in their own ability to successfully exercise influence over events that affect their lives (“self-efficacy”). Clearly, both concepts are closely related. These findings are consistent with literature focused on personality characteristics of patients who use <sc>cam</sc>. The most common themes in these studies suggest that <sc>cam</sc> users are more open (creative, imaginative, intelligent) than the general population ##REF##15530580##17##,##REF##12220087##18## and that they desire a more active role in decision-making ##REF##12220087##18##–##REF##15005956##20##.</p>", "<p>Despite the important recommendations that patients have provided regarding the role of health professionals in decisions related to conventional care and <sc>cam</sc>, other factors need to be considered as well. Treatment decision-making by patients is a process not limited to one point in time; it will depend on many different factors. In the prostate cancer study, we found that, within the 3-year follow-up period, 5 of the men eventually decided to use some form of conventional treatment.</p>", "<p>The need for effective, compassionate, open-minded, and respectful communication is probably the most important theme in the studies we have reviewed (for example, Shumay <italic>et al.</italic>\n##REF##11742609##6##, Montbriand ##REF##9494229##7##). Alleviating patient concerns about conventional cancer treatments, understanding the potential supportive role of <sc>cam</sc>, being aware of patient preferences, and the personality characteristics of patients related to decision-making is crucial. In several studies, poor communication was even mentioned as a reason for declining conventional treatment (for example, Shumay <italic>et al.</italic>\n##REF##11742609##6##). Understanding who these patients are and what their motivations are may help to improve communication. In addition, it is important to keep in mind that most patients want to discuss these issues with their physicians and prefer to stay in touch. The decision to decline treatment is not necessarily an indicator of distrust of the medical system and the care received to date, but can be a reflection of intensely personal factors. Accepting the challenge and recognizing and honouring the uniqueness of patients who decline conventional treatments will create opportunities for rich patient–provider relationships that will transform “problem” patients into partners in care.</p>" ]
[ "<title>Background</title>", "<p>Several studies have shown that a small but significant percentage of cancer patients decline one or more conventional cancer treatments and use complementary and alternative medicine (<sc>cam</sc>) instead.</p>", "<title>Objectives</title>", "<p>Here, drawing on the literature and on our own ongoing research, we describe why cancer patients decide to decline conventional cancer treatments, who those patients are, and the response by physicians to patients who make such decisions.</p>", "<title>Results</title>", "<p>Poor doctor–patient communication, the emotional impact of the cancer diagnosis, perceived severity of conventional treatment side effects, a high need for decision-making control, and strong beliefs in holistic healing appear to affect the decision by patients to decline some or all conventional cancer treatments. Many patients indicate that they value ongoing follow-up care from their oncologists provided that the oncologists respect their beliefs. Patients declining conventional treatments have a strong sense of internal control and prefer to make the final treatment decisions after considering the opinions of their doctors. Few studies have looked at the response by physicians to patients making such a decision. Where research has been done, it found that a tendency by doctors to dichotomize patient decisions as rational or irrational may interfere with the ability of the doctors to respond with sensitivity and understanding.</p>", "<title>Conclusions</title>", "<p>Declining conventional treatment is not necessarily an indicator of distrust of the medical system, but rather a reflection of many personal factors. Accepting and respecting such decisions may be instrumental in “keeping the door open.”</p>" ]
[]
[ "<title>Acknowledgments</title>", "<p>4. ACKNOWLEDGMENTS</p>", "<p>The research team responsible for this paper, established in 2000 as part of the Sociobehavioural Cancer Research Network of the Centre for Behavioural Research and Program Evaluation, University of Waterloo, has benefited from the support of the National Cancer Institute of Canada, with funds from the Canadian Cancer Society. The Cancer and CAM Research Team is one of five research teams devoted to studying the sociobehavioural aspects of cancer in order to better support patients and ultimately alleviate the burden of cancer.</p>" ]
[]
[ "<table-wrap id=\"tI-co15_s2ps101\" position=\"float\"><label>TABLE I</label><caption><p>Sociodemographic characteristics of prostate (<italic>n</italic> = 29) and breast (<italic>n</italic> = 33) cancer patients</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Characteristic</italic></th><th align=\"center\" colspan=\"2\" rowspan=\"1\"><italic>Patients (</italic>n<italic>) with cancer of</italic></th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>prostate</italic></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>breast</italic></th></tr></thead><tbody><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">Age</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 40–49</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 50–59</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 60–69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">≥70</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Marital status</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Married</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Married or living with a partner</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">19</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Other</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">Education level</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> High school or less</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Technical or some university</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> University degree or higher</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Employment status</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Employed or self-employed</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Retired</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Unemployed</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Top 3 conventional treatments declined</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Surgery</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Radiation therapy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Brachytherapy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Chemotherapy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">22</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Hormone therapy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">20</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tII-co15_s2ps101\" position=\"float\"><label>TABLE II</label><caption><p>Scores on the Multiple Health Locus of Control scale for prostate and breast cancer patients compared with normative scores for patients with a diagnosis of cancer</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Item</italic></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Group</italic></th><th align=\"center\" rowspan=\"1\" colspan=\"1\">n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Mean</italic></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>SD</italic></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>95% CI</italic></th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Internal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cancer diagnostic group <xref ref-type=\"table-fn\" rid=\"tfn1-co15_s2ps101\">a</xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">93</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.72</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.3 to 19.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer group</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.46</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25.1 to 29.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Breast cancer group</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.55</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.4 to 25.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Chance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cancer diagnostic group <xref ref-type=\"table-fn\" rid=\"tfn1-co15_s2ps101\">a</xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">93</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.3 to 21.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer group</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.58</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.4 to 17.4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Breast cancer group</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.42</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.7 to 14.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Doctors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cancer diagnostic group <xref ref-type=\"table-fn\" rid=\"tfn1-co15_s2ps101\">a</xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">93</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.39</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.4 to 16.4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer group</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.42</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.4 to 12.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Breast cancer group</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.85</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.5 to 10.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Powerful others</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cancer diagnostic group <xref ref-type=\"table-fn\" rid=\"tfn1-co15_s2ps101\">a</xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">93</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.96</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.1 to 11.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer group</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.55</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.1 to 9.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Breast cancer group</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.0 to 6.9</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><fn id=\"tfn1-co15_s2ps101\"><p><sup>a</sup> Wallston <italic>et al.</italic>\n##REF##7844739##10##.</p></fn><fn id=\"tfn2-co15_s2ps101\"><p>SD = standard deviation; CI = confidence interval.</p></fn></table-wrap-foot>" ]
[]
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[{"label": ["4"], "surname": ["Polit", "Hungler"], "given-names": ["DF", "BP"], "source": ["Nursing Research: Principles and Methods"], "edition": ["6th ed"], "publisher-loc": ["Philadelphia"], "publisher-name": ["Lippincott"], "year": ["1999"]}, {"label": ["11"], "surname": ["Jerusalem", "Schwarzer"], "given-names": ["M", "R"], "article-title": ["The General Self-Efficacy Scale [Web resource]"], "comment": ["n.p.: n.d. [Available at: "], "ext-link": ["userpage.fuberlin.de/~health/engscal.htm"]}, {"label": ["14"], "surname": ["Sackett", "Straus", "Richardson", "Rosenberg", "Haynes"], "given-names": ["DL", "SE", "WS", "W", "RB"], "source": ["Evidence-based Medicine: How to Practice and Teach"], "sc": ["ebm"], "edition": ["2nd ed"], "publisher-loc": ["London, U.K."], "publisher-name": ["Churchill Livingstone"], "year": ["2000"]}, {"label": ["16"], "surname": ["Kingston"], "given-names": ["R"], "article-title": ["When we use a word"], "source": ["BMJ"], "year": ["2003"], "volume": ["326"], "fpage": ["597"]}]
{ "acronym": [], "definition": [] }
20
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s101-s106
oa_package/7e/ef/PMC2528553.tar.gz
PMC2528554
18769572
[ "<title>1. INTRODUCTION</title>", "<p>The field of integrative oncology has emerged as a response both to cancer patients’ advocacy for holistic care and to an increasing evidence base for the safety and effectiveness of many complementary approaches, commonly called complementary and alternative medicine (<sc>cam</sc>). Cancer patients desire care that not only focuses on treating their disease, but also manages the course of their illness experience, optimizing health and enhancing well-being. Most cancer patients use complementary medicine alongside conventional medicine to meet these needs ##REF##12235222##1##–##UREF##0##4##. Complementary and alternative medicine includes whole medical systems (Traditional Chinese Medicine, among others), mind–body medicine (for example, meditation), biologically-based practices (natural health products, for instance), manipulative and body-based therapies (for example, massage), and energy therapies (<italic>qi gong,</italic> among others) ##UREF##1##5##.</p>", "<p>Integrative oncology uses both conventional medicine and <sc>cam</sc> to meet the needs of individual patients and focuses on the whole person ##UREF##2##6##. At the core of integrative oncology is the need for an evidence base to support the use of conventional and complementary treatments in a collaborative and synergistic manner. The nature of the evidence to guide the development of integrative oncology has, however, been given little attention. In the present paper, we discuss the need for evidence to support the integration of complementary therapies for integrative oncology care; we emphasize that the evidence base must be valid and must respect the underlying principles of individual complementary therapies and integrative oncology practice; and we suggest ways to begin developing that evidence base.</p>" ]
[ "<title>4.2.2 The Power of Mixed Methods</title>", "<p>Although quantitative research approaches have been well established in evaluating treatment interventions, the importance of qualitative research cannot be overlooked or dismissed as development begins of the evidence base for complementary therapies and integrative oncology. Qualitative research has amazing potential to explore, in depth, from a variety of perspectives (patients, oncology team, cancer care system), how integrative cancer treatment and care are experienced. Including qualitative inquiry in the evaluation of interventions should be an integral part of evidence-based medicine ##UREF##7##19##.</p>", "<p>Qualitative research aims to understand the nature of phenomena; it will prove crucial as exploration of the potential of integrative oncology for cancer care moves forward. For example, qualitative research is ideally suited to answer the question of what integrative oncology is, how it is being practiced, how it can best be practiced, and the benefits that are possible. Such exploratory work can help to elucidate the key components in integrative care and their synergistic relationships from the viewpoint of patients and of practitioners.</p>", "<p>Because qualitative research can fill an important niche in this field, it should, when possible, be nested within clinical trials or other quantitative designs ##REF##12652884##20##,##REF##12165185##21## as a form of mixed-methods research. Such a design represents a further modification of the <sc>rct</sc> method that addresses the requirements of internal, external, and model validity. This approach can help to elicit whether, why, and how patients benefit from a complex intervention and can explore relevant outcomes from a variety of perspectives.</p>" ]
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[ "<title>5. CONCLUSIONS</title>", "<p>As cancer patients increasingly turn to <sc>cam</sc> as a way to complement their cancer care, it is crucial that health care professionals become informed about the evidence base behind this group of practices ##REF##16459074##26## and, further, that they remain critical of the need for valid research. The assessment of validity for complementary therapies and integrative oncology alike should encompass internal, external, and model validity. Because it seems unlikely that any one study design can achieve optimal levels of each type of validity, health professionals and researchers need to be open to emerging models of evidence that are not necessarily aligned to traditional ideas of evidence in biomedicine. Programs of research have to include a variety of evidence types and treat all of them as legitimate. A whole-systems research framework is helpful to guide the development of these research programs.</p>" ]
[ "<title>Background</title>", "<p>Integrative oncology uses both conventional and complementary medicine to meet the needs of individual patients and to focus on the whole person. The core principles of integrative oncology include individualization, holism, dynamism, synergism, and collaboration, but the nature of the evidence to guide the development of integrative oncology has been given little attention.</p>", "<title>Objectives</title>", "<p>\n<list list-type=\"bullet\"><list-item><p>To discuss the need for evidence to support the integration of complementary therapies for integrative oncology care.</p></list-item><list-item><p>To emphasize that the evidence base must be valid and respect the underlying principles of individual complementary therapies and integrative oncology practice.</p></list-item><list-item><p>To suggest ways to begin developing the evidence base.</p></list-item></list></p>", "<title>Review and Discussion</title>", "<p>Although the evidence for safety and efficacy seems paramount for supporting the integration of an individual complementary therapy into mainstream cancer care, the need for evidence to support the overall practice of integrative oncology has to be considered as well.</p>", "<p>We argue that developing an evidence base for integrative oncology requires a contextual and comprehensive research approach that assesses a range of outcomes over a suitable period of time that the patient and the patient’s family, in addition to the health care providers, deem important.</p>", "<title>Conclusion</title>", "<p>A whole-systems framework to the development of the evidence base for integrative oncology can guide the development of evidence that respects the complex nature of many complementary and integrative practices and their underlying principles of care delivery.</p>" ]
[ "<title>2. WHAT IS INTEGRATIVE ONCOLOGY?</title>", "<p>The goal of integrative oncology is to support cancer patients and their families throughout the cancer journey by improving quality of life, ameliorating symptoms associated with conventional cancer care, alleviating distress, and in some cases, augmenting the effectiveness of conventional treatment ##REF##17511932##7##,##REF##16737670##8##. Mumber defines integrative oncology as “a comprehensive, evidence-based approach to cancer care that addresses all participants at all levels of their being and experience. It represents the next step in the evolution of cancer care in that it addresses the limitations of the current system while retaining the system’s successful features” ##UREF##3##9##.</p>", "<p>Core principles of integrative oncology include individualization, holism, dynamism, synergism, and collaboration. In integrative oncology, the focus of care is on the whole person, and the aim is to promote the innate ability of each person to heal. Integrative oncology is individualized for each cancer patient over time, as each patient presents with unique symptoms and context, and as the goals of treatment change over time. Integrative care is also about compassion and caring for an individual in a holistic manner that gives voice to the patient’s values and needs. Grounded in a truly respectful partnership between patient and practitioner, a therapeutic alliance is forged that honours the patient’s informed choices. This collaborative approach to cancer care assumes that conventional and complementary practitioners—and patients—contribute their knowledge, experience, and skills to the healing encounter ##REF##16737670##8##. In this context, a safe, knowledgeable, and dynamic cancer management plan is developed cooperatively, ensuring accurate monitoring and evaluation ##REF##17283742##10##. Further, and in contrast to the prevailing pharmacologic model, the this cancer care approach recognizes the potential for synergy when therapies are integrated, with outcomes far exceeding the sum of the outcomes of individual therapies.</p>", "<p>Integrative oncology is usually defined as an evidence-based discipline; however, we argue that the traditional (scientific) understanding of evidence needs to be revisited and expanded.</p>", "<title>3. THE NEED FOR EVIDENCE</title>", "<p>Integrative oncology makes a deliberate, yet fluid, distinction between complementary therapies, which are supported by evidence and used in combination with conventional cancer care, and alternative therapies, which are unproven and used as a replacement for conventional cancer care ##REF##17511932##7##,##REF##17024875##11##. When a strong evidence base is developed for some complementary therapies, they can potentially become part of integrative cancer care. For example, after a review of the available evidence, the Society for Integrative Oncology supports the use of acupuncture as a complementary therapy when cancer-related pain is poorly controlled ##REF##17511932##7##.</p>", "<p>The practice of integrative oncology therefore depends mainly on complementary therapies meeting standards of safety and, to a lesser extent, effectiveness (because the latter may be assessed from different perspectives in real-life situations). As a result, an understanding of what constitutes appropriate evidence is crucial to the foundation and future development of the field.</p>", "<p>Despite agreement on the need for evidence to support the integration of complementary therapies into conventional cancer care ##REF##17511932##7##,##UREF##3##9##,##REF##17024875##11##, the discussion regarding the type of evidence required and its purpose is entirely based on evidence from specific <sc>cam</sc> treatments. We argue that this line of thinking misses an important point. To begin the discussion, a distinction must first be made between evidence for complementary therapies and evidence for integrative oncology practice. The first issue concerns evidence that supports the safety and effectiveness of individual complementary therapies, thus determining their suitability for integration into mainstream care. The second—and often forgotten—aspect concerns evidence that supports the synergistic integration of complementary and conventional practices in a collaborative and supportive manner within cancer care.</p>", "<title>3.1 Nature of the Evidence Required for Complementary Therapies</title>", "<p>According to Stark, Hess, and Shaw ##UREF##4##12##, different levels of evidence are required for the safety and effectiveness of individual complementary therapies depending on the goals of treatment. These levels of evidence depend on study design and sample size, and they range from well-designed randomized controlled trials [<sc>rcts</sc> (level 1)] to preclinical <italic>in vitro</italic> and <italic>in vivo</italic> studies and traditional medicines (level 4). Level 1 evidence is required for the use of complementary therapies with anti-neoplastic goals, but lower levels of evidence, such as nonrandomized trials or observational studies, are acceptable for less-invasive procedures and preventive or supportive goals. However, this hierarchy does not capture the multidisciplinary, synergistic approach that characterizes complementary therapies and integrative oncology alike in comparison with conventional care ##REF##17283742##10##. Further, while addressing the need for evidence to support the individual integration of a complementary therapy into mainstream care, the need for evidence to support the overall practice of integrative oncology is often ignored.</p>", "<p>A need arises to revisit traditional notions of evidence as they apply to complementary therapies. Traditional research methods are challenged in the attempt to evaluate complementary therapies, because these methods cannot account for the fundamental issues of individualization, synergism, and holism ##REF##16737670##8##,##UREF##3##9##,##REF##16150375##13##. These problems are compounded for the evaluation of integrative oncology, which involves the synergistic use of treatments from various healing paradigms and a range of physiologic, emotional, social, and spiritual outcomes.</p>", "<title>3.2 Nature of the Evidence Required for Integration</title>", "<p>Further to answering whether a complementary therapy works and is safe, questions regarding the appropriateness of integration must be examined. To this end, it is critical to document the ways in which complementary therapies and conventional care are being integrated and the outcomes that are important and relevant.</p>", "<p>Integration can occur at many levels: individual, clinical, institutional, regulatory, or policy ##UREF##5##14##. Integration can also occur in many ways. For example, numerous patients are known to be integrating complementary therapies into their conventional care, but research is only starting to uncover how those patients make decisions regarding therapy selection, who is involved in the decision-making process, why the patients are integrating these therapies, and which outcomes are seen as relevant ##REF##17609997##15##. Alternatively, health care providers may be the ones suggesting integration for their patients. The process of evaluation and decision-making is likely different in the two scenarios, in part because the intent may differ. For the health care professional, for example, the utmost concern is patient safety; but for patients, a decision to use <sc>cam</sc> may be driven by an attempt to minimize potential side effects and to feel empowered ##REF##18769576##16##.</p>", "<p>“Integrating” must also be distinguished from “combining.” “Combining” is more akin to adding various therapies to a treatment plan without considering the overall picture. “Integrating” involves synergistically applying a range of treatments to address holistic treatment goals as they change over time and in accordance with patient needs and values. Currently, although the goals and philosophy underlying integrative oncology are well developed, practical knowledge is not available concerning the extent to which and the manner in which diverse therapies are being integrated or combined.</p>", "<p>Finally, integrative care seems to represent untapped opportunities (which so far remain understudied) for meeting the needs of cancer patients across the cancer trajectory.</p>", "<title>4. VALIDITY AS CRITERION FOR EVIDENCE</title>", "<p>As development of an evidence base begins both for individual complementary therapies and for integrative oncology, assurance is needed that the evidence base is valid—“validity” referring to the extent that appropriate research methods were used to support a conclusion regarding the efficacy or effectiveness of a therapy.</p>", "<p>“Validity” commonly includes internal validity and external validity. Model validity is separate from both of those, but is just as important. Model validity is often overlooked because of the biomedical focus of most health care research. It refers to the extent to which the research methods used have addressed the unique theory and therapeutic context of the intervention being assessed ##UREF##6##17##.</p>", "<p>Traditional clinical research methods have been developed to assess biomedical interventions, and thus model validity can typically be assumed. For research results to be valid in the case of integrative oncology, the research methods used must address the underlying principles of integrative oncology, such as its individualized, synergistic, holistic, and collaborative nature. The same is true for research regarding individual complementary therapies that are often based on assumptions contrary to biomedicine, such as the network of channels and blood vessels connected by <italic>qi</italic> (an essential fast-flowing substance full of vigour) in an approach using Traditional Chinese Medicine.</p>", "<title>4.1 Limitations of the RCT Design for Complementary Therapies and Integrative Oncology</title>", "<p>The double-blind <sc>rct</sc> is often upheld as the “gold standard” in clinical research, because of its strong internal validity arising from the ability to control for expected and unexpected bias, confounding factors, and error. However, it is impossible to achieve model validity while applying the blinded <sc>rct</sc> design to the practice of integrative oncology and many complementary therapies ##REF##16150375##13##.</p>", "<p>Sagar ##REF##16737670##8## highlights several of the key challenges in applying the <sc>rct</sc> design to the study of select complementary therapies. Examples include difficulties in determining appropriate placebos or sham treatments, the impossibility of double-blinding when the practitioner is part of the intervention, and problems respecting the individualized approach of many complementary practices. Acupuncture provides a good example, because the choice of an appropriate sham treatment for acupuncture has been an ongoing challenge ##REF##12184353##18##, not unlike that in determining an appropriate sham treatment for surgery in the realm of conventional care. Further, blinding patients and providers is difficult, because both are quite aware of whether needling has taken place, although single-blinding may be possible if simulated needling is used as a sham treatment. In practice, different needling protocols are developed for specific patients, depending on their unique symptoms and holistic context, and thus standardization of an acupuncture protocol for a <sc>rct</sc> is problematic if model validity is to be upheld.</p>", "<p>The same argument can easily be extended to integrative oncology practice, in which therapies from diverse philosophical backgrounds are combined, thus making model validity even more difficult to attain. In addition to the problems of defining a placebo, blinding, and standardization, the <sc>rct</sc> is not designed to measure the effect that each patient’s unique physical, social, and cultural context and corresponding reasons for integration may have on treatment outcomes. Further, the <sc>rct</sc> cannot assess the synergism that results from the integration of various therapies, coupled with the healing context and the clinical skills and expertise of the integrative team.</p>", "<title>4.2 Building Evidence for Integrative Oncology</title>", "<p>Developing an evidence base for integrative oncology requires a contextual and comprehensive research approach that assesses a range of outcomes over a suitable period of time that the patient, the patient’s family, and health care providers deem important. We next highlight some approaches that begin to meet the requirements of internal, external, and model validity. Readers should consult the papers referenced in this section for detailed descriptions of these approaches.</p>", "<title>4.2.1 Variations of the RCT Design</title>", "<p>Although the <sc>rct</sc> in its classical form cannot meet the requirements for model validity when applied to complementary therapies or integrative oncology, some variations have been suggested that address many of the shortcomings. For example, pragmatic <sc>rcts</sc> do not require standardization of the intervention and thus allow for the individualized nature of the treatments to be assessed. Preference <sc>rcts</sc> account for the effect that beliefs and preferences of patients for certain treatment types will have on treatment outcomes. In a preference <sc>rct</sc>, patients with treatment preferences receive their preferred treatment; patients who do not have a preference are randomized as usual.</p>", "<title>4.2.3 Whole-Systems Research</title>", "<p>Whichever research methods are ultimately adopted to study complementary therapies and integrative oncology, investigators must systematically capture the complexity inherent in these approaches to healing. A “whole-systems” research framework is helpful to conceptualize the important issues that need attention and to design step-wise programs of research that will answer the multifaceted questions.</p>", "<p>The notion of whole-systems research has recently been given attention in the literature, both with regard to complementary therapies ##REF##16150375##13##,##REF##12868250##22## and integrative oncology ##REF##17101757##23##,##REF##17609993##24##. In a general sense, the goal of whole-systems research “is to appropriately combine research designs and methods in a coherent research program, so that all aspects of an internally consistent approach to treatment, or a whole system, can be assessed. It acknowledges an individualized, patient centered and participatory approach to diagnosis and treatment and a process of healing that collaboratively combines patient and practitioner knowledge and skills, thus enhancing healing” ##REF##17101757##23##.</p>", "<p>The relevance of a whole-systems framework to the development of the evidence base for integrative oncology should be apparent. It can guide the development of evidence that respects the complex nature of many complementary and integrative practices and their underlying principles of care delivery ##REF##18638695##25##. A program of research guided by a whole-systems research framework would include <sc>rcts</sc>, variations of <sc>rcts</sc>, observational trials, and qualitative research, ideally mixing methods as appropriate, depending on the specific focus. A program of whole-systems research would thus produce research that collectively has strong validity.</p>" ]
[ "<title>Acknowledgments</title>", "<p>6. ACKNOWLEDGMENTS</p>", "<p>Dr. A. Leis is the Dr. Louis Schulman Cancer Research Chair; Ms. L. Weeks has received a Canada Graduate Scholarship (Doctoral) from the Canadian Institutes of Health Research and an Incentive Award from the Alberta Heritage Foundation for Medical Research; and Dr. M. Verhoef is a Canada Research Chair in Complementary Medicine. Co-led by Drs. Leis and Verhoef, the research team responsible for six of this issue’s articles including this one, was established in 2000 as part of the Sociobehavioural Cancer Research Network of the Centre for Behavioural Research and Program Evaluation, University of Waterloo, and has benefited from the support of the National Cancer Institute of Canada, with funds from the Canadian Cancer Society. The Cancer and CAM Research Team is one of five research teams devoted to studying the sociobehavioural aspects of cancer to better support patients and ultimately alleviate the burden of cancer.</p>" ]
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[{"label": ["4"], "surname": ["Leis", "Ferro"], "given-names": ["A", "M"], "article-title": ["Complementary and alternative medicine: responding to the needs of cancer patients"], "source": ["Oncol Exch"], "year": ["2006"], "volume": ["5"], "fpage": ["57"], "lpage": ["60"]}, {"label": ["5"], "collab": ["National Institutes of Health, National Center for Complementary and Alternative Medicine ("], "sc": ["nccam", "cam", "cam"], "article-title": ["Health information > "], "publisher-loc": ["Gaithersburg, MD"], "publisher-name": ["NCCAM"], "year": ["February 2008"], "comment": ["[Available at: "], "ext-link": ["nccam.nih.gov/health/whatiscam"]}, {"label": ["6"], "surname": ["Barraclough", "Barraclough"], "given-names": ["J", "J"], "article-title": ["Introducing the holistic approach to cancer care"], "source": ["Enhancing Cancer Care"], "publisher-loc": ["Oxford"], "publisher-name": ["Oxford University Press"], "year": ["2007"]}, {"label": ["9"], "surname": ["Mumber", "Rakel"], "given-names": ["MP", "D"], "article-title": ["Integrative oncology: an overview"], "source": ["Integrative Medicine"], "edition": ["2nd ed"], "publisher-loc": ["Philadelphia"], "publisher-name": ["Saunders Elsevier"], "year": ["2007"]}, {"label": ["12"], "surname": ["Stark", "Hess", "Shaw", "Mumber"], "given-names": ["N", "S", "E", "MP"], "article-title": ["Clinical research and evidence"], "source": ["Integrative Oncology: Principles and Practice"], "publisher-loc": ["London"], "publisher-name": ["Taylor and Francis"], "year": ["2005"]}, {"label": ["14"], "surname": ["Tataryn", "Verhoef"], "given-names": ["D", "MJ"], "article-title": ["Combining conventional, complementary and alternative health care: a vision of integration"], "source": ["Perspectives on Complementary and Alternative Health Care"], "publisher-loc": ["Ottawa"], "publisher-name": ["Health Canada"], "year": ["2001"]}, {"label": ["17"], "surname": ["Lewith", "Walach", "Jonas", "Lewith", "Walach", "Jonas"], "given-names": ["G", "H", "WB", "G", "H", "WB"], "article-title": ["Balanced research strategies for complementary and alternative medicine"], "source": ["Clinical Research in Complementary Therapies: Principles, Problems and Solutions"], "publisher-loc": ["Edinburgh"], "publisher-name": ["Churchill Livingstone"], "year": ["2002"]}, {"label": ["19"], "surname": ["Straus", "Richardson", "Glasziou", "Haynes"], "given-names": ["S", "WS", "P", "RB"], "source": ["Evidence-based Medicine: How to Practice and Teach "], "sc": ["ebm"], "publisher-loc": ["Edinburgh"], "publisher-name": ["Churchill Livingstone"], "year": ["2005"]}]
{ "acronym": [], "definition": [] }
26
CC BY
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2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s83-s87
oa_package/fd/1c/PMC2528554.tar.gz
PMC2528555
18769573
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[ "<title>Objective</title>", "<p>Cancer patients are increasingly known to use complementary medicine (<sc>cam</sc>) during conventional treatment, but data are limited on how Canadian oncology health professionals attempt to assist patients with their use of <sc>cam</sc> in the context of conventional cancer care. As part of a larger qualitative study assessing the perceptions of Canadian oncology health professionals regarding integrated breast cancer care, we undertook an exploration of current integrative practices of oncology health professionals.</p>", "<title>Design</title>", "<p>Using an interpretive description research design and a purposive sampling, we conducted a series of in-depth qualitative interviews with various oncology health professionals recruited from provincial cancer agencies, hospitals, integrative clinics, and private practice settings in four Canadian cities: Vancouver, Winnipeg, Montreal, and Halifax. A total of 16 oncology health professionals participated, including medical and radiation oncologists, nurses, and pharmacists.</p>", "<title>Results</title>", "<p>Findings highlighted two main strategies used by oncology health professionals to create a more integrative approach for cancer patients:\n<list list-type=\"bullet\"><list-item><p> acting as an integrative care guide, and</p></list-item><list-item><p> collaborating with other health professionals.</p></list-item></list></p>", "<title>Conclusions</title>", "<p>Although few clear standards of practice or guidance material were in place within their organizational settings, health professionals discussed some integrative roles that they had adopted, depending on interest, knowledge, and skills, in supporting patients with <sc>cam</sc> decisions. Given that cancer patients report that they want to be able to confer with their conventional health professionals, particularly their oncologists, about their <sc>cam</sc> use, health professionals who elect to adopt integrative practices are likely offering patients much-welcomed support.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/283/253\">http://www.current-oncology.com/index.php/oncology/article/view/283/253</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
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2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s110.es87-s110.es91
oa_package/27/9a/PMC2528555.tar.gz
PMC2528556
18769574
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[ "<p>Various surveys show that interest in complementary and alternative medicine (<sc>cam</sc>) is high among cancer patients. Patients want to explore all options that may help their treatment. Many <sc>cam</sc> modalities offer patients an active role in their self-care, and the resulting sense of empowerment is very appealing. On the other hand, many unscrupulous marketeers promote alternative cancer “cures,” targeting cancer patients who are particularly vulnerable. Some alternative therapies can hurt patients by delaying effective treatment or by causing adverse effects or detrimental interactions with other medications. It is not in the best interest of cancer patients if they cannot get appropriate guidance on the use of <sc>cam</sc> from the health care professionals who are part of their cancer care team.</p>", "<p>The Integrative Medicine Service at Memorial Sloan–Kettering Cancer Center in New York was established in 1999 to address patient interest in <sc>cam</sc>, to incorporate helpful complementary therapies into each patient’s overall treatment management, to guide patients in avoiding harmful alternative therapies, and to develop prospective research to evaluate the efficacy of <sc>cam</sc> modalities.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/276/238\">http://www.current-oncology.com/index.php/oncology/article/view/276/238</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
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no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s108.es68-s108.es71
oa_package/a7/a1/PMC2528556.tar.gz
PMC2528557
18769575
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[ "<title>Background</title>", "<p>Individuals have increasingly sought complementary therapies to enhance health and well-being during cancer, although little evidence of their effect is available.</p>", "<title>Objectives</title>", "<p>We investigated\n<list list-type=\"bullet\"><list-item><p> how an Iyengar yoga program affects the self-identified worst symptom in a group of participants.</p></list-item><list-item><p> whether quality of life, spiritual well-being, and mood disturbance change over the Iyengar yoga program and at 6 weeks after the program.</p></list-item><list-item><p> how, from a participant’s perspective, the Iyengar yoga program complements conventional cancer treatment.</p></list-item></list></p>", "<title>Patients and Methods</title>", "<p>This pre–post instrumental collective case study used a mixed methods design and was conducted at a private Iyengar yoga studio. The sample consisted of 24 volunteers (23 women, 1 man; 88% Caucasian; mean age: 49 years) who were currently on treatment or who had been treated for cancer within the previous 6 months, and who participated in ten 90-minute weekly Iyengar yoga classes.</p>", "<p>The main outcome measures were most-bothersome symptom (Measure Your Medical Outcome Profile 2 instrument), quality of life and spiritual well-being (Functional Assessment of Chronic Illness Therapy–General subscale and Spiritual subscale), and mood disturbance (Profile of Mood States–Short Form). Participant perspectives were obtained in qualitative interviews.</p>", "<title>Results</title>", "<p>Statistically significant improvements were reported in most-bothersome symptom (<italic>t</italic><sub>(23)</sub> = 5.242; <italic>p</italic> &lt; 0.001), quality of life (<italic>F</italic><sub>(2,46)</sub> = 14.5; <italic>p</italic> &lt; 0.001), spiritual well-being (<italic>F</italic><sub>(2,46)</sub> = 14.4; <italic>p</italic> &lt; 0.001), and mood disturbance (<italic>F</italic><sub>(2,46)</sub> = 10.8; <italic>p</italic> &lt; 0.001) during the program. At follow-up, quality of life (<italic>t</italic><sub>(21)</sub> = −3.7; <italic>p</italic> = 0.001) and mood disturbance (<italic>t</italic><sub>(21)</sub> = 2.4; <italic>p</italic> = 0.025) significantly improved over time. Categorical aggregation of the interview data showed that participants felt the program provided them with various benefits not included on the outcomes questionnaires.</p>", "<title>Conclusions</title>", "<p>Over the course of the Iyengar Yoga for Cancer program, participants reported an improvement in overall well-being. The program was also found to present participants with a holistic approach to care and to provide tools to effectively manage the demands of living with cancer and its treatment.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/284/280\">http://www.current-oncology.com/index.php/oncology/article/view/284/280</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s109.es72-s109.es78
oa_package/5b/5f/PMC2528557.tar.gz
PMC2528558
18769576
[ "<title>1. INTRODUCTION</title>", "<p>Since the late 1990s, a growing number of studies have focused on the engagement of cancer patients in the treatment decision-making process ##REF##10452420##1##–##REF##11533436##4##. The use of qualitative research methods has provided a greater understanding of how cancer patients make sense of the treatment recommendations provided by their health professionals, and of the process by which they seek additional information and evaluate the risks and benefits of available treatment options ##REF##12586585##5##–##REF##12787015##8##. Individuals living with cancer have varying needs regarding their preferred level of involvement in treatment decisions, and correspondingly, vary in the way in which they can be best supported by health professionals during the decision-making process ##REF##11838719##9##,##REF##9145723##10##. Although previous research has provided insight into the decision-support strategies required by patients faced with conventional treatment decisions, its applicability to patients making decisions about complementary and alternative medicine (<sc>cam</sc>) is questionable.</p>", "<p>The literature suggests that most Canadian cancer patients use <sc>cam</sc> at some point during their illness ##REF##16813510##11##,##REF##15035882##12##. It is therefore essential that oncology health professionals understand and acknowledge the unique contexts and processes that influence treatment decisions specific to <sc>cam</sc> for each patient. This acknowledgment is especially important given the emerging field of integrative oncology in North America, in which evidence-based <sc>cam</sc> therapies are gradually being integrated into mainstream cancer care ##REF##17511932##13##. Decision-support strategies that facilitate patients’ informed use of <sc>cam</sc> and full disclosure of <sc>cam</sc> use with health professionals are essential for the safe integration of <sc>cam</sc> with conventional cancer care.</p>", "<p>Canadian researchers have taken a leading role in examining the treatment decision-making process of cancer patients interested in <sc>cam</sc>\n##REF##17609997##14##–##REF##14528705##18##. This work has resulted in the development of several decision-making models that capture the complex interplay between key personal, social, and cultural factors and the cognitive processes that underlie the <sc>cam</sc> decision-making process. Although these models have been limited mainly to breast and prostate cancer and have yet to be empirically tested, they provide health professionals with insight into the experiences of patients making <sc>cam</sc> decisions, and they highlight moments during the cancer trajectory when patients may possibly benefit most from decision support.</p>", "<p>In the present paper, we begin our discussion by highlighting the highly complex, dynamic, and individualized nature of <sc>cam</sc> decisions, which results from the unique personal, social, and cultural contexts in which these decisions are embedded. We then draw on previous decision-making models and provide a summary of the main stages within the <sc>cam</sc> decision-making process (##FIG##0##Figure 1##):</p>", "<p> Taking stock of treatment options</p>", "<p> Gathering and evaluating <sc>cam</sc> information</p>", "<p> Making a decision</p>", "<p> Revisiting the decision</p>", "<p>This overview emphasizes the iterative nature of the <sc>cam</sc> decision-making process and how it unfolds across the cancer trajectory.</p>" ]
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[ "<title>2. DISCUSSION</title>", "<title>2.1 The Context of CAM Decisions</title>", "<p>The decision about whether to use <sc>cam</sc>, and if so, the type or types of <sc>cam</sc> to use, is influenced throughout the cancer trajectory by a myriad of factors that can be grouped as follows: sociodemographic and disease-related factors, psychological factors, and social factors.</p>", "<title>2.1.1 Sociodemographic and Disease-Related Factors</title>", "<p>At a basic level, specific demographic and disease-related factors have been found to be associated with <sc>cam</sc> use, including age (younger), sex (women), and socio-economic status (higher education and income) ##REF##16821087##19##–##REF##16282504##21##. These personal factors have been identified as being predictive of other self-care health behaviours and reflect health care access issues and the increased use of health care services by women ##REF##17939874##22##. Further, despite an increasing number of private health insurance plans providing coverage for specific <sc>cam</sc> practices (such as acupuncture and chiropractic treatment), many <sc>cam</sc> therapies are not covered through public or private health insurance. As a result, the decision to use <sc>cam</sc> can be an expensive undertaking for many patients, particularly those who are on disability as a result of their illness.</p>", "<p>In Canada’s multicultural society, it is also imperative to acknowledge the important influence that ethnicity may have on <sc>cam</sc> use. For a growing number of immigrants and indigenous peoples, traditional medical systems (for example, Traditional Chinese Medicine, Ayurveda, First Nations traditional healing) are the primary source of health care. As a result, many cancer patients arrive at initial consultations already using or interested in trying <sc>cam</sc> therapies that are not considered “alternative” within their ethnocultural community.</p>", "<p>Increased <sc>cam</sc> use has also been related to disease characteristics. It has been observed to be higher in breast and prostate cancer populations than in populations with other cancer diagnoses ##REF##14499031##20##,##REF##12230908##23##. The heightened interest in <sc>cam</sc> in these populations may be a consequence of the proactive nature of these patient groups with regard to advocacy and self-care (that is, support-group membership). Cancer patients with advanced disease have also been found to have a heightened interest in <sc>cam</sc>\n##UREF##0##24##, which may reflect their attempt to maintain hope when presented with a poor prognosis and limited conventional treatment options, coupled with a search for healing when cure is not possible. Lastly, <sc>cam</sc> therapies that require intensive time and energy, such as restrictive diets or frequent visits to a practitioner, may be impractical for patients undergoing active cancer treatment, particularly for those experiencing fatigue or other debilitating physical or psychological symptoms.</p>", "<title>2.1.2 Psychological Factors</title>", "<p>Equally influential in the <sc>cam</sc> decision-making process are psychological factors. For many individuals, the initial decision to explore <sc>cam</sc> treatment options arises from a strong internal locus of control (that is, the tendency to attribute event outcomes to one’s own control ##REF##5340840##25##) and a desire to be an active participant in treatment decisions ##REF##14528705##18##,##REF##14528706##26##–##REF##11858470##29##. In addition, for some patients, <sc>cam</sc> therapies may also provide the hope and optimism required to cope with the cancer journey ##REF##14528705##18##,##REF##11966834##30##. For others, their fears about death and dying may motivate their search for treatment options beyond conventional cancer care so that they can “cover all their bases” ##REF##14528705##18##,##REF##15803360##31##.</p>", "<title>2.1.3 Social Factors</title>", "<p>It is important to recognize how the personal and psychological factors associated with <sc>cam</sc> use are also embedded within a larger social context that legitimizes and reinforces the exploration and use of many <sc>cam</sc> therapies. For example, the increasing tendency of cancer patients to consider <sc>cam</sc> as a treatment option during their illness may reflect the currently persistent postmodern ideals of individualism, consumerism, and holism ##UREF##1##32##,##UREF##2##33##. In addition, an individual’s understandings of what constitutes appropriate treatment and how it can best be achieved are derived not only from personal experience, but also from social interaction and interface with cultural products—most notably the mass media ##UREF##1##32##. Information about <sc>cam</sc> is increasingly available and accessible through media sources ##REF##17624558##34##, which lend visibility and perceived legitimacy to this group of therapies and practices.</p>", "<p>Further, male and female cancer patients both describe family members, friends, and fellow cancer survivors to be highly influential in their <sc>cam</sc> decisions ##REF##10558372##15##,##REF##14672107##16##,##REF##17683580##35##,##REF##16725245##36##. Members of a patient’s social network can take on a variety of decision-support roles depending on a patient’s diagnosis and stage of illness, and the nature of their relationship with the patient ##REF##17683580##35##,##REF##16725245##36##. These roles include “interested bystander” (a person who listens and supports a patient’s <sc>cam</sc> decisions), “information gatherer and reviewer” (a person who helps collect and evaluate information), and “director” (a person who takes over the decision-making process on behalf of the patient) ##REF##16725245##36##.</p>", "<title>2.2 The CAM Decision-Making Process</title>", "<p>Unlike the many rational treatment decision-making models presented within the health care literature, the <sc>cam</sc> decision-making process has been described as a dynamic and iterative process that is highly variable across individuals ##REF##17609997##14##,##REF##10558372##15##,##REF##14528705##18##. Despite its complex, nonlinear, and individualized nature, some common stages of the <sc>cam</sc> decision-making process can be explicated.</p>", "<title>2.2.1 Taking Stock of Treatment Options</title>", "<p>Research has shown that the <sc>cam</sc> decision-making process begins immediately following a diagnosis of cancer ##REF##17609997##14##,##REF##10558372##15##,##REF##14528705##18##. Despite being emotionally overwhelmed by the news of their diagnosis, most cancer patients are eager to learn about the full spectrum of treatment options and often do not distinguish between conventional and complementary therapies ##REF##14528705##18##. At the time of diagnosis, patients are particularly interested in <sc>cam</sc> therapies that will enhance the effectiveness of their conventional treatment protocols and mediate potential side effects ##REF##17609997##14## such as fatigue, nausea and vomiting, and anxiety. However, the already complex decision about whether to use <sc>cam</sc> is challenged by concerns held by some patients and their health professionals regarding the potential risks posed by inappropriate <sc>cam</sc> use ##REF##17609997##14##,##REF##14672107##16##,##REF##15911931##37##.</p>", "<title>2.2.2 Gathering and Evaluating CAM Information</title>", "<p>During the initial phase of taking stock of available treatment options and identifying a personal interest in <sc>cam</sc>, cancer patients begin to gather and evaluate information about the possible role of <sc>cam</sc> in their cancer experience. Because the decision-making process is highly dependent on the patient’s unique contextual factors, the process of gathering and evaluating information is highly variable across individuals.</p>", "<p>For some cancer patients, particularly those who feel overwhelmed by their diagnosis, the information gathering and evaluation phase is a passive process in which they seek information only about the <sc>cam</sc> therapies that they have had previous experience with or that are recommended by a trusted health professional, family member, or friend ##REF##17609997##14##,##REF##10558372##15##,##REF##14528705##18##. Other cancer patients take on a more active role in which they engage in an extensive and iterative information-seeking and evaluation process related to a diverse range of <sc>cam</sc> therapies. This process often continues throughout their cancer journey and is revisited at key milestones, such as at the end of conventional cancer treatment and at diagnosis of recurrence ##REF##17609997##14##,##REF##14528705##18##,##REF##17683580##35##. For these individuals, the search for <sc>cam</sc> information is motivated by their information needs, including the potential risks and benefits of <sc>cam</sc> use, the likelihood of negative interactions of specific <sc>cam</sc> therapies (typically natural health products) with conventional treatments (that is, chemotherapy, radiation, hormone therapy), the appropriate timing of <sc>cam</sc> use in the conventional cancer treatment trajectory, and the availability and financial cost of specific therapies ##REF##17609997##14##,##REF##10558372##15##,##REF##14528705##18##.</p>", "<p>The type of evidence privileged by cancer patients in making <sc>cam</sc> decisions varies widely and includes professional advice, the scientific literature, anecdotes about <sc>cam</sc> use from social networks, the Internet, and previous personal experiences with <sc>cam</sc>\n##REF##17609997##14##,##REF##17683580##35##,##REF##18048882##38##. As a result, patients seek information about <sc>cam</sc> from a multitude of sources, although there is a preference to seek assistance from trusted individuals who are perceived as being credible, such as oncologists, family physicians, or regulated <sc>cam</sc> practitioners (a naturopathic physician, for instance) ##REF##17609997##14##,##REF##14672107##16##,##REF##16618687##39##. Whether these health professionals have the training, knowledge, or interest to discuss <sc>cam</sc> therapy options with cancer patients, however, is discussed in elsewhere in this issue ##REF##18769581##40##.</p>", "<p>The information gathering and evaluation phase can be an anxiety-provoking experience. Some individuals are able to control the amount of information they obtain about <sc>cam</sc> by restricting their search to a limited number of therapies or by avoiding certain resources, such as the Internet, but others report feeling overwhelmed by the amount of <sc>cam</sc> information they acquire ##REF##17609997##14##,##REF##14528705##18##. Some patients struggle to make sense of the often contradictory information that exists about <sc>cam</sc>\n##REF##17609997##14##,##REF##17944766##41##, and they report being particularly distressed about the lack of consensus between and among their <sc>cam</sc> and conventional health professionals about the implications of <sc>cam</sc> use. This conflict causes profound anxiety in some cancer patients who are fearful of making the “wrong” treatment decision that could have potentially serious consequences regarding their survival.</p>", "<title>2.2.3 Making a CAM Decision</title>", "<p>How cancer patients ultimately reach a decision about <sc>cam</sc> varies considerably between individuals and along the cancer trajectory. This complex process, labelled “bridging the gap” by Balneaves <italic>et al.</italic>\n##REF##17609997##14##, involves cancer patients attempting to make sense of the disparate advice and information they have gathered about <sc>cam</sc> while reflecting on their personal beliefs about cancer, treatment, and healing.</p>", "<p>In our previous work, we identified three different types of <sc>cam</sc> decisions.</p>", "<p>First, individuals in the midst of conventional treatment who are experiencing high anxiety and conflict often “take it one step at a time” and postpone their <sc>cam</sc> decisions to later in the cancer trajectory when they have more energy to reflect on a broader spectrum of treatments. This delay is particularly evident in patients who have received limited support from their oncology health professionals in the <sc>cam</sc> decision-making process. The <sc>cam</sc> therapies chosen by these patients are typically those that fall within the realm of supportive care (for example, massage, relaxation therapy) and have been associated with positive psychosocial outcomes ##REF##17609997##14##,##REF##14528705##18##.</p>", "<p>Second, cancer patients who have a high level of trust in the conventional health care system engage in a “playing it safe” decision-making process in which the advice of their oncologists is privileged throughout the cancer trajectory. Only <sc>cam</sc> therapies that can be easily incorporated into their conventional treatment protocol are chosen. Frequently, these patients perceive their cancer diagnosis to be “too serious to play around with” and are hesitant to use any therapies, particularly natural health products, that may negatively interact with their conventional treatment ##REF##17609997##14##,##REF##7720049##42##.</p>", "<p>In contrast, a third group of cancer patients is able to “bring it all together” and make treatment decisions that incorporate <sc>cam</sc> as part of their treatment plan with minimal conflict and anxiety. These individuals often report having a life-long commitment to <sc>cam</sc> use that precedes their diagnosis, and they believe that <sc>cam</sc> therapies are natural, supportive of the body’s innate ability to heal, and better able to holistically address physiologic and psychosocial needs than conventional care can ##REF##17609997##14##,##REF##17683580##35##,##REF##16470237##43##. However, some of these patients have described feeling “pushed” towards <sc>cam</sc> as result of their beliefs about conventional cancer treatments being “toxic,” “poisonous,” or immunosuppressive ##REF##16282504##21##,##REF##15911931##37##. Still others have turned toward <sc>cam</sc> because of their dissatisfaction with conventional care, including the quality and quantity of their interactions with health professionals, the adverse effects of conventional treatment, and their experiences with ineffective therapies ##REF##14672107##16##,##REF##17683580##35##,##REF##15911931##37##,##REF##16880427##44##,##REF##12470448##45##. These latter patients are most at risk of abandoning conventional care in lieu of alternative treatments.</p>", "<title>2.2.4 Revisiting the CAM Decision</title>", "<p>As patients move through the cancer trajectory and reach the end of their conventional treatment protocol, many revisit the <sc>cam</sc> decisions made following their diagnosis and during adjuvant treatment. For some individuals, this reflection is a consequence of feeling as if they have “fallen off the cliff” as they lose the frequent contact they have had with their oncology health professionals during active treatment ##REF##17283742##46##. Adding <sc>cam</sc> therapies to their health care repertoire allows these individuals to feel as if they are “still doing something” and helping their bodies recover from the trauma of chemotherapy and radiation. Other cancer patients see the end of conventional treatment as opening the door to specific <sc>cam</sc> therapies, especially natural health products, which were discouraged while they were undergoing treatment because of fears of negative interactions ##REF##17609997##14##. Lastly, the completion of conventional treatment also liberates patients from what can be an extensive commitment of time and physical and emotional energy; it gives them the opportunity to explore new treatment options that were too overwhelming to consider at the beginning of the illness experience.</p>", "<p>Cancer patients also revisit their <sc>cam</sc> decisions in response to new information received regarding disease progression and prognosis ##REF##17609997##14##,##REF##14528705##18##. The identification of a recurrence or metastases can result in some individuals returning to the taking-stock phase of the decision-making process to reconsider their <sc>cam</sc> decisions and perhaps expand their search for <sc>cam</sc> therapies, including more alternative forms of treatment. Others interpret disease progression as a sign of ineffectiveness, and they withdraw or significantly alter their <sc>cam</sc> regimen. Conversely, results suggestive of remission or tumour regression can encourage some individuals to maintain or increase their use of <sc>cam</sc>.</p>", "<p>Lastly, the growing field of <sc>cam</sc> research is providing new data on the efficacy and safety of specific <sc>cam</sc> therapies on almost a daily basis. This information is rapidly translated to cancer patients and oncology health professionals through the media, scientific journals, and research-based databases [for example, Natural Standards (<ext-link ext-link-type=\"uri\" xlink:href=\"www.naturalstandard.com\">www.naturalstandard.com</ext-link>)]. For individuals for whom scientific evidence is an important consideration in their treatment decisions, such information may encourage their exploration of promising <sc>cam</sc> therapies or their withdrawal from products or practices suggested to be ineffective or potentially harmful ##REF##17609997##14##,##REF##18048882##38##.</p>" ]
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[ "<title>Objective</title>", "<p>In this paper, we set out to describe the personal and social contexts of treatment decisions made by cancer patients concerning complementary and alternative medicine (<sc>cam</sc>) and also the process through which cancer patients reach <sc>cam</sc> decisions throughout the cancer trajectory.</p>", "<title>Methods</title>", "<p>We selected and reviewed a variety of <sc>cam</sc> decision-making models published in the past 10 years within the Canadian health literature.</p>", "<title>Results</title>", "<p>The <sc>cam</sc> decision-making process is influenced by a variety of sociodemographic, disease-related, psychological, and social factors. We reviewed four main phases of the <sc>cam</sc> decision-making process:</p>", "<p> Taking stock of treatment options</p>", "<p> Gathering and evaluating <sc>cam</sc> information</p>", "<p> Making <sc>cam</sc> decisions</p>", "<p> Revisiting the <sc>cam</sc> decision</p>", "<p>Immediately following diagnosis, cancer patients become interested in taking stock of the full spectrum of conventional and <sc>cam</sc> treatment options that may enhance the effectiveness of their treatment and mediate potential side effects. Information about <sc>cam</sc> is then gathered from numerous information sources that vary in terms of credibility and scientific legitimacy, and is evaluated. When making a decision regarding <sc>cam</sc> options, patients attempt to make sense of the diverse information obtained, while acknowledging their beliefs and values. The <sc>cam</sc> decision is often revisited at key milestones, such as the end of conventional treatment and when additional information about disease, prognosis, and treatment is obtained.</p>", "<title>Conclusions</title>", "<p>The <sc>cam</sc> decision-making process is a dynamic and iterative process that is influenced by a complex array of personal and social factors. Oncology health professionals need to be prepared to offer decision support related to <sc>cam</sc> throughout the cancer trajectory.</p>" ]
[ "<title>3. SUMMARY</title>", "<p>Our review of the <sc>cam</sc> decision-making process (see ##FIG##0##Figure 1##) highlights the fact that <sc>cam</sc> decisions are highly individualized, complicated, and multifaceted, and that they involve dynamic processes that vary throughout the cancer trajectory. The decision to use—or not to use—<sc>cam</sc> is not a one-time whimsical decision; instead, it is a decision that leads cancer patients to reflect on their unique personal and social context and to ponder how <sc>cam</sc> may fit with their values, beliefs, and specific health care needs. As the individual and social contexts of patients change, the appropriateness of select <sc>cam</sc> therapies in their treatment regimen also changes. Decisions about <sc>cam</sc> are not static; rather, they are dynamic entities that require assessment and follow-up by health professionals throughout a patient’s illness.</p>", "<p>Recognizing that <sc>cam</sc> decision-making can be an anxiety-laden experience for patients suggests the need for ongoing decision and information support by oncology health professionals from diagnosis through survivorship—and for some individuals, through end of life. Research indicates that <sc>cam</sc> decisions coincide with patients’ decisions about conventional treatment, with many individuals regarding conventional and <sc>cam</sc> therapies as part of the same continuum of care ##REF##17609997##14##,##REF##14528705##18##. As a consequence, cancer patients often assume that discussions about treatment options with their oncology health professionals will include both conventional and <sc>cam</sc> therapies. However, oncology health professionals are often hampered in these types of discussions because of the limited evidence that supports the safety and efficacy of many <sc>cam</sc> therapies in the context of conventional cancer care and because of the lack of integration of <sc>cam</sc> education into their professional training programs ##REF##12020190##47##,##REF##14722590##48##.</p>", "<p>Despite the need for additional research and professional education on <sc>cam</sc>, it is essential that oncology health professionals begin a dialogue with patients that focuses not just on individual <sc>cam</sc> therapies, but also on how to make treatment decisions that acknowledge scientific evidence and each patient’s beliefs, values, and sociocultural contexts.</p>", "<p>A dialogue regarding <sc>cam</sc>-related treatment decisions would ideally be an ongoing discussion that corresponds with key milestones in a patient’s illness, such as diagnosis, end of conventional treatment, and recurrence or metastasis. A key element of patient-centered decision support is an assessment of an individual’s preferred level of engagement in the decision-making process and preferred degree of involvement of their oncology health professionals. Further, recognizing the individual contexts of patients, including pre-existing beliefs about healing, previous experiences with conventional medicine and <sc>cam</sc>, and the influence of significant others and the surrounding community, is also essential. Assessing patient goals related to <sc>cam</sc> use is also important and can illuminate important belief systems and experiences that need to be acknowledged in tailoring decision support. Although some patients may be interested in using <sc>cam</sc> for curative reasons and to address the side effects of conventional treatment, other patients may be pursuing <sc>cam</sc> to preserve hope in the face of a terminal prognosis. In addition, acknowledging the potential barriers to <sc>cam</sc> use can help patients consider the fuller implications of using <sc>cam</sc> therapies beyond the physiologic effect and their own physical limitations in using a diverse range of therapies.</p>", "<p>Verhoef, Boon, and Page ##REF##18769581##40## provide further suggestions regarding how oncology health professionals can communicate with patients about <sc>cam</sc>. As research in this field continues, we can expect to see evidence-based decision-support strategies emerge, such as decision-support aids and counselling strategies that will assist patients and professionals alike in the <sc>cam</sc> decision-making process.</p>" ]
[ "<title>Acknowledgments</title>", "<p>4. ACKNOWLEDGMENTS</p>", "<p>The research team responsible for this paper, established in 2000 as part of the Sociobehavioural Cancer Research Network of the Centre for Behavioural Research and Program Evaluation, University of Water-loo, has benefited from the support of the National Cancer Institute of Canada, with funds from the Canadian Cancer Society. The Cancer and CAM Research Team is one of five research teams devoted to studying the sociobehavioural aspects of cancer to better support patients and ultimately alleviate the burden of cancer.</p>", "<p>A Canadian Cancer Society Research Scientist Award (funded by the Prostate Cancer Research Initiative) and grant funding from the Canadian Institutes of Health Research (<sc>cihr</sc>) and the Canadian Breast Cancer Research Alliance help to support Dr. Balneaves’s work, and a Canada Graduate Scholarship (Doctoral) from <sc>cihr</sc> and an Incentive Award from the Alberta Heritage Foundation for Medical Research help to support Ms. Weeks’s work.</p>" ]
[ "<fig id=\"f1-co15_s2ps094\" position=\"float\"><label>FIGURE 1</label><caption><p>The complementary and alternative medicine (<sc>cam</sc>) decision-making process.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"co15_s2ps094f1\"/>" ]
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[{"label": ["24"], "surname": ["Broom", "Tovey"], "given-names": ["A", "P"], "article-title": ["The dialectical tension between individuation and depersonalization in cancer patients\u2019 mediation of complementary, alternative and biomedical cancer treatments"], "source": ["Sociology"], "year": ["2007"], "volume": ["41"], "fpage": ["1021"], "lpage": ["39"]}, {"label": ["32"], "surname": ["Lupton", "Lupton"], "given-names": ["D", "D"], "article-title": ["Theoretical perspectives on medicine and society"], "source": ["Medicine As Culture"], "edition": ["2nd ed"], "publisher-loc": ["London"], "publisher-name": ["Sage"], "year": ["1994"], "fpage": ["5"], "lpage": ["21"]}, {"label": ["33"], "surname": ["Seale"], "given-names": ["C"], "source": ["Health and Media"], "publisher-loc": ["London"], "publisher-name": ["Sage"], "year": ["2003"]}]
{ "acronym": [], "definition": [] }
48
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s94-s100
oa_package/c0/9d/PMC2528558.tar.gz
PMC2528559
18769577
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[ "<p>Research on the factors that promote healing of the body through mind and spirit is at a very early stage. Reliance on experimental designs seems premature; we need much more exploratory research to identify relevant variables and useful therapeutic approaches before applying to them the same methods used to evaluate drugs. The Healing Journey is a program that has been in operation since 1982 at the Princess Margaret Hospital, Toronto, Ontario. Observational data collection, followed by qualitative analysis has demonstrated benefits for many cancer patients.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/269/252\">http://www.current-oncology.com/index.php/oncology/article/view/269/252</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s107.es37-s107.es41
oa_package/84/a0/PMC2528559.tar.gz
PMC2528560
18769578
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[ "<p>Many oncology patients are empowering themselves to self-treat with herbs, nutritional supplements, and mind–body techniques. Other practitioners, such as acupuncturists, are becoming involved in the supportive care of cancer patients. Government research agencies are supporting studies that evaluate complementary therapies. This educational article provides an overview of the challenges in designing appropriate studies of complementary and alternative therapies, evaluating the results, and regulating implementation of useful therapies.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/273/242\">http://www.current-oncology.com/index.php/oncology/article/view/273/242</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s107.es53-s108.es63
oa_package/87/49/PMC2528560.tar.gz
PMC2528561
18769579
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[ "<p>Established in 1993 after a 2-year consultation between professionals and cancer patients, the Lynda Jackson Macmillan Centre (<sc>ljmc</sc>) has been a catalyst for change in the United Kingdom. The Centre began with a small core staff in a purpose-built building next to a cancer centre, networking with outreach workers in 12 surrounding hospitals, with a mission to improve information, communication, and support for cancer patients. Since 1996, the <sc>ljmc</sc> model has been adopted and developed by the charity Macmillan Cancer Support and has been spread to more than 60 units across the United Kingdom and Australia.</p>", "<p>Introducing complementary therapies (<sc>cam</sc>s) to a cancer centre was a particular early challenge. Establishing a shared understanding of the role of complementary therapies and developing nationally accredited written information about them, credible recruitment and governance procedures for therapy practitioners, agreed outcome measures, and peer-reviewed evaluation and research have all been important in engaging cancer physicians and managers; however, charitable funding is still required to support free access to most complementary therapies.</p>", "<p>An integrated supportive care service for cancer patients begins with a shift in the culture of cancer treatment organizations, moving from a professional-centred to a patient-centred agenda. Real reach and impact requires “new” ideas and services to be integrated into the routine practice of the cancer care delivery organizations. A key lesson learned over the last 15 years is that an integrated support centre must continually adapt to be viable. Sustaining meaningful user guidance is a particular challenge. Support for self-management and the testing and development of <sc>cam</sc> services are growing parts of the portfolio.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/275/282\">http://www.current-oncology.com/index.php/oncology/article/view/275/282</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s108.es64-s108.es67
oa_package/86/60/PMC2528561.tar.gz
PMC2528562
18769580
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[ "<title>YOUR PART IN THE DISCUSSION</title>", "<p>Readers are invited to evaluate this special supplement by answering a short survey on the <italic>Current Oncology</italic> Web site. The editors appreciate your interest in this supplement and will similarly appreciate receiving your comments. We would like to publish those comments on the Web site; the option of anonymity for specific comments is available.</p>" ]
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[ "<p>We are pleased to present this special supplement of <italic>Current Oncology,</italic> which is generously financed by a grant from the Lotte and John Hecht Memorial Foundation. Integrative oncology is both a science and a philosophy that focuses on the complexity of the health of cancer patients and proposes a multitude of approaches to accompany the conventional therapies of surgery, chemotherapy, molecular therapeutics, and radiotherapy to facilitate health. As such, integrative oncology involves thinking outside the box, and so we are indeed fortunate to have attracted a plethora of manuscripts from innovative leaders both of Canadian and of international cancer treatment and control services.</p>", "<p>In line with the modern approach to media communications, we have “integrated” this issue with manuscripts published on the <italic>Current Oncology</italic> Web site. Readers will also have the opportunity to view and listen to slide presentations submitted to the Integrating Wellness into Cancer Care Conference held at the University of Toronto, October 4–5, 2007. The conference was organized by Dr. Paul Fortin in memory of his wife Dr. Veronique Benk. Veronique was a radiation oncologist, clinician, and researcher who specialized in breast cancer, and she was devoted to her patients. Her personal experience of breast cancer and myeloid leukemia was transformative, and she embraced a wider approach to cancer treatment. That approach prioritized state-of-the-art medical care with a new emphasis on spirituality, wellness, and quality of life. The conference was sponsored by a non-restricted educational grant from the Lotte and John Hecht Memorial Foundation, CV Technologies, Astra/Zeneca Pharmaceuticals, Pfizer Pharmaceuticals, Novartis Pharmaceuticals, Wellspring, and the University of Toronto Department of Radiation Oncology, with special thanks to Drs. Mary Gospodarowicz and Pamela Catton.</p>", "<title>PURSUING THE INTEGRATIVE PATH</title>", "<p>So, what is <sc>cam</sc>?</p>", "<p>Complementary and alternative medicine (<sc>cam</sc>) is an umbrella term encompassing a group of diverse medical and health care systems, practices, and products that are not always considered part of conventional medicine ##UREF##0##1##. Yet in traditional healing systems, the power of the mind and other non-pharmaceutical interventions are integral to the treatment of ill health.</p>", "<p>Promotion, maintenance, monitoring, or restoration of health is the goal of <sc>cam</sc> use. According to the U.S. National Cancer Institute, <sc>cam</sc> includes whole medical systems (for example, Traditional Chinese Medicine), mind–body medicine (meditation, for instance), biologically-based practices (natural health products, among others), manipulative and body-based therapies (for example, massage), and energy therapies (<italic>qi gong,</italic> for instance) ##UREF##0##1##.</p>", "<p>An approach is called “complementary” when it is used adjunctively to conventional treatments, with the intent to enhance the body’s natural abilities to heal. Modalities used instead of conventional treatments are labelled “alternative” therapies. The term “alternative” means that treatments outside of conventional medicine are used to treat the disease. Depending on intent, some therapies can be considered either complementary or alternative. It is therefore important always to clarify the intent of the patient considering <sc>cam</sc> use. Studies have consistently found that most cancer patients use <sc>cam</sc> as a complement to their conventional treatment ##REF##10893280##2##,##UREF##1##3##.</p>", "<p>The lack of consensus around terminology and definitions makes it difficult to accurately assess the prevalence of <sc>cam</sc> use. For example, the list of products and therapies designated as <sc>cam</sc> continually changes as therapies that are proved to be safe and effective are integrated into conventional health care and as new, untested ones emerge ##REF##17093910##4##.</p>", "<p>An increasing number of people living with cancer are using therapies in addition to those prescribed by conventional health care providers ##REF##11331323##5##,##REF##14767750##6##. In the literature, an explosion of <sc>cam</sc> surveys has suggested that utilization rates by cancer patients fall between 40% and 60% ##REF##15699021##7##. For example, with the inclusion of prayer in the <sc>cam</sc> list, the prevalence of <sc>cam</sc> use in the United States is estimated at 62%; minus prayer, it is about 36% ##REF##15188733##8##.</p>", "<p>Integrative oncology does not usually incorporate prayer into its definition of therapies, preferring to classify prayer in a religious domain, but the concept of prayer as a mind–body intervention illustrates the challenge of defining a <sc>cam</sc> therapy ##REF##10030348##9##. Throughout the cancer trajectory, higher use of <sc>cam</sc> is consistently found during chemotherapy to mitigate adverse effects, after conventional cancer treatment to boost energy, during survivorship to foster wellness, and during the last months of life to control symptoms ##REF##12175424##10##.</p>", "<p>For this special issue, the term “<sc>cam</sc>” is being used because of its worldwide recognizability as a label linked to traditional medicine. Given the specific focus of this issue on integrative medicine, <sc>cam</sc> uniquely refers to the complementary side—in other words, to something used in conjunction with conventional oncology treatments. The powerful synergy of the holistic approach of complementary medicine together with biomedical cancer treatments is central to the purpose of integrative oncology. The combination permits dysfunctional physical, mental, emotional, and spiritual symptoms to be treated and thereby fully addresses the healing needs of cancer patients in a tailored fashion.</p>", "<title>IN THIS SUPPLEMENT</title>", "<p>In his article, Dr. Simon Sutcliffe discusses the importance of combining science and evidence-based medicine with individual and societal values and of integrating values into a process of holistic care. To quote Sutcliffe, the goal is ultimately “to achieve a responsive, efficient, effective, and sustainable system to improve health and control cancer (as a process, not as an event).” He expands his argument that complex problems require a more sophisticated approach than can be achieved through scientific reductionism, and that the multifaceted perspectives of the patient must be part of the decision-making process in designing health policy.</p>", "<p>Drs. Jacqueline Bender and Alejandro Jadad extend the perspective of patients’ values and education through individuals socializing over the Internet. They discuss the potential for empowerment and how that empowerment may modify the relationships of patients with their health care providers. Dr. Alastair Cunningham emphasizes the existential crisis that patients endure when they receive a diagnosis of cancer and speaks of the importance of psychological healing as part of the process of restoring health. And Dr. Mary Vachon expands on the notion of spirituality and meaning for cancer patients in her article “The Soul’s Wisdom: Stories of Living and Dying” (e-manuscript on the Web).</p>", "<p>How is outcome to be evaluated in an integrative oncology program? Dr. Stephen Sagar discusses various health outcome domains and patient satisfaction, and points to some validated measurement tools. For a detailed source of measurement tools, readers are also directed to the new online IN-CAM Outcomes Database (<ext-link ext-link-type=\"uri\" xlink:href=\"www.outcomesdatabase.org\">www.outcomesdatabase.org</ext-link>) organized by Dr. Marja Verhoef.</p>", "<p>Dr. Sagar and Dr. Raimond Wong together provide an educational article on integrative oncology research and regulation (e-manuscript on the Web). Further e-manuscripts recount the experiences of two international integrative oncology programs: Dr. Jane Maher, Chief Medical Officer of MacMillan Cancer Support, describes the Lynda Jackson Macmillan Centre at Mount Vernon Hospital in Northwood, United Kingdom, and Dr. Gary Deng describes the Integrative Medicine Service at Memorial Sloan–Kettering Hospital, headed by Dr. Barrie Cassileth, in New York City, United States. Both centres have pioneered similar models in other countries.</p>", "<p>Attendees of the University of Toronto Integrating Wellness into Cancer Care Conference participated in a workshop titled “How to Put Wellness on the Prescription Pad”, and in another e-manuscript, Dr. Fortin presents a summary of the discussions that took place.</p>", "<p>A group of manuscripts from the Cancer, Complementary and Alternative Medicine (<sc>ccam</sc>) team, a multidisciplinary group of Canadian scientists, presents some of their work in evaluating the role of complementary therapies for cancer care.</p>", "<p>Dr. Ann Leis discusses the scope of integrative oncology and the need for evidence to support the integration of complementary therapies into cancer care. She concludes that “a whole-systems framework to the development of the evidence base for integrative oncology can guide the development of evidence that respects the complex nature of many complementary and integrative practices and their underlying principles of care delivery.”</p>", "<p>In her manuscript “Talking to Cancer Patients About Complementary Therapies,” Marja Verhoef concludes that discussing <sc>cam</sc> with patients is the physician’s responsibility and that it will facilitate evidence-based, patient-centred cancer care.</p>", "<p>Dr. Lynda Balneaves uses research from the Canadian health literature to address the issue of patient decision-making. In her discussion, she says that “decision to use, or not to use, <sc>cam</sc> is not a one-time whimsical decision; instead, it is a decision that leads cancer patients to reflect on their unique personal and social context and to ponder how <sc>cam</sc> may fit with their values, beliefs, and specific health care needs. As the individual and social contexts of patients change, the appropriateness of select <sc>cam</sc> therapies in their treatment regimen also changes. Decisions about <sc>cam</sc> are not static; rather, they are dynamic entities that require assessment and follow-up by health professionals throughout a patient’s illness.”</p>", "<p>Why do some patients decline conventional evidence-based therapies and pursue alternative non-proven options? Dr. Verhoef concludes that “poor doctor–patient communication, the emotional impact of the cancer diagnosis, perceived severity of conventional treatment side effects, a high need for decision-making control, and strong beliefs in holistic healing appear to affect the decision by patients to decline some or all conventional cancer treatments.” The “tendency by doctors to dichotomize patient decisions as rational or irrational may interfere with the ability of the doctors to respond with sensitivity and understanding,” she continues.</p>", "<p>Doctor of naturopathic medicine Dugald Seeley, together with Dr. Doreen Oneschuk, discusses the important topic of interactions of natural health products with biomedical cancer treatments.</p>", "<p>These subjects require knowledgeable physicians and a modification of medical school curricula, a task that is being undertaken by the CAM in UME Project (<ext-link ext-link-type=\"uri\" xlink:href=\"www.caminume.ca/about.html\">www.caminume.ca/about.html</ext-link>) and the Consortium of Academic Health Centers for Integrative Medicine (<ext-link ext-link-type=\"uri\" xlink:href=\"www.imconsortium.org\">www.imconsortium.org</ext-link>).</p>", "<p>What, then, are the current integrative practices of Canadian health care professionals? Dr. Alison Brazier uses an interpretive–description research design, with a series of in-depth qualitative interviews, to highlight two main strategies: acting as an integrative cancer guide and collaborating with other health care professionals.</p>", "<p>Mind–body techniques derived from Eastern mysticism have become an important part of some integrative oncology programs. In a research paper that evaluates an Iyengar yoga program, Ms. Meghan Duncan finds an overall improvement in the well-being of cancer patients (e-manuscript on the Web). This research contributes further to the evidence that some techniques derived from Eastern spirituality can help some patients cope with cancer and its treatment.</p>", "<title>THE WAY FORWARD</title>", "<p>The future of complementary therapies lies in mainstream medicine, but only if those therapies are based on scientific understanding and evidence of effectiveness. A willingness to discard therapies that fail to be proved effective in clinical studies is vital. Accepted therapies must also be seen to be safe and cost-effective.</p>", "<p>A comprehensive cancer program should integrate surgery, chemotherapy, radiotherapy, and targeted molecular therapies with meaningful psycho-spiritual, psychological, and physical supportive therapies, and an investigative program of botanicals. New technology is facilitating the quality-controlled preparation of simple and complex mixtures of phytochemicals that are being investigated as biologic response modifiers. International collaboration between North American, European, and Asian universities and interested pharmaceutical companies is encouraging the development of multi-targeted therapies using traditional herbs. During a recent visit by S.M.S. to Fudan University in Shanghai, China, that institution ratified a collaborative research agreement with the M.D. Anderson Hospital (the largest international cancer centre) and signed a new collaborative agreement with the Institut Gustav Roussy (Europe’s largest cancer centre), under the umbrella of integrative oncology. Video extracts of the Shanghai conference of the Society for Integrative Oncology can be found on the <italic>Current Oncology</italic> Web site. Further information on the Society for Integrative Oncology and its conferences (including abstracts from its recent conference held in Shanghai) can be found at <ext-link ext-link-type=\"uri\" xlink:href=\"www.integrativeonc.org\">www.integrativeonc.org</ext-link>.</p>" ]
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[{"label": ["1"], "collab": ["National Institutes of Health, National Center for Complementary and Alternative Medicine ("], "sc": ["nccam", "cam", "cam"], "article-title": ["Health information > "], "publisher-loc": ["Gaithersburg, MD"], "publisher-name": ["NCCAM"], "year": ["February 2008"], "comment": ["[Available at: "], "ext-link": ["nccam.nih.gov/health/whatiscam"]}, {"label": ["3"], "surname": ["Lewith", "Walach", "Jonas", "Lewith", "Walach", "Jonas"], "given-names": ["G", "H", "WB", "G", "H", "WB"], "article-title": ["Balanced research strategies for complementary and alternative medicine"], "source": ["Clinical Research in Complementary Therapies: Principles, Problems and Solutions"], "publisher-loc": ["Edinburgh"], "publisher-name": ["Churchill Livingstone"], "year": ["2002"]}]
{ "acronym": [], "definition": [] }
10
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s71-s73
oa_package/8f/9c/PMC2528562.tar.gz
PMC2528563
18769581
[ "<title>1. INTRODUCTION</title>", "<p>Studies report that most patients undergoing cancer treatment also choose to use selected forms of complementary and alternative medicine (<sc>cam</sc>), including natural health products ##REF##10893280##1##,##REF##10833691##2##. Reasons cited by cancer patients for <sc>cam</sc> use include treating the cancer, managing treatment side effects, enhancing quality of life and well-being, boosting the immune system, maintaining hope, and having more control over their cancer care ##REF##10833691##2##–##REF##16282504##4##.</p>", "<p>It is important that physicians treating cancer patients know whether their patients are using <sc>cam</sc> and, if so, why. First, physicians need to know because of the possibility of direct adverse events associated with the use of <sc>cam</sc>. Second, interaction effects between conventional medicine and <sc>cam</sc> are possible, and the harms and benefits of <sc>cam</sc> may be misattributed to conventional treatment and thus may complicate treatment regimens. Third, patients may delay the use of conventional treatment when using <sc>cam</sc>. Finally, knowing why patients are using <sc>cam</sc> may provide important information about beliefs, values, expectations, and hopes on the part of the patient and will facilitate building a trusting relationship that will enhance the delivery of patient-centred cancer care.</p>", "<p>However, research has shown that 40%–77% of patients who use <sc>cam</sc> therapies do not disclose their use of or interest in <sc>cam</sc>, or their desire to use <sc>cam</sc>, to their physicians because of concerns that the physicians will react negatively or will dismiss their questions ##REF##15561518##5##–##REF##10386489##7##. Patients may also think that physicians do not need to know that they are using <sc>cam</sc>, because the patients may believe that <sc>cam</sc> therapies are natural, completely safe, and not within the physicians’ scope of practice. Finally, patients often do not tell, simply because physicians do not ask about <sc>cam</sc> use.</p>", "<p>In this paper, we discuss the ethical and legal obligations of physicians to discuss <sc>cam</sc> use in an oncology setting, and we provide practical advice on how patient–provider communication about <sc>cam</sc> can be improved.</p>" ]
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[ "<title>2. DISCUSSION</title>", "<title>2.1 Obligation of Physicians to Discuss CAM</title>", "<p>Physicians have both ethical and legal obligations to their patients, including the obligation to respect patient autonomy. Operationally, respecting patient autonomy in the context of treatment decision-making means allowing patients to make choices ##REF##12650001##8##. However, choice is meaningless if it is not made in light of all relevant information and advice ##REF##12649999##9##. Although patients are the ones who must make the treatment decisions, physicians have the duty to inform patients about all therapeutic options, including <sc>cam</sc>. This means that physicians must be prepared to provide information and advice about</p>", "<p> benefits and likely outcomes of treatment,</p>", "<p> risks involved in treatment,</p>", "<p> possibility and probability of complications, and</p>", "<p> side effects and alternative treatment options.</p>", "<p>This information is needed to meet the traditional ethical principles of non-maleficence (do no harm) and beneficence (offer a benefit) ##REF##16679948##10##. Specifically for <sc>cam</sc>, the obligation to provide information and advice means that where risks are unknown and benefits are uncertain, it is necessary to highlight this absence of information.</p>", "<p>Canadian courts have been very liberal and expansive in interpreting the disclosure obligations of physicians. In the context of informed consent, physicians are legally and ethically required to provide patients with detailed information about the evidence (for and against) the possible efficacy of treatments and to discuss costs, risks, and how a given treatment compares with other therapeutic options ##REF##12141221##11##,##REF##11606143##12##. The informed consent approach applies whether the recommended treatment is labelled biomedical or <sc>cam</sc>, and it raises the question of whether physicians must discuss <sc>cam</sc> treatment options to fulfil the informed consent requirement to compare a given treatment option with other therapeutic options.</p>", "<p>It has been argued that an exploration of <sc>cam</sc> treatment options with patients is necessary, especially when information about <sc>cam</sc> options will be material—that is, significant—to a decision about a conventional treatment. Thus, if a patient’s decision about pursuing a particular biomedical treatment is likely to be influenced by knowing about specific <sc>cam</sc> treatments (including evidence of their safety, efficacy, and cost), then a physician has an obligation to include a discussion of these <sc>cam</sc> options as part of the informed consent process ##REF##11606143##12##,##REF##17309810##13##. Given the widespread use of <sc>cam</sc> among Canadians diagnosed with cancer, it appears reasonable to assume that <sc>cam</sc> options will be material to many patients. In addition, perhaps the only way to determine if <sc>cam</sc> options are relevant factors in the decision-making processes of patients is to open a dialogue about the issues.</p>", "<p>There is increasing consensus in the literature about the importance of fully disclosing detailed information about the risks and benefits of <sc>cam</sc> interventions, including clear explanations of what is not known about them ##REF##12141221##11##–##REF##17309810##13##. The focus appears to be on protecting patients from physicians that promote <sc>cam</sc> products and therapies beyond what is believed by others to be supported by scientific evidence. Legally, physicians are required to practice in accordance with the “standard of care,” which generally refers to “the level of care the average and prudent health care professional in a given community would provide” ##REF##17309810##13##. This standard changes over time and across cultural contexts. Although therapies with scientific evidence of safety and efficacy are unlikely to be judged outside the standard of care, scientific evidence is not the only criterion upon which such judgments are made.</p>", "<p>In today’s culture of evidence-based practice, scientific evidence is becoming increasingly important ##REF##12141221##11##, but clinical judgment and patient values are important, too ##UREF##0##14##. Thus, physicians who provide or recommend <sc>cam</sc> therapies for which there is little evidence could leave themselves open to charges of medical malpractice ##REF##12141221##11##,##REF##17309810##13##. In contrast, requirements to fully discuss <sc>cam</sc> options as “alternative” treatment options when recommending a conventional biomedical treatment may soon become standard practice.</p>", "<p>Adams <italic>et al.</italic>\n##REF##12379066##15## identified a wide range of patient-and physician-related factors that affect decision-making and subsequent use of <sc>cam</sc>, including</p>", "<p> severity of the illness,</p>", "<p> curability with conventional treatment,</p>", "<p> side effects of conventional treatment,</p>", "<p> quality of evidence of safety and efficacy of <sc>cam</sc>,</p>", "<p> degree of understanding of risks and benefits,</p>", "<p> knowing and voluntary acceptance of risks by the patient, and</p>", "<p> commitment to <sc>cam</sc> use by the patient.</p>", "<p>Clearly, the need for physicians to assist patients in treatment decision-making is high and requires more than being informed about <sc>cam</sc>. Physicians also need to have effective (and non-judgmental) communication skills to manage the discussion. Patient–physician communication plays a crucial role, because these issues are best resolved by means of shared decision-making between patient and physician, where sufficient information is exchanged to create a consensus approach to deciding on the optimal clinical course.</p>", "<title>2.2 How to Discuss CAM with Patients</title>", "<p>It is becoming increasingly clear that patients have many legitimate needs and concerns that are not being met by conventional medicine. By adhering to the ethical criteria for informed decision-making and by honouring patient autonomy, physicians should be able to engage in open discussions with their patients about <sc>cam</sc> and to enable their patients to make sound decisions ##REF##11795082##6##.</p>", "<p>Not communicating with patients about <sc>cam</sc> may not only result in decreased trust within the therapeutic relationship, but also in selection by the patients of harmful, ineffective, and costly <sc>cam</sc> therapies. Patients who use <sc>cam</sc> may have unidentified needs or may be dissatisfied with the conventional care they are receiving. Once the issue of <sc>cam</sc> use is raised, the unidentified needs or dissatisfaction may come to the forefront and be addressed. However, the fact that relatively few physicians talk with their patients about <sc>cam</sc> suggests that these conversations are not easy for physicians. Lack of training in <sc>cam</sc>, limited training in communication skills, limited knowledge about <sc>cam</sc>, lack of scientific evidence about the risks and benefits of <sc>cam</sc>, and skepticism towards <sc>cam</sc> all appear to prevent physician engagement in such discussions.</p>", "<p>Tasaki ##REF##12112481##16## found that patients identified these major barriers to successful discussions of <sc>cam</sc>:</p>", "<p> perceived indifference or opposition towards <sc>cam</sc> by the physician,</p>", "<p> emphasis on scientific evidence by the physician, and</p>", "<p> anticipation on the part of the patient of a negative response from the physician.</p>", "<p>Initiating communication about <sc>cam</sc> is crucial. ##TAB##0##Table I## summarizes a number of suggestions on how to encourage patients to talk about <sc>cam</sc>. Foley ##REF##12100100##19## underscores the need for such conversations by talking about “the need for us as oncology professionals to ‘Seek first to understand,’ to be open and to learn from our patients to serve them better.”</p>", "<p>Eisenberg ##REF##9214254##20## was one of the first to propose a step-by-step strategy that conventionally trained physicians could use to proactively discuss <sc>cam</sc> use. This strategy involves a formal discussion of the treatment preferences and expectations of patients, the maintenance of symptom diaries, and follow-up visits to monitor for potentially harmful situations. In the absence of medical and legal guidelines, the proposed management plan emphasizes patient safety, the need for documentation in patient records, and the importance of shared decision-making. Although this strategy may be impractical and cumbersome in practice, Eisenberg should be credited for highlighting essential elements in physician–patient communication and for focusing on documentation and follow-up.</p>", "<p>Informally, the five major steps to intervention—“ask, advise, assess, assist, and arrange” (5 A’s) ##REF##16049088##21##—have been mentioned as important guidelines for communicating about <sc>cam</sc>, but a more focused approach has been presented by Cohen <italic>et al.</italic>\n##REF##17309810##13##. They suggest exploration (of the patient’s main issues), validation (acknowledge and commend the patient for seeking to resolve symptoms and improve health), empathy (with the patient’s desire to do everything possible), evaluation (consult with colleagues and other experts, and consult reputable sources of information), communication (share findings with the patient), and documentation of the conversations with the patient and of the patient’s progress as previously highlighted by Eisenberg ##REF##9214254##20##.</p>", "<p>Yet another perspective is provided by Frenkel <italic>et al.</italic>\n##REF##15813155##22##. These authors suggested that, to help cancer patients be truly informed and autonomous, physicians need to identify the patient’s beliefs, fears, hopes, and expectations; learn which conventional treatments have been tried, have failed, or have been rejected and why; make sure the patient understands the prognostic factors associated with his or her stage of disease, plus the potential benefits and harms of conventional medicine; acknowledge the patient’s spiritual and religious values and beliefs to understand how these affect health care choices; and assess the level of support that the patient has from friends, family, and community.</p>", "<p>These frameworks approach the issue of communication with patients from slightly different angles, yet all are important, and physicians will most likely use elements of all approaches depending on the particular situation.</p>", "<p>Implicitly, all models suggest that <sc>cam</sc> use should be inquired about from the beginning of contact with the patient, ideally before the patient starts using <sc>cam</sc>. This emphasis suggests that <sc>cam</sc> use should be made a regular part of history-taking. Physicians therefore need effective communication skills to fulfil a variety of roles, including collecting medical histories, answering patients’ questions, developing interpersonal relations, and suggesting treatment ##REF##12112481##16##. Although this need seems obvious, it is yet another demand on physicians working in often busy and stressful situations.</p>", "<p>Lastly, it is important to consider that, although disclosure of <sc>cam</sc> is essential, successful communication hinges on supporting patient autonomy even when the patient is making the decision to use a therapy of which a physician does not approve.</p>", "<title>2.3 Current and Future Trends in Patient–Provider CAM Discussion</title>", "<p>The importance of talking with patients about <sc>cam</sc> therapies is currently receiving much attention. Recently, the National Center for Complementary and Alternative Medicine (<sc>nccam</sc>) in the United States started the Time to Talk campaign ##UREF##3##23##, urging health care providers to talk about <sc>cam</sc>. The <sc>nccam</sc> Web site also includes tips on how to talk with patients. In addition, the <italic>British Medical Journal</italic> recently published a challenging editorial, “Wham, bam, thank you <sc>cam</sc>” ##REF##17901515##24##, highlighting the need to discuss <sc>cam</sc>; however, the author’s question, “Alternative medicine is wildly popular ... but what are we supposed to do about it?” raises the challenge of finding relevant, evidence-based information.</p>", "<p>Uncovering evidence-based information is especially difficult given the large number and heterogeneity of <sc>cam</sc> interventions. As a result, only a limited number of interventions have been adequately tested. Limited time in which to learn about <sc>cam</sc> and to discuss <sc>cam</sc>-related issues in patient consultations that are often already too short to address all patients’ concerns poses yet another challenge for physicians.</p>", "<p>Currently, no single resource contains comprehensive summaries of the evidence base of all <sc>cam</sc> treatments relevant for patients diagnosed with cancer. In addition, the available evidence changes almost daily. However, helpful information can be found in a number of places. ##TAB##1##Table II## includes a list of evidence-based <sc>cam</sc> resources that may help physicians when talking to patients about <sc>cam</sc>. Most are Web sites, because these are much easier to access (and are updated more regularly) than are books and articles. We recommend that physicians track the sources they access and find helpful, so that those sources are readily available when needed. Information on how to evaluate the wide range of <sc>cam</sc> information sources on the Web is available from <sc>nccam</sc> in the United States ##UREF##4##25##.</p>", "<p>Because patients may see <sc>cam</sc> practitioners for cancer and cancer-related symptoms, it is also beneficial to be informed about the <sc>cam</sc> practitioners in the local area who are seeing cancer patients. The Prince of Wales Foundation for Integrated Health in the United Kingdom has published several guides for patients using <sc>cam</sc>\n##UREF##5##26##. These guides include helpful information on finding <sc>cam</sc> practitioners and asking the right questions about those practitioners. An important aspect to assess is whether a given <sc>cam</sc> profession is regulated and whether a specific practitioner has adequate credentials.</p>", "<p>It will be important in the future to ensure that <sc>cam</sc> is a topic in medical education, because all graduating physicians will encounter this issue in their practice. The Canadian <sc>cam</sc> in <sc>ume</sc> (undergraduate medical education) Web site provides useful resources for those involved in teaching medical students about <sc>cam</sc>\n##UREF##1##17##. For physicians already in practice, continuing medical education may be a solution; however, most important is what can be learned from talking with patients: not only what they use, but also what their questions are.</p>" ]
[ "<title>3. CONCLUSIONS</title>", "<p>Many people have already been using <sc>cam</sc> before a cancer diagnosis, and they consider it to be part of their health care. It is important to note that it is not possible to provide evidence-based, patient-centred care without engaging in a discussion of <sc>cam</sc>, including an exploration of the patient’s beliefs. Patients may be reluctant to discuss <sc>cam</sc> because of a fear of rejection or because of their beliefs about the complete safety of <sc>cam</sc>. It is therefore important that physicians initiate discussion of the topic—ideally, early in the relationship (for example, during initial history-taking), before patients have made any decisions about <sc>cam</sc> treatments. It truly is time to talk about <sc>cam</sc> with patients.</p>" ]
[ "<title>Background</title>", "<p>To ensure the safety and effectiveness of cancer management, it is important for physicians treating cancer patients to know whether their patients are using complementary and alternative medicine (<sc>cam</sc>) and if so, why.</p>", "<title>Objective</title>", "<p>Here, we discuss the ethical and legal obligations of physicians to discuss <sc>cam</sc> use in an oncology setting, and we provide practical advice on how patient–provider communication about <sc>cam</sc> can be improved.</p>", "<title>Results</title>", "<p>Physicians have both ethical and legal obligations to their patients, including the obligation to respect patient autonomy. This latter obligation extends to use of <sc>cam</sc> by patients and needs to be addressed beginning early in the patient–provider relationship. Because lack of education in this field and lack of time during patient consultations are barriers to talking with patients about <sc>cam</sc>, we provide resources to facilitate such discussions. These resources include suggestions on how to discuss the topic of <sc>cam</sc> and a wide range of information sources.</p>", "<title>Conclusions</title>", "<p>Discussing <sc>cam</sc> with patients is the physician’s responsibility, and such discussion will facilitate evidence-based, patient-centred cancer care.</p>" ]
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[ "<title>Acknowledgments</title>", "<p>4. ACKNOWLEDGMENTS</p>", "<p>The research team responsible for this paper, established in 2000 as part of the Sociobehavioural Cancer Research Network of the Centre for Behavioural Research and Program Evaluation, University of Waterloo, has benefited from the support of the National Cancer Institute of Canada, with funds from the Canadian Cancer Society. The Cancer and CAM Research Team is one of five research teams devoted to studying the sociobehavioural aspects of cancer to better support patients and ultimately alleviate the burden of cancer.</p>" ]
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[ "<table-wrap id=\"tI-co15_s2ps088\" position=\"float\"><label>TABLE I</label><caption><p>Communicating with patients: keeping the door open##UREF##1##17##,##UREF##2##18##</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>What follows is a list of possible questions and issues that physicians can raise. Obviously, it is neither possible nor necessary to address all areas; however, even one good question may be the key to either opening the door to discussing <sc>cam</sc> or keeping the door open.</italic></th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1. Always ask about complementary and alternative medicine (<sc>cam</sc>) use—for example, “What else are you doing to take care of your cancer?” Ask in an open, non-judgmental way, and avoid using labels such as quackery, unscientific, and so on.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2. Watch for “non disclosing” clues: “You have read a lot about this. Have you seen other types of practitioners?”</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3. Give permission for the patient to raise the topic by asking, “Many of my patients are interested in trying complementary therapies. Have you used any other therapies for this problem?”</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">4. Check with patients about their explanatory models: “What do you think is causing your symptoms [or cancer (because many patients have strong opinions of causes of cancer)]?”</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5. Seek more information from patients and other sources: “Do you have any articles you can share with me?”</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> a. Be prepared for patients doing their own research.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> b. Be aware of what they are being told about <sc>cam</sc>.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6. Explore why patients are using <sc>cam</sc>, and learn about their beliefs and values. It is important to consider that</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> a. a great deal more than evidence goes into a patient’s decision to use <sc>cam</sc>.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> b. for many patients, care (enhancing well-being, easing suffering) is as important as cure.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">7. Discuss the patient’s treatment preferences and expectations.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">8. Review issues of efficiency and safety with respect to <sc>cam</sc>.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">9. Be frank about your level of understanding or knowledge. It is okay not to know everything about <sc>cam</sc>.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10. Support the patient in efforts to obtain answers to important questions about risk and benefit. Ask yourself:</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> a. Is the <sc>cam</sc> therapy really dangerous?</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> b. Does it prohibit necessary medical care?</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> c. Can you work within the patient’s belief system to provide good care?</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  If the answer to the last question is yes, the next steps include negotiation and education. If the answer is no, the next step would be to arrive at a mutually acceptable course of action.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">11. Discussing <sc>cam</sc> use does not mean that you are endorsing or promoting <sc>cam</sc> use.</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tII-co15_s2ps088\" position=\"float\"><label>TABLE II</label><caption><p>Resource books and Web sites</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>The Desktop Guide to Complementary and Alternative Medicine: An Evidence-based Approach</italic></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Ernst E, Pittler MH, Wider B, editors.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 2nd edition, 2006</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 480 pages, paperback</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Integrative Medicine: Principles for Practice</italic></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> (Chapter 23, pp. 535–549)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Kligler B, Lee R.2004</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 700 pages, hardcover</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Complementary and Alternative Medicine Secrets: Q&amp;As about Integrating CAM Therapies into Clinical Practice</italic></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> (Chapters 54 and 55, pp. 363–388)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Kohatsu W.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 2002</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 456 pages, paperback</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Integrative Medicine</italic></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(Section 13, pp. 809–899; evidence for all treatments is rated)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Rakel D.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 2nd edition, 2007</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 1238 pages, hardcover</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>The Oxford Handbook of Complementary Medicine</italic></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ernst E, Pittler MH, Wider B, Boddy K. </td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 2008</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 512 pages, paperback</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CAMline</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> <ext-link ext-link-type=\"uri\" xlink:href=\"www.camline.ca\">www.camline.ca</ext-link></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Center for Health and Healing</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> (a service of Beth Israel Medical Center in New York) </td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> <ext-link ext-link-type=\"uri\" xlink:href=\"www.healthandhealingny.org\">www.healthandhealingny.org</ext-link></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">National Center for Complementary and Alternative Medicine (<sc>nccam</sc>)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> <ext-link ext-link-type=\"uri\" xlink:href=\"nccam.nih.gov/health\">nccam.nih.gov/health</ext-link></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Natural Medicines Comprehensive Database</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> <ext-link ext-link-type=\"uri\" xlink:href=\"www.naturaldatabase.com\">www.naturaldatabase.com</ext-link></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Natural Medicines Comprehensive Database—Clinical Management Series</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> <ext-link ext-link-type=\"uri\" xlink:href=\"www.naturaldatabase.com/(S(st2arzb2hbi2v355rtipno2p))/nd/ClinicalMngt.aspx?cs=&amp;s=;ND\">www.naturaldatabase.com/(S(st2arzb2hbi2v355rtipno2p))/nd/ClinicalMngt.aspx?cs=&amp;s=ND</ext-link></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Natural Standard Database</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> <ext-link ext-link-type=\"uri\" xlink:href=\"www.naturalstandard.com\">www.naturalstandard.com</ext-link></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Turning Research into Practice (<sc>trip</sc>)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> database<ext-link ext-link-type=\"uri\" xlink:href=\"www.tripdatabase.com\">www.tripdatabase.com</ext-link></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">The University of Texas MD Anderson Cancer CenterComplementary/Integrative Medicine Education Resources</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> <ext-link ext-link-type=\"uri\" xlink:href=\"www.mdanderson.org/departments/cimer\">www.mdanderson.org/departments/cimer</ext-link></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Memorial Sloan–Kettering Cancer Center</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> <ext-link ext-link-type=\"uri\" xlink:href=\"www.mskcc.org/mskcc/html/44.cfm\">www.mskcc.org/mskcc/html/44.cfm</ext-link></td></tr></tbody></table></table-wrap>" ]
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[{"label": ["14"], "surname": ["Sackett", "Strauss", "Richardson", "Rosenberg", "Haynes"], "given-names": ["D", "S", "W", "W", "R"], "source": ["Evidence-based Medicine. How to Practice and Teach EBM"], "publisher-loc": ["Toronto"], "publisher-name": ["Churchill Livingstone"], "year": ["2000"]}, {"label": ["17"], "collab": ["Community Health Sciences, University of Calgary"], "article-title": ["Complementary and Alternative Medicine Issues in Undergraduate Medical Education [Web resource]"], "publisher-loc": ["Calgary"], "publisher-name": ["University of Calgary"], "year": ["2003"], "comment": ["[Available at: "], "ext-link": ["www.caminume.ca"]}, {"label": ["18"], "surname": ["Coulehan", "Block"], "given-names": ["JL", "MR"], "source": ["The Medical Interview. Mastering Skills for Clinical Practice"], "edition": ["5th ed"], "publisher-loc": ["Philadelphia"], "publisher-name": ["F.A. Davis Company"], "year": ["2006"], "fpage": ["301"], "lpage": ["18"]}, {"label": ["23"], "collab": ["National Institutes of Health, National Centre for Complementary and Alternative Medicine ("], "sc": ["nccam"], "article-title": ["Time to Talk [Web page]"], "publisher-loc": ["Bethesda"], "publisher-name": ["NCCAM"], "comment": ["n.d. [Available at: "], "ext-link": ["nccam.nih.gov/timetotalk"]}, {"label": ["25"], "collab": ["National Institutes of Health, National Centre for Complementary and Alternative Medicine ("], "sc": ["nccam"], "article-title": ["10 Things to Know About Evaluating Medical Resources on the Web [Web page]"], "publisher-loc": ["Bethesda"], "publisher-name": ["NCCAM"], "comment": ["[available at: "], "ext-link": ["nccam.nih.gov/health/webresources"]}, {"label": ["26"], "surname": ["Pinder", "Pedro", "Theodorou", "Treacy", "Miller"], "given-names": ["M", "L", "G", "K", "W"], "source": ["Complementary Healthcare: A Guide for Patients"], "publisher-loc": ["London, U.K."], "publisher-name": ["The Prince of Wales\u2019s Foundation for Integrated Health"], "year": ["2005"], "comment": ["[Available online at: "], "ext-link": ["www.fih.org.uk/document.rm?id=19"]}]
{ "acronym": [], "definition": [] }
26
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s88-s93
oa_package/cf/94/PMC2528563.tar.gz
PMC2528564
18769582
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[ "<p>Almost since its inception, the Internet has been used by ordinary people to connect with peers and to exchange health-related information and support. With the rapid development of software applications deliberately designed to facilitate social interaction, a new era is dawning in which patients and their loved ones can collaboratively build knowledge related to coping with illness, while meeting their mutual supportive care needs in a timely way, regardless of location. In this article, we provide background information on the use of “one-to-one” (for example, e-mail), “one-to-many” (for example, e-mail lists), and “many-to-many” (for example, message boards and chat rooms, and more recently, applications associated with Web 2.0) computer-mediated communication to nurture health-related social networks and online supportive care. We also discuss research that has investigated the use of social networks by patients, highlight opportunities for health professionals in this area, and describe new advances that are fuelling this new era of collaboration in the management of cancer.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/270/236\">http://www.current-oncology.com/index.php/oncology/article/view/270/236</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s107.es42-s107.es47
oa_package/9e/ce/PMC2528564.tar.gz
PMC2528565
18769583
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[ "<p>The Integrating Wellness into Cancer Care conference was held at the University of Toronto, October 4–5, 2007, and was dedicated to the memory of the late Dr. Véronique Benk. This article summarizes the workshops at that conference.</p>", "<p>The notion of wellness and an integrated approach should be introduced from the outset as part of the cancer patient’s management. Having wellness as part of the treatment sets a standard for taking care of the patient’s emotional, spiritual, physical, and nutritional needs, and for providing information on complementary therapies. A focus on holistic supportive care during treatment and survivorship is important.</p>", "<p>The whole medical team should support an integrative program. Referral to an education program and one-to-one assessments by a point person such as an advanced nurse practitioner, a social worker, or a psychological counsellor with appropriate special training should be mandatory. The concept of a pathfinder or cancer guide was discussed.</p>" ]
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[ "<fn-group><fn><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.current-oncology.com/index.php/oncology/article/view/277/239\">http://www.current-oncology.com/index.php/oncology/article/view/277/239</ext-link></p></fn><fn><p>*With the increasing national and international popularity and exposure of Current Oncology, the queue of excellent submissions continues to lengthen. After substantial consideration, the journal’s management has determined that the best way to manage this abundance is to move to a “hybrid” of combined print and electronic publication, with every e-manuscript being supported by a full print abstract and key words, and of course, indexing in PubMed for international recognition.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s109.es79-s109.es80
oa_package/30/a3/PMC2528565.tar.gz
PMC2528566
18769584
[ "<title>1. INTRODUCTION</title>", "<p>Integrative oncology focuses on the roles of complementary therapies such as meditation and other mind–body approaches, music therapy, massage and other touch therapies, acupuncture, natural health products (botanicals, for example), nutrition, fitness therapies, and more. Its goal is to increase the effectiveness of conventional cancer treatment programs, to reduce symptoms, and to improve quality of life for cancer patients. The Consortium of Academic Health Centers for Integrative Medicine defines integrative medicine as “the practice of medicine that reaffirms the importance of the relationship between practitioner and patient, focuses on the whole person, is informed by evidence, and makes use of all appropriate therapeutic approaches, healthcare professionals and disciplines to achieve optimal health and healing.”</p>" ]
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[ "<p>Integrative oncology focuses on the roles of complementary therapies to increase the effectiveness of conventional cancer treatment programs by improving defined outcomes such as symptom control, quality of life, rehabilitation, and prevention of recurrence. Implementation of integrative oncology programs should be based on the best evidence and must continually be evaluated to ensure quality, optimization of techniques, collection of new data, and cost-effectiveness. Useful domains that can be evaluated include symptom control, adherence to treatment protocols, quality of life, individual outcomes, prevention, rehabilitation, potential advantages of a whole-systems health approach, and economics of health services.</p>" ]
[ "<title>2. IMPLEMENTING INTEGRATIVE ONCOLOGY</title>", "<p>Implementation of integrative oncology programs should be based on the best evidence and must continually be evaluated to ensure quality, optimization of techniques, collection of new data, and cost-effectiveness. Useful domains that can be evaluated include symptom control, adherence to treatment protocols, quality of life, individual outcomes, prevention, rehabilitation, potential advantages of a whole-systems health approach, and economics of health services.</p>", "<title>2.1 Symptom Control</title>", "<p>Various modalities play a role in symptom control. These include mind–body therapies (meditation, group support, cognitive behavioural therapy, spirituality, yoga, tai chi, <italic>qi gong,</italic> labyrinth walking), manipulative and physical therapies (massage, acupuncture), biofield therapies (polarity, healing touch, Reiki), natural health products (botanicals), nutrition interventions (phytoceuticals, diet adjustment), and ethnic health systems (Traditional Chinese Medicine, Ayurveda, Native American Medicine) ##REF##16737670##1##. Current evidence for safety and effectiveness has been summarized by the Society for Integrative Oncology ##REF##17511932##2##. Visual analogue scales are often used to measure such symptoms as pain, anxiety, and tension at baseline and after treatment. Validated assessment procedures are required to select and triage specific patients to an appropriate therapy program.</p>", "<p>An integrative medicine program provides opportunities to connect with the patient, enhance trust, and communicate information in a supportive environment. These factors increase adherence to treatment protocols and can improve treatment response and survival ##REF##2299375##3##,##REF##15465065##4##. Positive expectation, appreciation of meaning, and a sense of optimism should be enhanced within the context of factual knowledge ##REF##16131282##5##,##REF##16455221##6##.</p>", "<p>A National Health System report from the United Kingdom encourages positive expectation in the delivery of health care. A pessimistic approach can encourage the nocebo effect, which can result in poor coping skills, disempowerment, and adverse health outcomes ##REF##10448203##7##. The aim of the report was to assess the nature and extent of the placebo effect and to consider how it may be harnessed within the National Health System to improve the quality of care. The authors concluded that the existing evidence justifies the use of strategies to enhance expectancies—specifically, to enhance accurate expectations by the patients about medical procedures and how to cope with them. In addition, evidence encourages the use of the positive effects of expectation to enhance the skills of patients for self-management of their illness, to improve communication concerning health issues with health care providers, and to enhance belief in the benefits of effective medical treatments.</p>", "<title>2.2 Patient-Focused Approach</title>", "<p>Patients that choose complementary or alternative therapies require guidance. Such guidance is often not available in cancer treatment organizations because many of the staff are not trained in integrative oncology and because information resources have not been critically examined by a qualified person for credibility. Cancer “guide,” “navigator,” or “pathfinder” is an important role for an appropriately trained health care professional. Although many patients are simply seeking empowerment and trying to do everything possible, some patients have important psychosocial issues that need to be diagnosed and managed appropriately. These issues can include cultural beliefs, heightened anxiety, fear, anger, advanced and uncontrolled symptom burden, poor satisfaction with conventional medicine, communication breakdown, and misinformation that includes bogus therapies found on the Internet ##REF##16282504##8##,##UREF##0##9##.</p>", "<p>The evaluation of integrative health care models is becoming increasingly important, and an appropriate set of outcome measures is required. Dr. Marja Verhoef has proposed these questions ##REF##16332188##10##:</p>", "<p>How do we identify the manner in which cancer patients phrase and frame the beneficial outcomes of their experience?</p>", "<p>What are the recommendations that can be made for an appropriate outcome measures package to evaluate integrative health care?</p>", "<p>Through interviews and focus groups, Verhoef identified six types of benefit: physical well-being, change in physiologic indicators, improved emotional well-being, personal transformation, feelings of connectedness, global state of well-being, and cure. Patients’ goals included improvement in their state of being, freedom from cancer, increased energy, more effective pain management, and achievement of an improved quality of life.</p>", "<p>The evaluation of medical practice should use quantitative and qualitative techniques alike. Contemporary medicine is assumed to be conceptually based on modern science; however, the summation of quantity and quality is impossible to evaluate using only scientific parameters. Integrative medicine is postulated to be comprehensive in its fundamental doctrine, emphasizing a holistic approach, including technical, artistic, social, religious, philosophic, and ethical elements. The personal preferences of patients for outcome as defined by qualitative experiences is emphasized. The World Health Organization suggests that health be evaluated from the viewpoint of the disability-adjusted life expectancy, which is expressed using both quantity (life expectancy) and quality of life. One of the challenges of assessing the utility of integrative oncology is the expression of joint measurements of quantitative and qualitative outcomes. Medical quantity and quality are in reality mixed and fused together, both in treatment and in outcomes.</p>", "<title>2.2.1 Quantitative Evaluation of an Intervention for an Individual</title>", "<p>The N-of-1 randomized controlled trial was designed to evaluate the contribution to the therapeutic process of an intervention as compared with either a placebo (expectation) effect or regression to the mean. The patient participates in choosing the intervention. Distinguishing the components of healing is probably not an important issue if the intervention is safe and inexpensive. It may not be applicable to the cancer patient when conditions change rapidly. In addition, the resources and expenses of implementing this type of evaluation must be taken into account. That being said, the N-of-1 trial may have a place in the evaluation of some treatment programs ##REF##15673992##11##,##REF##16679928##12##.</p>", "<title>2.2.2 Qualitative Evaluation of an Intervention for an Individual</title>", "<p>A major benefit of integrative therapies is their potential to improve quality of life (<sc>qol</sc>). Evaluation of <sc>qol</sc> is now a standard of clinical investigation that is routinely used in addition to measurement of tumour response. An improvement in symptom control, coping, and function enhances rehabilitation and restoration of a normal lifestyle. Nevertheless, an understanding of what <sc>qol</sc> is and how it should be evaluated is a very difficult matter.</p>", "<p>A fundamental part of the definition of a high <sc>qol</sc> is a large degree of freedom in thinking and behaviour that includes personal subjective feelings. As a result, the cornerstones of science—which include objectivity, universality, reproducibility, and logical consistency—can no longer be totally applied. Evaluation of medical practices in terms of <sc>qol</sc> is non-scientific in this respect, because those scientific characteristics are not preserved ##REF##16998695##13##. The fact that so many <sc>qol</sc> evaluation parameters have been proposed ultimately suggests that none are reliable. Unless a logical and scientific way of assessing personal feelings is established, <sc>qol</sc> simply cannot be evaluated using scientific analysis and numeric expression.</p>", "<p>Because <sc>qol</sc> may vary between individuals according to their primary problems and values, individual and personalized <sc>qol</sc> criteria may be more appropriate for measuring outcomes. The Measure Yourself Medical Outcome Profile was developed to allow patients to set their own criteria for <sc>qol</sc> outcomes. This measure was developed further for cancer patients in the form of the validated Measure Yourself Concerns and Wellbeing assessment protocol, which specifically evaluates outcome in cancer supportive care that includes complementary therapies ##REF##15233501##14##,##REF##17352970##15## (##TAB##0##Table I##).</p>", "<p>The term ‘‘healing enhancement’’ (Mayo Clinic, Rochester, MN, U.S.A.) was coined to identify the goals of an emerging paradigm that focuses on all aspects of the patient’s experience—mind, body, and spirit ##REF##17950176##16##. That subjective experience of healing was investigated in a multi-method pilot study ##REF##17950179##17##: Before and after each of 6 weekly healing sessions, perception of well-being and client experience were assessed in 15 clients by the EuroQol, the Measure Yourself Concerns and Wellbeing index, and a client satisfaction tool. Qualitative methods included focus groups that explored the perceived effects of healing in more depth. Over the course of healing, quantitative data showed perceived significant improvements in concerns or problems for which clients wanted help and in distressing symptoms of anxiety and depression. Qualitative analysis showed that clients sought help mainly for psychological and emotional concerns. Clients attributed many of the quantitative improvements to healing itself. The study suggested that clients and healers perceive healing to have a range of benefits, particularly in terms of coping with cancer.</p>", "<p>Research to date indicates that patients who are involved in their own care and who take an active role in their treatment feel better and have an improved recovery. Other studies have shown that depression predicts cancer incidence or progression, although not all studies agree. Rosenbaum <italic>et al.</italic>\n##REF##14991388##18## discussed this effect in more detail. A randomized study that compared <sc>qol</sc> according to the survival of patients in the adjuvant and metastatic settings noted that an increase in <sc>qol</sc> included improved mood, appetite, and well-being, and hence facilitated longer survival ##REF##11078489##19##. That finding suggests that <sc>qol</sc> may also play a role in determining a patient’s length of life. Resources and programs that are shown to improve <sc>qol</sc>, such as the Stanford Cancer Supportive Care Program, promote a more meaningful and longer lifespan for those struggling with cancer.</p>", "<p>Because <sc>qol</sc> involves subjective measurement in domains such as the psychological, functional, and social, it can be difficult to assess. Unlike readily quantifiable measures, such as weight or tumour size, <sc>qol</sc> is not as easily defined. Interpretation of <sc>qol</sc> differs with each person and is influenced by factors such as culture, age, and life experience. The patient evaluations from the Stanford Cancer Supportive Care Program suggest that participants received several benefits from the programs, such as an increase in sense of well-being, reduced feelings of stress, an increase in energy, more restful sleep, and an increase in hopefulness and empowerment—all ingredients that improve <sc>qol</sc>\n##REF##14991388##18##.</p>", "<title>2.3 Systems Approach to Health Care</title>", "<p>Integrative oncology recognizes a systems approach to health care. A program of coordinated appropriate interventions may be more effective than individual therapies. The holistic approach to health is a maturing tautology that is now binding the sciences from molecular biology to psychosocial and health services research ##REF##17373844##20##. Systems approaches, by taking the myriad of connections into account, are more suited for addressing chronic, long-term improvements. In many chronic diseases, the body system may not have been healthy over a prolonged period of time before the symptoms appeared.</p>", "<p>Only short-term improvements can be obtained by aiming at a particular subsystem related to the symptoms. Overall sustained improvement can be obtained only through multiple interventions. A multi-pronged approach encourages homeostasis to return to the whole physiologic organism. For example, diabetes care involves the interaction of nutrition, exercise, stress management, and education—not just the prescription of insulin. Instead of disease management, health promotion becomes the aim of the intervention.</p>", "<p>Whole-systems research explores effectiveness through observational studies that combine qualitative and quantitative approaches such as pattern of interactions, context, process, philosophy, and outcomes. It deals with “real life” scenarios and recognizes multiple inseparable components, model validity, effectiveness, and external validity ##REF##17101757##21##,##REF##17101758##22##. A systems analysis can reveal new approaches to improve the current situation of ever-increasing health care costs.</p>", "<title>2.4 Health Economics</title>", "<p>The financial implications of integrative oncology programs require evaluation with respect to functional outcomes, in-kind contributions, safety, restoration of function, and efficiency of rehabilitation ##REF##15750380##23##. The self-empowerment of individuals using complementary therapies can have life-long advantages for self–health care. Short-term measurements of outcome may not reflect lifetime benefits ##REF##17030296##24##. Using complementary therapies over the long term, together with judicious temporary pharmacologic therapies, could have cost-effectiveness advantages. For example, short-term administration of antidepressant drugs may give the patient adequate function to learn cognitive behavioural techniques that will become the source of a maintenance regimen after withdrawal of the pharmaceuticals.</p>", "<p>Measuring <sc>qol</sc> with respect to financial cost is methodologically challenging. Quality-adjusted life year (<sc>qaly</sc>) is a quantitative measurement that may allow for a financial comparison between various interventions and their related health outcomes. The main problem is to be able to compare “apples with oranges” (to use a common expression). The ability to compare directly the dollar cost of various health outcomes is attractive to the decision-maker. However, the use of the <sc>qaly</sc> for this purpose has severe limitations, and these limitations must be widely understood ##REF##12591784##25##. A key question is who is to make the subjective choices that determine the <sc>qaly</sc>? Is it health professionals? The general public? Or patients who have experience of the particular medical condition and treatment? Other problems include the hypothetical nature of the situations being responded to, which therefore may not accurately reflect real human decisions, and the influence that the length of the illness and the way in which the questions are asked influence the valuations made. Finally, <sc>qaly</sc>s are likely to undervalue health care because they do not capture the wider benefits.</p>", "<p>The evidence for cost advantages of complementary therapies is limited because the research is at an early stage ##REF##17173105##26##,##REF##16223821##27##. Many outcomes studies are not well controlled, and determining whether outcome is attributable to the intervention, expectation, regression to the mean, or a combination of these factors is difficult. This difficulty has led to much controversy and many unwarranted claims ##UREF##1##28##,##UREF##2##29##. However, uncertain evidence of effectiveness does not necessarily preclude a positive recommendation in a guideline, and original modelling of cost-effectiveness can be part of guideline development ##REF##16223798##30##.</p>", "<title>3. CHALLENGES</title>", "<p>Integrating complementary therapies into conventional cancer care poses a number of challenges. What is required is an integration of the best approaches to cancer care with a more cohesive move forward based on collaboration and open-mindedness. Skepticism comes from lack of knowledge and training, and the result is a reluctance to refer patients to <sc>cam</sc> therapists ##REF##16648094##31##. However, implementation of an evidence-based integrative medicine program within an academic setting can be successful. A survey of one large academic institution with a Chair for Holistic and Integrative Medicine (Wake Forest University Baptist Medical Center, Winston–Salem, NC, U.S.A.) concluded that strong interest is present among medical staff. Future studies will need to assess the cost-effectiveness of such services, their financial sustainability, and their effect on patient satisfaction, health, and quality of life ##REF##17291340##32##.</p>", "<p>Most mainstream and complementary medicine practitioners agree that patients should take an active role in their care, that an understanding of treatment and its goals should be established between the patient and practitioners, and that the culture should encourage patients to take responsibility for their health ##REF##17210511##33##. We need to focus on the “effectiveness gaps” in the treatment of cancer patients. For example, emesis, fatigue, neuropathy, anxiety, insomnia, and cachexia are some symptoms that continue to show problematic responses to conventional therapies. Drawing together services and developing expertise in integrative health care requires leadership to promote team-building. However, without collaboration and support, such visions are limited. Without audit and evaluation of outcomes, they will not be accepted by mainstream practitioners. Championing integrative oncology focuses on the value of the vision and the public acknowledgment of that value. A willingness to measure and act upon the results of clinical outcomes studies is more important than are policies based on outdated cynicism (##TAB##1##Table II##) ##REF##15984224##34##.</p>" ]
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[ "<table-wrap id=\"tI-co15_s2ps078\" position=\"float\"><label>TABLE I</label><caption><p>Measure Yourself Concerns and Wellbeing assessment form <xref ref-type=\"table-fn\" rid=\"tfn1-co15_s2ps078\">a</xref></p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Concern or problem 1:</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Not bothering me at all</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" rowspan=\"1\" colspan=\"1\">Bothers me greatly</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">How would you rate your general feeling of well being now?</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"right\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">As good as it can be</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" rowspan=\"1\" colspan=\"1\">As bad as it can be</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Other things affecting your health; changes you have made yourself</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">What has been most important to you?</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tII-co15_s2ps078\" position=\"float\"><label>TABLE II</label><caption><p>Champion ideas for <sc>cam</sc> and cancer care (adapted from Mackereth and Stringer, 2005 ##REF##15984224##34##)</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Encourage researchers to be sensitive to the vulnerabilities of people affected by cancer and mindful of their values and concerns.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Collaborate with cancer interest groups, education and communication specialists, researchers, other national and international programs.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Provide leadership training and support to assist practitioner innovation, to build dynamic services, and to disseminate their work.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Improve information and access to <sc>cam</sc> therapies within public cancer services.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Provide greater equity in funding service provision and research.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Involve users, with the goal of providing cost-effective, accountable, and responsive services.</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ensure safety of therapies and credentialing of practitioners.</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><fn id=\"tfn1-co15_s2ps078\"><p><sup>a</sup> Available at <ext-link ext-link-type=\"uri\" xlink:href=\"www.bristol.ac.uk/hsrc/research/other/mymop/index.html\">www.bristol.ac.uk/hsrc/research/other/mymop/index.html</ext-link> (registration required)</p></fn></table-wrap-foot>" ]
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[{"label": ["9"], "surname": ["Sagar", "Abrams", "Weil"], "given-names": ["S", "D", "A"], "article-title": ["Alternative therapies as primary treatments for cancer"], "source": ["Oxford Textbook of Integrative Oncology"], "publisher-loc": ["Oxford"], "publisher-name": ["Oxford University Press"], "year": ["2008"]}, {"label": ["28"], "surname": ["Richardson", "Garrow", "Ernst", "Thompson"], "given-names": ["J", "J", "E", "T"], "article-title": ["The Smallwood Report on the role of "], "sc": ["cam", "nhs"], "source": ["Focus Altern Complement Ther"], "year": ["2006"], "volume": ["11"], "fpage": ["14"], "lpage": ["18"]}, {"label": ["29"], "surname": ["Ernst"], "given-names": ["E"], "article-title": ["The \u201cSmallwood report\u201d: method or madness?"], "source": ["Br J Gen Prac"], "year": ["2006"], "volume": ["56"], "fpage": ["64"], "lpage": ["5"]}]
{ "acronym": [], "definition": [] }
34
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s78-s82
oa_package/43/d8/PMC2528566.tar.gz
PMC2528567
18769585
[ "<title>1. INTRODUCTION</title>", "<p>Integrative medicine brings together the care philosophies inherent in biomedicine—commonly conceived as Western, “evidence-based,” and rooted in science—and holistic medicine—often described in alternative or complementary medicine terms and based strongly in values or beliefs. Not uncommonly, this integration is obscured by debate as to which paradigm is “right” or “correct.” However, such a distinction is largely without value if the goal is to justify “care and decision-making” according to science (biomedicine) or to human values and beliefs (holistic medicine).</p>", "<p>Science informs the process of decision-making and human values place scientific evidence within the context of the individual and society. In integrative care, not only are practices based in science and complementary medicine brought together, also brought together are the philosophies of analytic and deductive reasoning based in evidence (science) and the contextualization of science within the beliefs of individuals and society based on invention, judgment, and values (logic). A satisfactory outcome for the person needing care is confidence in the decision or decisions because information was made available, because the processes of assembling information were codified to allow for alignment with beliefs, and because the rationale for care formulated within the values of the individual was such that the eventual decision is compelling and consistent for that person.</p>" ]
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[ "<title>2. DISCUSSION</title>", "<title>2.1 Life, Death, and Human Values</title>", "<p>In his treatise on the health of individuals and populations, Cairns notes that one cannot interpret health (life), without first being knowledgeable about illness and death ##UREF##0##1##. In ##FIG##0##Figure 1##, attention is drawn to the unchanging pattern of human mortality from the earliest of available evidence to the mid-19th century, preceding the Industrial Revolution in the Westernized world. By contrast, the profound change in mortality following the Industrial Revolution (post-1850) is shown in the population survival plot for England in the 1990s (##FIG##0##Figure 1##). The illustration demonstrates certain points:</p>", "<p> The power of interventions to change survival—in this case, interventions largely related to the introduction of public health measures and, to a lesser, more recent degree, interventions based in medical treatment</p>", "<p> The value of observations validated through scientific methods as a basis for improvements in survival</p>", "<p> The factual basis for interventions designed to achieve longevity through informed, valid, applied health policy</p>", "<p>In large part, interventions designed to control communicable diseases (pathogen A causes disease B, which can be controlled by intervention or interventions C) have resulted in avoidance of premature death, with resultant longevity. This benefit is based on a proposition that values longevity.</p>", "<p>Through observation, deductive reasoning, intervention, validation through scientific method, analysis of outcome, and application to practice through policy, the population has, over one and one-half centuries, gained on average an extra 30 years of life expectancy from birth. However, in Westernized, high-income economies, cancer and most chronic diseases causing premature death are not communicable diseases for which straightforward interventions are available. Accordingly, the value set for communicable disease (longevity through avoidance of premature death) is not necessarily the same as that set for cancer or non-communicable diseases, in which functionality and quality determinants may take greater precedence than does longevity. Furthermore, the existing organization of health services based on an acute reversible illness model does not necessarily align well with the management of interventions to ameliorate the personal and societal impacts of chronic disease.</p>", "<p>In ##FIG##1##Figure 2##, domains of scientific enquiry are related to effects on humanity according to the value proposition applied to the preferred outcome. The illustration draws recognition to the roles of individual values and societal values in determining the expected role of the intervention, thereby highlighting the dichotomy of the individual and society as consumer and beneficiary and funder of health care in a publicly funded health system.</p>", "<title>2.2 The Cancer Control Problem</title>", "<p>The population burden of cancer continues to increase, primarily as a function of the increase in population numbers and the structure of increasing age of our society. Cancer is principally a disease of aging, the rate increasing substantially after the age of 60 years. The rising burden of cancer draws attention to several issues:</p>", "<p> The effect of our interventions is insufficient to offset the increasing incidence consequent upon aging and population growth.</p>", "<p> The burden of cancer is not borne equally by individuals or populations: disparities exist within and across communities and populations.</p>", "<p> The interpretation and the effect of the burden has different meanings, all encompassed within the context of health need. That is,</p>", "<p> “burden” as a statement of medically necessary care,</p>", "<p> “burden” as a personal or societal consequence of illness,</p>", "<p>“burden” as an expression of capacity to benefit from intervention,</p>", "<p>“burden” as the capacity to afford necessary health care, and</p>", "<p> “burden” as the expression of inequality to achieve the gains of intervention.</p>", "<p>The interpretation of burden has both a personal and societal context and value proposition. Not-withstanding the definition or value placed on burden, the problem is not solely that the burden is rising—that is a fact. The problem is what can be done about the rising burden, individually and societally. A further point of importance is that the variation in health outcomes across communities and populations has as much, if not more, to do with variations in exposure to risk factors (environment, occupation, smoking, diet, obesity, nutrition, and exercise, among others) as with variations in access to interventional care.</p>", "<title>2.3 Cancer As a Process, Not an Event</title>", "<p>Molecular medicine has identified cancer as a genetic disease. Thus, the transformation of the healthy cell through a series of events that lead to a cancer cell with the properties of invasion and metastasis that may lead to death is underwritten in serial and cumulative changes within the genome of the cell. These are measurable biologic changes that correlate with predisposition, expression, sensitivity or resistance to therapies, and the prognosis of cancer—a concept underlying aspects of the uniqueness of cancer to the individual and to the personalization of care.</p>", "<p>If we accept cancer as a process, arising in health and, if unperturbed, resulting in illness, disability, and death, then the strategy for cancer control must address the process, not just the disease. In the case of symptomatic cancer, the process is well advanced and the opportunities for intervention limited to treatment or palliation.</p>", "<p>In the context of addressing the process of cancer, the definition and the nature of interventions related to cancer control assume importance ##UREF##1##2##. Cancer control encompasses</p>", "<p> cancer treatment: treatment interventions for individuals with an established diagnosis of cancer, commonly delivered through hospitals or cancer centres, focused on the treatment episode and the requirement for acute hospital-based services.</p>", "<p> cancer care: integrated programs across care settings (hospital, community, home) by networks of providers, with the goal of coordinated, continuous services for the individual or individuals experiencing cancer and needing care.</p>", "<p> cancer control: interventions directed to the healthy (at risk), those experiencing cancer, and those who are cured (survivors) or dying of their cancer. The interventions engage multiple sectors (health, education, and transport, among others) and are ultimately attempting to achieve a responsive, efficient, effective, and sustainable system to improve health and control cancer (as a process, not as an event).</p>", "<p>Within each of these contexts of cancer intervention, values attached to the delivery of treatment, care, or control will be relevant both from an individual and a societal perspective. What ultimately drives the decisions regarding provision and access to interventions? How is this expressed from the perspective of the public, the patient, the care provider, and the funder?</p>", "<p>Factors of relevance relate to the quality of the service (processes, outcomes), safety (incidents, errors, adverse events), accessibility (distance, costs), availability (timeliness), cost-efficiency and cost-effectiveness, satisfaction and sustainability (individually and societally), and attachment of individual and societal values and judgments to the foregoing parameters.</p>", "<p>The premise underlying reductionism is that complex problems can be solved by dividing them into smaller—indeed individual—parts and by so doing, represent the whole by the sum of the individual parts. Scientific method would naturally lead us in the direction of the necessity to control all variation other than the characteristics and performance related to a single attribute or a small number of attributes. The current pursuit of targeted therapies is an illustration of that ideology, and it has been remarkably successful in a number of circumstances—for example, imatinib and tyrosine kinase inhibition in chronic myeloid leukemia and gastrointestinal stromal tumours; rituximab for CD20<sup>+</sup> lymphoma; trastuzumab for breast cancer that is positive for human epidermal growth factor receptor. Yet notwithstanding these remarkable examples, reductionism has profound limitations as a strategy to explain and treat disease:</p>", "<p> It focuses on a single factor as cause.</p>", "<p> It emphasizes static control of function rather than dynamic management of a range of function.</p>", "<p> It implies a one-target, one-risk-factor approach to disease management.</p>", "<p> It takes a piecemeal approach to the multiple problems inherent in a chronic disease process.</p>", "<p>Although inappropriately simplistic, reductionism aligns more to the management of communicable disease (pathogen A causes disease B, necessitating intervention C) than to the more complex gene–environment interactions characterizing non-communicable diseases including cancer.</p>", "<p>Reductionism is a tool to investigate cause-and-effect through the scientific method, but other than in unique circumstances, it is incapable of addressing the whole simply as the sum of the parts within a system that is characterized by multiple biologic processes, multiple genes within multiple pathways, multiple targets, and multiple organ systems (tumour, tissue micro-environment, organ, host, and host environment). Even if a reductionist approach were to be employed to “dissect” the multiple, individual pathways underlying the cause and expression of cancer, would the establishment of therapeutic benefit through current clinical trial methods be feasible or practical? Current oncologic practice would suggest that only 5% of novel therapeutics actually enter clinical application, with an expected investment in excess of $800 million for each successful drug candidate achieving approval ##REF##15286737##3##.</p>", "<p>Challenges within the reductionist approach have stimulated the concept of a systems perspective to biology and health. In this construct, the holistic and composite characteristics of the problem are recognized, and the integration and interplay of relevant attributes are explored as an explanatory basis for observations. A relevant metaphor would be that “the forest cannot be explained by the study of individual trees.” The systems perspective would follow the principle that behaviour is explained by the system as a whole, not by the sum of the parts; that rarely is there a magic bullet for a unique, single target that will address health and illness; that many targets and many functions will be relevant to the control of biologic networks that include both cancer cells and normal cells; that time, space, and context will be relevant to how networks behave and respond; and that health is better defined by robustness, adaptability, and homeodynamism than by normalcy, control, and homeostasis ##UREF##2##4##.</p>", "<title>2.4 The Concept of Integrated Health</title>", "<p>Accordingly, how might health be viewed from a systems perspective?</p>", "<p>A definition that would align to this concept would be “Health is the extent to which an individual or group is able, on the one hand, to realize aspirations and satisfy needs, and on the other hand, to change and cope with the environment. Health is, therefore, seen as a resource for everyday life, not the objective of living: it is a positive concept emphasizing social and personal resources as well as physical capacities” (cited in Young 1998 ##UREF##3##5##).</p>", "<p>This concept describes health in the context of functional potential: the individual as an asset whose function can be maximized and dysfunction minimized through planned mitigating interventions, recognizing that health risks (risk factors) are largely created and maintained by social systems, that the magnitude of risk exposure is a function of socio-economic disparities and psychosocial gradients, and that the development of sustained health or illness plays out over extended periods of time, crossing many stages of human development (prenatal to late adult, environmental, and socio-economic political domains) ##UREF##4##6##.</p>", "<p>In this framework, disparities in health outcomes and in psychological adjustment to varying health states, rather than being attributable to discrete causes and actions, are determined by cumulative, compounding early-life events reflective of genes, biology, and environment embedded within the individual’s make-up, sustained by social, cultural, and economic forums, and affected by biologic, psychosocial, socio-economic, cultural, and physical environments. In each of these contexts, evidence established through the scientific method contributes to the development of a conceptual model for health and illness and the resultant response of health policies, practices, and systems ##UREF##4##6##.</p>" ]
[ "<title>3. CONCLUSIONS</title>", "<p>Integrative care may be most aptly understood from the perspective of the beneficiary of care, the patient, rather than from the perspective of the care provider (the biomedical, complementary, or alternative medicine practitioner). In whose hands is the decision-making process placed? Which tools and information sources are made available to assist with the decision? How are the processes of reasoning codified—be they scientific, evidence-based, or debated—to provide a basis for decision-making that is aligned to individual beliefs? And how are arguments for interventions placed within the values and the priorities of the individuals?</p>", "<p>Good decisions are made with the full confidence of the patient. Confidence requires the information to be sound, the rationale compelling, and the judgment to be fully aligned to patient needs and values. The inputs to the decision, be they scientific, holistic, complementary, or belief-based are all relevant inasmuch as trust in the inputs will lead to trust in the decisions.</p>" ]
[ "<p>The burden of cancer continues to increase globally, with substantial personal, societal, and economic consequences. Population growth and aging underlie this increase—a reflection of the effect of population health interventions in the last two centuries. Much of this gain has come through observation, derivation of evidence, and rigorous application of valid science to the public, both healthy and affected by diseases such as cancer. Increasingly, molecular medicine will affect the knowledge of cause and the personalization of therapy. However, science informs the decision-making process and places evidence within the beliefs of individuals and society as they relate to innovation, judgment, and values—the “logic” underlying alignment of conventional and complementary (holistic) care as a basis for compelling, consistent, and confident decisions.</p>" ]
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[ "<fig id=\"f1-co15_s2ps074\" position=\"float\"><label>FIGURE 1</label><caption><p>Patterns of survival in Breslau (17th century), Liverpool (19th century), and modern England (late 20th century). From Cairns 1997 ##UREF##0##1##, p. 21, reproduced with permission.</p></caption></fig>", "<fig id=\"f2-co15_s2ps074\" position=\"float\"><label>FIGURE 2</label><caption><p>Domains of scientific enquiry.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"co15_s2ps074f1\"/>", "<graphic xlink:href=\"co15_s2ps074f2\"/>" ]
[]
[{"label": ["1"], "surname": ["Cairns"], "given-names": ["J"], "source": ["Matters of Life and Death"], "publisher-loc": ["Princeton"], "publisher-name": ["Princeton University Press"], "year": ["1997"]}, {"label": ["2"], "surname": ["Caron", "De Civita", "Law", "Brault"], "given-names": ["L", "M", "S", "I"], "source": ["Cancer Control Interventions in Selected Jurisdictions: Design, Governance, and Implementation"], "comment": ["Monograph "], "sc": ["aetmi"], "publisher-loc": ["Montreal"], "publisher-name": ["Agence d\u2019\u00e9valuation des technologies et des modes d\u2019intervention en sant\u00e9"], "year": ["2008"], "fpage": ["339"]}, {"label": ["4"], "surname": ["Ahn", "Tewan", "Poon", "Phillips"], "given-names": ["AC", "M", "CS", "RS"], "article-title": ["The limits of reductionism in medicine: could systems biology offer an alternative?"], "source": ["PLoS Med"], "year": ["2006"], "volume": ["3"], "fpage": ["709"], "lpage": ["13"]}, {"label": ["5"], "surname": ["Young"], "given-names": ["TK"], "source": ["Population Health: Concepts and Methods"], "publisher-loc": ["New York"], "publisher-name": ["Oxford University Press"], "year": ["1998"]}, {"label": ["6"], "surname": ["Halfon", "Hochstein"], "given-names": ["N", "M"], "article-title": ["Life course health development: an integrated framework for developing health, policy and research"], "source": ["Milbank Q"], "year": ["2002"], "volume": ["80"], "fpage": ["1"], "lpage": ["33"]}]
{ "acronym": [], "definition": [] }
6
CC BY
no
2022-01-12 16:56:35
Curr Oncol. 2008 Aug; 15(Suppl 2):s74-s77
oa_package/2a/01/PMC2528567.tar.gz
PMC2528895
18765444
[ "<title>Introduction</title>", "<p>Direct to consumer advertising is a major component of drug promotion in the United States; manufacturers spent an estimated $4.24bn (£2.28bn; €2.88bn) in 2005—a 330% increase since 1996.##REF##17699817##1## The merits of direct to consumer advertising have been extensively debated, which has led to differing regulations across countries.##REF##9929095##2##\n##REF##15855439##3## Regulatory disputes continue worldwide, with ongoing debate about the introduction of direct to consumer advertising in the European Union and Canada; at the same time, the US Senate has recently considered legislation prohibiting such advertising during the first two years after the release of a new drug.##UREF##0##4##\n##UREF##1##5##\n##REF##17476002##6## Although the debate includes a broad range of concerns, many assertions assume that direct to consumer advertising increases the use of particular types of drugs. For example, proponents argue that it increases use of effective treatments for undertreated conditions, such as depression.##REF##9929095##2## Opponents, however, suggest that it drives up demand for newer drugs with higher costs, marginal benefits, and unknown safety profiles.##REF##15855439##3##</p>", "<p>Both sides of the argument assume that direct to consumer advertising increases use. However, the effectiveness of drug advertising campaigns is unclear and no extant studies use a concurrent control group and quantify the impact on use of marketed drugs.##REF##18205555##7##\n##REF##16076787##8## Previous uncontrolled longitudinal studies have found that expenditure on direct to consumer advertising was associated with higher sales of antidepressants, proton pump inhibitors, antihistamines, and nasal sprays but non-significant or very small association with sales of statins and cyclo-oxygenase-2 selective non-steroidal anti-inflammatory drugs.##UREF##2##9##\n##REF##15550797##10##\n##UREF##3##11##\n##REF##16966735##12## How these associations might be confounded by selection bias is unclear from these previous studies. For example, drugs with a larger pool of potential users or that are more innovative are more likely to be promoted through both direct to consumer advertising and physician directed campaigns using detailing, journal advertisements, and free samples.##UREF##4##13## Moreover, previous studies have not controlled for pre-advertising trends in use or evaluated comparable markets that are unexposed to such advertising.</p>", "<p>In the absence of firm evidence describing the effect of direct to consumer advertising on use of prescription drugs, policy makers in the United States and New Zealand have permitted it whereas their counterparts in Europe, Canada, and Australia have prohibited it. The extent of benefits or harms attributable to direct to consumer advertising will be directly proportional to how effectively it increases use of particular advertised drugs and at what cost. We studied the impact of US direct to consumer advertising campaigns on Canadian prescribing rates for three heavily marketed drugs by using a controlled longitudinal study design. Because Canadians are regularly exposed to “illicit” English language direct to consumer advertising from the United States, we hypothesised that these campaigns would increase use of the marketed drugs in English speaking Canadian provinces. For any campaigns associated with increased use in our Canadian analysis, we examined US Medicaid data without a control group to investigate whether the effects were greater with increased exposure to direct to consumer advertising.</p>" ]
[ "<title>Methods</title>", "<title>Study setting</title>", "<p>Examination of US data alone to delineate the impact of direct to consumer advertising is limited by two factors. Firstly, near universal exposure to advertising makes it almost impossible to find a comparable unexposed control group within the United States.##UREF##5##14## Secondly, manufacturers start many direct to consumer advertising campaigns shortly after the launch of a drug—precisely when detailing to physicians and coverage in the medical literature are likely to be at their highest. We sought to limit these threats to validity by examining the impact of US direct to consumer advertising campaigns on Canadian patterns of drug use in provinces with and without substantial exposure to such advertising—that is, in predominantly English speaking provinces compared with predominantly French speaking Quebec. For drugs for which we found an impact on Canadian prescribing rates, we used data from nationwide US Medicaid programmes to assess whether a dose-response relation might exist between greater exposure to direct to consumer advertising in the United States and more marked increases in drug use.</p>", "<p>Although Canada prohibits direct to consumer advertising that includes both a brand name and indications, substantial cross border exposure to US advertising occurs through cable and satellite television, radio, print media, and internet advertising.##REF##12952801##15## Statistics Canada estimates that around 30% of television watched by English speaking Canadians is foreign sourced, most of which is probably US cable and satellite stations.##UREF##6##16## Previous Canadian survey work suggested that more than 85% of English speaking patients had seen drug advertisements in the previous year and half had seen advertisements for six or more different products.##REF##12952801##15## Moreover, their primary care physicians filled nearly three quarters of patients’ requests for specific drugs.##REF##12952801##15## Thus, English speaking Canadians are regularly exposed to considerable amounts of US advertising and have the means to obtain advertised drugs.</p>", "<title>Data sources</title>", "<p>Our primary analysis used monthly drug use data from the nationally representative CompuScript audit from IMS Health Canada, an independent health information company, from January 2002 to December 2006. This audit uses a panel of approximately 2700 pharmacies (roughly 34% of all community pharmacies in Canada) to estimate total Canadian use of each drug. The major outcome of interest was the number of dispensed prescriptions of each drug per 10 000 residents per month. To calculate these rates, we used population estimates from Statistics Canada.##UREF##7##17## We also obtained IMS Health Canada data estimating Canadian expenditure on detailing and distribution of free samples for the study drugs, to assess whether other marketing increased coincidently with US direct to consumer advertising. We found no evidence of such changes. Our analysis in the United States used quarterly data from 50 US Medicaid programmes.##UREF##8##18## Using state level enrolment numbers, we calculated dispensed prescription rates per 10 000 Medicaid enrolees per quarter.##UREF##9##19## These data provide estimates up to the end of 2005, when many patients were transferred to the new Medicare Drug Benefit.</p>", "<p>The start month and total spending on US direct to consumer advertising campaigns came from TNS Media Intelligence. The dataset tracks advertising and estimates expenditure across several media, including television, radio, and print media, and has been used in previous research on direct to consumer advertising.##UREF##2##9## We also searched the Vanderbilt Television news archive to ascertain when particular drugs were advertised during major US national news broadcasts.##UREF##10##20## Finally, we assessed whether manufacturers aired television advertising in Canada mentioning a brand name by reviewing the databases of Eloda, an independent company that provides monitoring and verification services for North American advertising.</p>", "<title>Study drugs</title>", "<p>Differences exist between the United States and Canada in terms of availability and approval dates for drugs.##REF##16472888##21## Consequently, we sought out drugs that were included in US marketing campaigns started between January 2003 and December 2005; not advertised on Canadian television with a brand name; and approved for use in Canada before US advertising, to allow estimation of the marginal effect of direct to consumer advertising on prescribing.</p>", "<p>On the basis of these characteristics, we identified three study drugs. The first eligible drug was etanercept (Enbrel), a biological agent approved in Canada for the treatment of symptom refractory rheumatoid arthritis. Direct to consumer advertising for etanercept started in January 2003, and US network news advertising started in March 2003.##UREF##10##20## The second eligible drug was mometasone (Nasonex), an inhaled nasal steroid spray for symptoms of allergy. Direct to consumer advertising for mometasone started in December 2004, and the Vanderbilt database showed extensive US news advertising starting the same month.##UREF##10##20## Thirdly, tegaserod (Zelnorm) is a serotonin receptor agonist approved for the treatment of constipation predominant irritable bowel syndrome in women. When released, it was the only drug approved specifically for this indication in Canada. Although direct to consumer advertising began in February 2003, tegaserod’s most influential and major campaign first aired in August 2003 and featured memorable written messages such as “I feel better” on actresses’ stomachs.##REF##17476002##6## This later campaign was considered very successful from a marketing perspective, and even won major advertising industry awards, before the drug was withdrawn in both Canada and the United States owing to concern about cardiac side effects.##REF##17476002##6##\n##UREF##11##22## The Vanderbilt database indicates that US newscast advertising for tegaserod occurred in 1-12 September 2003 and subsequently in March 2004.##UREF##10##20##</p>", "<title>Analysis</title>", "<p>We used regional differences in exposure to investigate the impact of direct to consumer advertising. As all US advertising was in English, we hypothesised that changes in prescribing in Canada would be concentrated in predominantly English speaking provinces. Although French speaking Canadians watch a similar amount of television, they view much less foreign sourced television, estimated at less than 5% of all viewing.##UREF##6##16## Consequently, we analysed the difference in prescribing rates between predominantly English speaking provinces (n=8) and Quebec, where French is the mother tongue for more than 80% of the population.##UREF##12##23## Quebec is also attractive as a control as it has one of the least restrictive public drug formularies in Canada but has comparable universal health insurance coverage, age, sex, and income profiles to the other provinces.##UREF##13##24##\n##UREF##14##25##</p>", "<p>We used interrupted time series analysis, one of the strongest quasi-experimental designs available, to examine longitudinal changes in Canadian prescribing rates.##REF##12174032##26## Firstly, we calculated the difference in the prescribing rate per 10 000 population by subtracting the rate in French speaking provinces from that in English speaking provinces. We then fitted time series models to test whether a statistically significant change occurred in the level or trend of the difference after the start of US advertising or US national network news advertising, controlling for the pre-direct to consumer advertising level and trend. This method simultaneously controlled for any pre-advertising differences in the absolute level of prescribing between the provinces as well as any differences in pre-advertising temporal trends related to changes in the rates of prescribing between provinces. We also did a sensitivity analysis using the ratio of English and French prescribing rates instead of the difference. The results and interpretation of this analysis (not shown) were consistent with those shown below. For drugs that showed any significant impact of direct to consumer advertising in Canada, we did a sensitivity analysis using data from US Medicaid programmes. We also did this with an interrupted time series analysis but without an “unexposed” US control group. Although this method is uncontrolled compared with the Canadian analyses, it still controlled for pre-direct to consumer advertising trends in drug use. We used a generalised least squares model allowing for a first order autoregressive correlation between consecutive months or quarters and excluded the advertising start month in Canada. We validated our use of this autocorrelation structure by using likelihood ratio tests. Moreover, alternative models with no or longer autocorrelation structures led to results with very similar estimates and identical interpretations.</p>" ]
[ "<title>Results</title>", "<p>Table 1 describes the US advertising campaigns and Canadian approval dates for the three study drugs. All three drugs had large direct to consumer advertising expenditures, ranging from US$194 million to $314 million during the study period. Pre-advertising trends in use for each of the study drugs were generally comparable between English speaking and French speaking provinces (figs 1, 2 and 3). We found that US direct to consumer advertising led to increased Canadian prescribing rates for only one of the three drugs, tegaserod.</p>", "<title>Etanercept</title>", "<p>Figure 1 shows the times series of monthly prescribing rates of etanercept in Canada, which were very similar in both language regions. We found that advertising had no statistically significant impact on the level or trend of differences in prescribing rate between English speaking and French speaking provinces (level change −0.18 prescriptions per 10 000 population, 95% confidence interval −0.39 to 0.04, P=0.10; trend change −0.03 prescriptions per 10 000 population per month, −0.06 to 0.003, P=0.07).</p>", "<title>Mometasone</title>", "<p>Figure 2 shows the monthly prescribing rates for mometasone in English speaking and French speaking Canadian provinces. As with etanercept, we saw no clinically important or statistically significant change in the level or trend of differences in prescribing rate between English speaking and French speaking provinces (level change −3.61 prescriptions per 10 000 population, −10.51 to 3.29, P=0.30; trend change −0.08 prescriptions per 10 000 population per month, −0.57 to 0.40, P=0.73).</p>", "<title>Tegaserod</title>", "<p>In contrast to the first two drugs described, US direct to consumer advertising for tegaserod seemed to have a strong influence on Canadian prescribing. Figure 3 shows the monthly prescribing rates for tegaserod. The February 2003 campaign, which contained no US network news advertising, had no significant impact on prescribing rates and was incorporated into the pre-advertising period. In contrast, a level increase of 0.56 prescriptions per 10 000 population (0.37 to 0.76, P&lt;0.001) in the difference in prescribing rate between English speaking and French speaking provinces occurred immediately after the August 2003 campaign. We found no statistically significant change in trend (−0.003 prescriptions per 10 000 population per month, −0.03 to 0.02, P=0.77). Overall, this represents an estimated 42% increase in the first month after direct to consumer advertising. However, this difference did not persist despite continued advertising throughout the study period. Within two years of direct to consumer advertising, prescribing rates were again virtually identical between English speaking and French speaking regions.</p>", "<p>Using the same start date for direct to consumer advertising, we found a similar increase in Medicaid prescription rates of tegaserod. Figure 4 shows that the pre-advertising upward trend in tegaserod use was substantially higher in US Medicaid than in Canada. After national network news direct to consumer advertising, we saw an increase in the level of prescribing in the United States; the number of prescriptions per 10 000 enrolees increased by 5.70 (3.65 to 7.75, P&lt;0.001). As in Canada, we found no statistically significant change in prescribing trends (−0.62 prescriptions per 10 000 enrolees per quarter, −1.52 to 0.27, P=0.15). Overall, the estimated increase in prescribing in the first quarter of direct to consumer advertising was 56% higher than would have been expected and greater than the 42% increase seen in Canada.</p>" ]
[ "<title>Discussion</title>", "<p>During the past decade, drug manufacturers have substantially increased spending on direct to consumer advertising.##REF##17699817##1## To our knowledge, this study is the first analysis that uses a concurrent control group to evaluate the impact of such advertising on use of specific drugs. We found that for two of three drugs the US direct to consumer advertising had no apparent impact on Canadian prescribing rates, and for one drug (tegaserod) we saw a short lived effect. These mixed findings are surprising, as we included several expensive advertising campaigns that were highly recalled by consumers.##UREF##15##27##\n##UREF##16##28## Our empirical results raise important questions about whether and how prescribing trends for specific drugs respond to advertising directed at consumers. Thus, they have important implications for the ongoing debate about the benefits and harms of direct to consumer advertising.</p>", "<title>Possible explanations</title>", "<p>We believe that the differential responses to direct to consumer advertising that we saw may be related to the characteristics of the drugs examined. Although all of the study drugs are primarily used for relieving symptoms, they differ in important ways. For example, etanercept requires referral to a specialist and intravenous administration, making the pathway between direct to consumer advertising and drug use complicated. Thus, the effect of advertising probably differs substantially from that of drugs prescribed predominantly in primary care settings. Furthermore, tegaserod, unlike the other study drugs, was the only drug approved for its indication in Canada.##UREF##17##29##\n##UREF##18##30## In contrast, the other drugs studied all had competitors within the same drug class. In such markets, direct to consumer advertising might protect against drops in levels of use, rather than expanding use. Other characteristics, such as effectiveness, may also be important. A meta-analysis of short term placebo controlled trials of tegaserod indicates that the number needed to treat for one patient to have some improvement in their gastrointestinal symptoms is about 17, suggesting that most patients trying tegaserod for the first time were unlikely to derive symptomatic benefit.##UREF##18##30## This may explain, in part, why the changes in use for this drug were short lived.</p>", "<p>Our results also suggest that when direct to consumer advertising does increase use, a dose-response relation with the level of exposure to advertising exists. Our results in US Medicaid programmes estimated a larger increase than in Canada, in both absolute and percentage terms. Although the immediate change in use in the United States was larger than in Canada, assessing the comparative long term effect of advertising in the United States is difficult, because no concurrent control group is available. Nevertheless, the observed US Medicaid prescribing rates returned to the pre-direct to consumer advertising trend around the same time as in Canada (mid-2005). Furthermore, use of tegaserod was both higher and growing faster in Medicaid before direct to consumer advertising, suggesting that other factors were driving these differential trends. For example, we cannot rule out between country differences in physician directed marketing activities.##REF##14567371##31##</p>", "<title>Strengths and limitations</title>", "<p>The major strength of our study is the use of a strong quasi-experimental design with a comparable and concurrent control group. Moreover, our study design controlled for difference in both pre-existing level and trend and explicitly considered the timing of advertising campaigns. This method controls for differences in characteristics between language regions of Canada that remained constant or changed predictably over time, such as culture or patterns of general medical practice. Indeed, other differences such as variation in provincial drug reimbursement plans would bias our results only if they coincidentally changed when the individual direct to consumer advertising campaigns started. We could find no evidence that this occurred for any of the drugs studied. None the less, exclusion from provincial formularies might constrain the effects of successful advertising campaigns. However, most private insurance plans in Canada do not have formularies and cover most of the population.##UREF##19##32## Moreover, although Ontario and Alberta both excluded tegaserod from their public drug programmes, the effect of direct to consumer advertising was apparent in both provinces (data not shown).</p>", "<p>The study has other limitations. Firstly, generalising beyond the three drugs that met our inclusion criteria is difficult. Secondly, we do not have information on whether these drugs were subject to disease awareness advertising by companies that did not mention the brand name. However, this would bias our results only if it was similarly timed, and we found no indication for mometasone or etanercept of increased use coincident with branded direct to consumer advertising, thus making it unlikely. Thirdly, variation in drug coverage, the overall health system, culture, levels of exposure to advertising, or television viewing patterns might result in the effect of direct to consumer advertising differing between drugs and between countries. However, the percentage increase in and duration of effect for tegaserod was similar in both countries. In terms of drug coverage, more than 60% of Canadians are covered by generally unrestrictive employer based private drug plans, and less than 20% of these plans use formularies to limit access to specific drugs.##UREF##19##32## Although the Canadian and US health systems vary substantially, studies indicate similar access to primary care and willingness of physicians to fulfil patients’ requests for specific prescription drugs in the two countries.##REF##12952801##15##\n##UREF##20##33## Overall, the striking initial effect of direct to consumer advertising on tegaserod prescribing rates provides evidence that exposure to US advertising is sufficient to influence Canadian prescribing.</p>", "<title>Implications</title>", "<p>The implications of our analysis are threefold. Firstly, it indicates that illicit cross border exposure to direct to consumer advertising has the potential to modify drug use, even where such advertising is technically prohibited. As advertising over global mediums such as the internet increases, this phenomenon may grow in importance. Secondly, to our knowledge, these results are the strongest evidence that direct to consumer advertising can increase use of a drug that was removed from the market as a result of concerns about safety. Finally, our findings suggest that the impact of direct to consumer advertising campaigns is mixed, as they seem to work for some drugs and not others. If the overall impact of direct to consumer advertising is limited or variable, then a substantial portion of expenditure on such advertising—borne by governments, insurers, and patients in the form of higher costs or by companies as reduced profits—may be better spent elsewhere. Previous commentary may have overemphasised the impact of direct to consumer advertising for many individual drugs for which evidence that it increases use is either weak or non-existent.##REF##9929095##2## Until we better understand how direct to consumer advertising modifies prescribing for particular drugs, debates about its positive and negative consequences will continue to be based on conjecture rather than strong evidence.</p>" ]
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[ "<p><bold>Objective</bold> To assess the impact of direct to consumer advertising of prescription drugs in the United States on Canadian prescribing rates for three heavily marketed drugs—etanercept, mometasone, and tegaserod.</p>", "<p><bold>Design</bold> Controlled quasi-experimental study using interrupted time series analysis.</p>", "<p><bold>Population</bold> Representative sample of 2700 Canadian pharmacies and prescription data from 50 US Medicaid programmes.</p>", "<p><bold>Main outcome measures</bold> Differences in number of filled prescriptions per 10 000 population per month between English speaking and French speaking (control) Canadian provinces before and after the start of direct to consumer advertising in the United States.</p>", "<p><bold>Results</bold> Spending on direct to consumer advertising for study drugs ranged from $194m to $314m (£104m-£169m; €131m-€212m) over the study period. Prescription rates for etanercept and mometasone did not increase in English speaking provinces relative to French speaking controls after the start of direct to consumer advertising. In contrast, tegaserod prescriptions increased 42% (0.56 prescriptions/10 000 residents, 95% confidence interval 0.37 to 0.76) in English speaking provinces immediately after the start of US direct to consumer advertising. Uncontrolled analysis of US Medicaid data showed a larger 56% increase in tegaserod prescriptions. However, this increase did not persist over time in either country, despite continued advertising.</p>", "<p><bold>Conclusions</bold> Exposure to US direct to consumer advertising transiently influenced both Canadian and US prescribing rates for tegaserod, a drug later withdrawn owing to safety concerns. The impact of direct to consumer advertising on drug use seems to be highly variable and probably depends on the characteristics of the advertised drug, the level of exposure to direct to consumer advertising, and the cultural context.</p>" ]
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[ "<p><bold>Cite this as:</bold>\n<italic>BMJ</italic> 2008;337:a1055</p>" ]
[ "<fig id=\"fig1\" position=\"float\"><caption><p><bold>Fig 1</bold> Number of etanercept prescriptions per 10 000 population per month in Canadian provinces that are predominantly English speaking (n=8) or French speaking (n=1). Vertical line indicates start of US advertising in January 2003. Difference between rates shown at bottom of chart; fitted trend line shows predicted differences from interrupted time series regression. DTCA=direct to consumer advertising</p></caption></fig>", "<fig id=\"fig2\" position=\"float\"><caption><p><bold>Fig 2</bold> Number of mometasone prescriptions per 10 000 population per month in Canadian provinces that are predominantly English speaking (n=8) or French speaking (n=1). Vertical line indicates start of US advertising in December 2004. Difference between rates shown at bottom of chart; fitted trend line shows predicted differences from interrupted time series regression</p></caption></fig>", "<fig id=\"fig3\" position=\"float\"><caption><p><bold>Fig 3</bold> Number of tegaserod prescriptions per 10 000 population per month in Canadian provinces that are predominantly English speaking (n=8) or French speaking (n=1). Vertical lines indicate start of US advertising in February 2003 and start of new TV advertising campaign in August 2003. Difference between rates shown at bottom of chart; fitted trend line shows predicted differences from interrupted time series regression. DTCA=direct to consumer advertising</p></caption></fig>", "<fig id=\"fig4\" position=\"float\"><caption><p><bold>Fig 4</bold> Number of tegaserod prescriptions per 10 000 enrolees per quarter in US Medicaid programmes. Vertical line indicates start of new TV advertising campaign in third quarter of 2003. Fitted trend line represents fitted interrupted time series analysis for rate of use in Medicaid</p></caption></fig>" ]
[ "<table-wrap id=\"tbl1\" position=\"float\"><caption><p>US approval and advertising dates and Canadian approval dates for study drugs</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"2\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Drug</th><th colspan=\"3\" rowspan=\"1\" align=\"left\" valign=\"bottom\">United States</th><th colspan=\"1\" rowspan=\"2\" align=\"left\" valign=\"bottom\"/><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Canada</th></tr><tr><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Generic name</th><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Brand name</th><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Approval</th><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Advertising start</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">DTCA spending to 2006 ($m)</th><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Approval</th></tr></thead><tbody><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Etanercept</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Enbrel</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">November 1998</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">January 2003</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">$194</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">December 2000</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Mometasone</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Nasonex</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">October 1997</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">December 2004</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">$235</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">July 1998</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Tegaserod</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Zelnorm</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">July 2002</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">February 2003</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">$314</td><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">March 2002</td></tr></tbody></table></table-wrap>" ]
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[ "<boxed-text position=\"float\" content-type=\"style3\"><sec><title>What is already known on this topic</title><list list-type=\"simple\"><list-item><p>Although direct to consumer advertising (DTCA) of prescription drugs remains controversial, no controlled studies have investigated its impact on prescribing</p></list-item><list-item><p>In the absence of such evidence, both opponents and proponents of DTCA have generally assumed it to be highly effective at increasing the use of advertised drugs</p></list-item></list></sec><sec><title>What this study adds</title><list list-type=\"simple\"><list-item><p>DTCA campaigns seem to have mixed effectiveness; drug use did not increase for two of three drugs studied</p></list-item><list-item><p>Despite prohibitions, DTCA can influence prescribing across national borders</p></list-item><list-item><p>The drug (tegaserod) for which use increased with DTCA was eventually withdrawn owing to safety concerns</p></list-item></list></sec></boxed-text>" ]
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[ "<table-wrap-foot><p>DTCA=direct to consumer advertising.</p><p>Start dates and US advertising values are from TNS Media Intelligence. Data include spending on network and cable television, magazine, newspaper, radio, and billboard advertising.</p></table-wrap-foot>", "<fn-group><fn><p>We are grateful to IMS Health Canada, ESI Canada, and Eloda for providing data for this research. We also thank Alyce Adams, Katherine Swartz, Cory Cowan, Ellen Aquilina, Véronique Laramée, Katy Backes Kozhimannil, and Rebecca Anhang-Price for valuable assistance.</p></fn><fn fn-type=\"participating-researchers\"><p>Contributors: MRL participated in the design of the study protocol, data collection, statistical analysis, and drafting and revision of the manuscript. SRM participated in the design of the study protocol, data collection, and drafting and revision of the manuscript. SBS participated in the design of the study protocol and drafting and revision of the manuscript. All authors accept full responsibility for the work and/or the conduct of the study, had access to the data, and controlled the decision to publish. MRL is the guarantor.</p></fn><fn fn-type=\"financial-disclosure\"><p>Funding: MRL is supported by the Thomas O Pyle Fellowship and the Fellowship in Pharmaceutical Policy at Harvard Medical School and by a Social Sciences and Humanities Research Council of Canada doctoral fellowship. SRM receives salary support from the Alberta Heritage Foundation for Medical Research (health scholar) and the Canadian Institutes of Health Research (new investigator). SBS is an investigator in the HMO Research Network Centers for Education and Research on Therapeutics, supported by the Agency for Healthcare Research and Quality (grant No. U18HS010391). The funding and data sources for this study had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.</p></fn><fn fn-type=\"conflict\"><p>Competing interests: None declared.</p></fn><fn><p>Ethical approval: The research was appraised and granted an exemption from review by the Harvard Pilgrim Health Care Human Studies Committee.</p></fn><fn><p>Provenance and peer review: Not commissioned; externally peer reviewed.</p></fn></fn-group>" ]
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{ "acronym": [], "definition": [] }
33
CC BY
no
2022-01-12 21:41:47
BMJ. 2008 Sep 2; 337:a1055
oa_package/e5/04/PMC2528895.tar.gz
PMC2528896
18765450
[ "<title>Introduction</title>", "<p>Fever is a normal part of childhood illness, affecting around 70% of preschool children yearly.##REF##15897210##1## It can be miserable for the child, cause anxiety for parents,##REF##8892420##2## and be expensive for health services. Up to 40% of preschool children see a health professional for a febrile illness each year.##REF##15897210##1## Although fever is considered by many to be an advantageous evolutionary byproduct of the host response to infection, and as such should not be treated,##REF##1681362##3## the use of antipyretics is widespread. The reasons for treating fever are contested and not necessarily evidence based but include minimising discomfort, controlling the fever, and preventing febrile convulsions.</p>", "<p>Options for treating fever include physical measures (taking cool fluids and dressing lightly) and the antipyretic drugs paracetamol (acetaminophen) and ibuprofen. Evidence for physical measures is now redundant as it mostly pertains to tepid sponging,##UREF##0##4## which is no longer recommended.##UREF##1##5## Paracetamol and ibuprofen have both been shown to be superior to placebo##REF##1941390##6##\n##REF##2663318##7##\n##REF##4878632##8## and ibuprofen superior to paracetamol##REF##15184213##9## for the relief of fever. Given that the drugs have different mechanisms of action##REF##11566461##10## it is possible that they are more effective together than when used alone, but the evidence to date is sparse and conflicting. Five published trials##REF##11262983##11##\n##REF##16464962##12##\n##REF##16461878##13##\n##REF##16515705##14##\n##UREF##2##15## mostly tested the effects of single doses at selected time points (which can arbitrarily advantage one drug because of the difference in times to maximum effect##REF##1505153##16##), were largely done in secondary care, and reached conflicting conclusions. Recently published UK guidelines##UREF##1##5## advise the use of either drug (no preference stated) for children with fever who are unwell or distressed and state that owing to the lack of evidence the drugs should not be used together or alternately.</p>", "<p>We carried out a community based, three arm, blinded, randomised controlled trial to investigate the relative clinical effectiveness of multiple doses (as used for most episodes of fever) of paracetamol plus ibuprofen compared with either drug alone. Our investigation into the relative cost effectiveness is reported in an accompanying paper.##UREF##3##17##</p>" ]
[ "<title>Methods</title>", "<p>We recruited and followed up children between January 2005 and May 2007 using three strategies: local, remote, and community. We invited all NHS organisations providing primary care services in Bristol to assist with recruitment to the trial, including NHS Direct, the walk-in centres, all general practices, the general practitioner out of hours cooperatives, and the emergency department of the Bristol Royal Hospital for Children.</p>", "<p>During local recruitment the NHS sites invited parents of appropriately aged children to discuss the study with our research nurses, who were present in the waiting rooms. In the remote strategy, clinicians faxed the details of potentially eligible children to the trial administrator, who notified the research nurses. In the community strategy, parents were invited to contact the trial directly by telephone. The telephone number was promoted during local and remote recruitment and in local newspaper and radio advertisements. When parents made contact, the trial administrator notified the research nurses of potentially eligible children.</p>", "<p>Once aware of potentially eligible children identified through any of the recruitment strategies, research nurses contacted parents by telephone to arrange a meeting (usually at home) to explain the trial fully and to verify eligibility.</p>", "<title>Participants</title>", "<p>We included children if they were aged between 6 months and 6 years and were unwell with a temperature of at least 37.8°C and up to 41.0°C as a result of illnesses that could be managed at home. We excluded children if they required hospital admission; were clinically dehydrated; had recently participated in another trial; had previously participated in PITCH; had a known intolerance, allergy, or contraindication to a trial drug##UREF##4##18##; had a chronic neurological, cardiac, pulmonary (except asthma), liver, or renal disease; or had parents who could not read or write in English. We followed up children at 24 and 48 hours and at day 5.</p>", "<title>Randomisation</title>", "<p>After written informed consent had been obtained and the baseline questionnaire completed, the research nurse telephoned a remote, automated randomisation service. Allocation to one of three trial arms (paracetamol plus ibuprofen, paracetamol alone, ibuprofen alone) was minimised##REF##9694748##19## by age (6-17 months <italic>v</italic> 18-71 months), severity of fever (37.8°C to 38.9°C <italic>v</italic> 39.0°C to 41.0°C), discomfort scale (“normal” or “not quite normal” <italic>v</italic> “some distress” or “very distressed”), previous duration of fever (≤24 hours <italic>v</italic> &gt;24 hours), and current antibiotic use (yes <italic>v</italic> no).</p>", "<title>Intervention</title>", "<p>Parents were given standardised verbal and written advice on the appropriate use of loose clothing and encouraging children to take cool fluids. The intervention was the provision of, and advice to give, the study drugs for up to 48 hours: paracetamol every 4-6 hours (maximum of four doses in 24 hours) and ibuprofen every 6-8 hours (maximum of three doses in 24 hours). Parents, research nurses, and investigators were blinded to treatment allocation by the use of identically matched placebo drugs. All parents received two medicine bottles; either both active or one containing the active drug and the other placebo. Given the differences in dosing, the parents were aware of which was paracetamol/placebo and which was ibuprofen/placebo. All liquid suspensions were sugar-free and supplied in licensed containers with child resistant caps. The dose of drug was determined by the child’s weight: paracetamol 15 mg/kg per dose and ibuprofen 10 mg/kg per dose. At the baseline visit and before randomisation the research nurse weighed the child, undressed to one layer, using scales approved for use in children (Seca, UK). Randomisation was abandoned if weight could not be established and administration of the study drug was deemed unsafe. The research nurse initially calculated the volume of suspension per dose (to the nearest 0.5 ml), which was confirmed during randomisation. The bottles of active drug contained the standard concentrations: 120 mg of paracetamol per 5 ml and 100 mg of ibuprofen per 5 ml.</p>", "<p>The first doses were given in the presence of the research nurse and were timed to coincide with the child’s next due dose of drug—that is, at least four hours after the last dose of paracetamol and six hours after that of ibuprofen, and were never such that the maximum number of doses over a 24 hour period was exceeded. The order in which the first drug was given was determined randomly. We recorded the time that the drug was swallowed and designated that as time zero. The first four hours, after children were observed to be given the drugs and before any further drug was given, was regarded as the “efficacy period.” We asked the parents to give the drugs regularly from four to 24 hours (“proactive period”). Figure 1 describes the intervention period for the first 24 hours. We asked parents to give the drugs between 24 and 48 hours in response to their child’s symptoms (“reactive period”). At 48 hours we retrieved the study drugs and advised the parents to use over the counter preparations as required until day 5.</p>", "<title>Outcomes</title>", "<p>We timed all outcomes in relation to the administration of the first drug doses. The primary outcomes were the number of minutes without fever (&lt;37.2°C) in the first four hours and the proportion of children reported as being normal on the discomfort scale at 48 hours. Secondary outcomes were collected at three time points. In the first 24 hours we recorded the time to temperature first falling below 37.2°C (fever clearance), the time spent without fever over 24 hours, and the proportion of children without fever associated symptoms: discomfort, reduced activity, reduced appetite, and disturbed sleep. At 48 hours and day 5 we obtained data on fever associated symptoms and temperature measured by parents. At all time points we asked parents about adverse effects.</p>", "<p>We measured time without fever using a technique similar to that in a previous study.##REF##1463951##20## Using a data logger (OM-CP-RTDTEMP110; Omega Engineering, Stamford, CT) connected to an axillary temperature probe, we measured and recorded temperature every 30 seconds. Parents were asked to help their child keep the logger on for 24 hours. With support from research nurses the parents completed symptom diaries on discomfort, sleep, appetite, and activity using ordered categorical scales. Parents were asked to enter the value best representing their child’s state at the time of recording or in the previous 10 minutes. They also recorded adverse effects (defined as new symptoms or worsening of pre-existing symptoms##UREF##5##21##) and temperature, which they measured with a standard digital axillary thermometer.</p>", "<title>Sample size</title>", "<p>In the original protocol the target difference for the time spent without fever in the first four hours was 30 minutes (with an estimated standard deviation of 80 minutes##REF##1463951##20##) and that for the binary outcome of being rated normal on the discomfort scale at 48 hours was 60% compared with 75% (equivalent to an odds ratio of 2.0). To detect the latter comparison with 90% power and a two sided α of 0.027 (allowing for multiple comparisons between the combined therapy group and each of the two single therapy groups##UREF##6##22##) we required a total sample size of 747 children. Difficulties with recruitment led to the addition of the remote and community methods and a reduced achievable sample size. For time without fever we estimated a revised standard deviation of 50 minutes on the basis of the first 50 children (independent of allocation group). Along with a revised 80% power, we determined that a total sample size of 180 would allow the detection of the original target difference of 30 minutes. Sensitivity to differences in discomfort was, however, reduced, with odds ratios of only 4 or more being detectable.</p>", "<title>Statistical analyses</title>", "<p>We obtained descriptive statistics to characterise children, assess baseline comparability, and compare side effects. Comparative analyses were done in Stata 9 on an intention to treat basis using linear or logistic regression and adjusting for minimisation variables. Primary comparisons were between paracetamol plus ibuprofen and either drug alone, and secondary comparisons were between paracetamol and ibuprofen, using Dunnett’s and Tukey’s adjustments, respectively, for multiple comparisons.##UREF##6##22## For all “time without fever” analyses we regarded as valid only biologically plausible temperatures of more than 33°C and less than 45°C. In regression models we used the proportion of valid time under the fever threshold (with results converted into minutes or hours for presentational purposes) and we weighted these according to the amount of valid data. Secondary analyses included additional adjustment for factors showing possible imbalance at baseline and preplanned exploratory analyses for differential effects of paracetamol plus ibuprofen compared with paracetamol alone or ibuprofen alone for baseline age, temperature, discomfort, antibiotic use, and presence of otitis media. We selected otitis media because affected children might experience enhanced effects for both fever and pain.</p>" ]
[ "<title>Results</title>", "<p>Thirty five primary care sites in Bristol agreed to take part in the trial: NHS Direct, one walk-in centre, 30 general practices, two general practitioner out of hours cooperatives, and the emergency department of the Bristol Royal Hospital for Children. Figure 2 shows the numbers of children recruited through the three different methods. Overall, 4515 contacts were made, of which 3477 children were ineligible, most commonly (89%) because of insufficient fever. The remaining 1038 children were potentially eligible, but the temperature criterion before randomisation could not be verified in 882 because the parents did not want to commit to the study or had concerns about the drugs (669 declined) or the parents saw a clinician but left without contacting the study team (213 missed). No parent declined at randomisation, and attrition was minimal. Deviations from the protocol occurred; in the first 24 hours (23 hours and 40 minutes), 13 (7) children received an erroneous fifth dose of paracetamol and similarly 18 (13) children an erroneous fourth dose of ibuprofen. In four children, clinicians and parents but not research staff were unblinded to treatment allocation.</p>", "<title>Descriptive results</title>", "<p>The groups were comparable at baseline, although potentially influential differences existed for sex, method of recruitment, and activity (table 1). Since additional adjustment for these variables had negligible effects in all analyses only minimisation variables were adjusted for in the comparative analyses. Nearly all the children were unwell, with more than 90% experiencing discomfort, reduced activity, abnormal appetite, or abnormal sleep (table 1).</p>", "<p>The median time between randomisation and giving the first dose of study drug was eight minutes for paracetamol plus ibuprofen and nine minutes for paracetamol and for ibuprofen. The mean number of valid minutes for time without fever (temperature &gt;33°C and &lt;45°C) in the first four hours (240 minutes) was 219 for children receiving paracetamol, 211 for ibuprofen, and 202 for paracetamol plus ibuprofen. The respective times over 24 hours (1440 minutes) were 1078, 1029, and 1051 minutes. For time without fever in the first four valid hours (and the corresponding secondary outcome within 24 valid hours), children receiving paracetamol plus ibuprofen spent more time without fever than those given ibuprofen and, in turn, those given paracetamol (table 2). Fever clearance was faster in children given paracetamol plus ibuprofen than in those given paracetamol but was similar for those given ibuprofen. Children given paracetamol plus ibuprofen spent less time with fever over 24 hours than those given either drug alone. A suggestion was that more fever associated symptoms had normalised in children given ibuprofen than the other treatments at 24 and 48 hours, but by day 5 these trends had largely disappeared.</p>", "<title>Comparative analyses</title>", "<title>Primary outcomes</title>", "<p>Strong evidence was found of more time spent without fever in the first four hours among children given paracetamol plus ibuprofen than those given paracetamol, and likewise for children given ibuprofen than those given paracetamol (table 3). Moreover, both point estimates exceeded the 30 minute target difference, as did the lower confidence limit for the primary comparison. The confidence interval and P value suggest little difference between giving paracetamol plus ibuprofen and giving ibuprofen alone.</p>", "<p>The low power for fever associated discomfort at 48 hours was reflected by the large P values and wide confidence intervals for all three comparisons, although the largest point estimate and upper confidence limit favoured ibuprofen over paracetamol. The lowest P value from subgroup analyses for the primary outcomes was 0.14.</p>", "<title>Secondary outcomes</title>", "<p>The comparison of fever clearance was consistent with the primary outcome for time without fever: strong evidence suggested that paracetamol plus ibuprofen had a faster effect than paracetamol alone, and ibuprofen alone had a faster effect than paracetamol alone (table 4). Giving paracetamol plus ibuprofen over 24 hours increased time without fever by 4.4 hours compared with paracetamol and by 2.5 hours compared with ibuprofen.</p>", "<p>No consistent evidence of effect for fever associated symptoms from 24 hours to day 5 was seen, but odds ratios tended to favour ibuprofen more than the other treatments at 24 and 48 hours (data not shown).</p>", "<title>Mean temperature by treatment group</title>", "<p>Figure 3 shows the mean temperature every 15 minutes by treatment group with the proportion of children febrile at corresponding two hourly time points. Ibuprofen and paracetamol plus ibuprofen reduced children’s temperatures faster and for longer than paracetamol in the first four hours, and paracetamol plus ibuprofen was superior to either drug alone in reducing mean temperatures over 24 hours. A rise in mean temperature was seen for children in the ibuprofen group, which then fell just after six hours, coinciding with the earliest time that parents were advised that a second dose of ibuprofen could be given. This rise may have been prevented in the other groups by paracetamol, which could have been given at four hours.</p>", "<p>The mean temperatures in the graph are lower than might be expected biologically. This could be explained by the choice of axillary thermometry, which is known to record temperatures around 0.8°C lower than rectal digital thermometers,##REF##10784539##23## or by the liberal definition of valid temperature used in this study, or both. A sensitivity analysis excluding temperatures below 35°C raised the mean temperatures but not the relative positions of the group means.</p>", "<title>Relation between discomfort and temperature</title>", "<p>Given the low power for treatment effects on discomfort, a repeated measure analysis was used to explore the relation between all discomfort measures recorded across up to eight time points to 48 hours and their coinciding mean digital axillary thermometer measures. The mean temperatures were 36.4°C for children who scored normal on the discomfort scale, 37.2°C for those who scored not quite normal, 38.1°C for those who scored some pain or distress, and 38.3°C for those who scored crying or very distressed.</p>", "<title>Adverse effects</title>", "<p>The most common adverse effects were diarrhoea and vomiting, which were equally distributed between groups (table 5). The overall number of children experiencing adverse events was, however, too small to make meaningful comparisons between treatments. Five children were admitted to hospital (constituting serious adverse events##UREF##5##21##): one child in the paracetamol group, three in the ibuprofen group, and one in the paracetamol plus ibuprofen group. On independent review none was considered to be related to the study process or drugs.</p>", "<title>Dosing of study drugs</title>", "<p>All 52 children in each of the three groups were given, as per protocol, their first dose of study drug under nurse supervision (table 6). The recommended maximum four doses of paracetamol or placebo in the first 24 hours was received by 65% of children given paracetamol, 46% given ibuprofen, and 42% given paracetamol plus ibuprofen, with this recommended maximum exceeded by 12%, 6%, and 8%, respectively. The corresponding percentages receiving the recommended maximum three doses of ibuprofen or placebo in 24 hours were 73%, 75%, and 71% and those exceeding this recommended maximum were 13%, 12%, and 13%. All percentages were much lower at 48 hours.</p>", "<title>Blinding</title>", "<p>The success of blinding was assessed at the nurse’s visit at 48 hours, when parents were asked to guess treatment allocation. Taking “I don’t know” responses to either drug as failure to guess correctly, allocation was guessed correctly by 16 (31%) parents in the paracetamol group, 17 (33%) in the ibuprofen group, and 9 (17%) in the paracetamol plus ibuprofen group, compared with the 33% expected by chance. Excluding “I don’t know” responses increased these percentages to 50% (32 parents), 53% (n=32), and 43% (n=21), respectively.</p>" ]
[ "<title>Discussion</title>", "<p>In febrile children we found strong evidence of faster time to fever clearance and more prolonged time without fever in the first four hours favouring the use of paracetamol plus ibuprofen and ibuprofen over paracetamol, but no evidence of any difference between paracetamol plus ibuprofen and ibuprofen alone. In the first 24 hours strong evidence suggested more time without fever favouring paracetamol plus ibuprofen over either drug alone. We found no evidence of differences in fever associated discomfort at 48 hours. The frequency of adverse effects did not seem to differ between groups.</p>", "<title>Comparison with existing literature</title>", "<p>Using continuous thermometry we compared the effects of two antipyretics combined with either drug alone using maximum licensed, repeated doses in children recruited from and managed in the community. Previous studies have recruited from secondary care,##REF##11262983##11##\n##REF##16464962##12##\n##REF##16515705##14##\n##UREF##2##15## investigated the effects of single doses,##REF##16464962##12##\n##REF##16515705##14## and did not use continuous thermometry. The finding that ibuprofen was found to be more effective than paracetamol in the first four hours is consistent with the literature.##REF##15184213##9##</p>", "<title>Strengths and limitations of the study</title>", "<p>The study has four main strengths. Firstly, its internal validity: randomisation was concealed, nurses and investigators were blinded to allocation, and attrition was minimal. Secondly, the intervention and follow-up periods were long enough to enable a fair comparison between multiple doses of antipyretics with differing times to maximum effect.##REF##1505153##16## Thirdly, we used continuous thermometry to generate the objective and intuitive outcome of time without fever. Finally, we recruited and followed up children in the community, where most cases of fever are managed.</p>", "<p>We are aware of five possible weaknesses of the study. Firstly, because we had no placebo only group our data cannot inform the decision on whether to use antipyretics. This was a deliberate design decision as we thought that parents would not have participated if there had been a placebo only group. This judgment is supported by the fact that over 80% of parents in the study said that they would not have participated in such a trial. Three previous trials have, however, shown that paracetamol and ibuprofen given separately are more effective than placebo,##REF##1941390##6##\n##REF##2663318##7##\n##REF##4878632##8## and one trial found that paracetamol is more effective at relieving fever than unwrapping children.##REF##1463951##20##</p>", "<p>Secondly, the recruited sample did not give sufficient power to detect plausible differences in discomfort. This is disappointing, given the importance of this question to the public and research community. Other research has, however, suggested that the use of two drugs combined compared with one alone does confer additional benefit on symptoms##REF##16461878##13## and we did find a relation between increasing discomfort and worsening fever, suggesting that with adequate power the effects on symptoms might have followed those of temperature.</p>", "<p>Thirdly, an axillary temperature of 37.8°C might not be regarded as denoting fever. Since no agreed definition of fever or how to measure temperature exists,##REF##14741143##24## to a limited extent its selection was arbitrary. For example, disagreement between thermometer types and measurement sites means this could represent a rectal temperature of as much as 39.7°C.##REF##10784539##23## Temperature is such a dynamic variable that although many children did not meet our criterion for temperature before randomisation, most were already being treated for a febrile illness and their parents and doctors thought that treatment with up to two drugs was warranted. The mean temperature at baseline was 38.5°C (table 1), a temperature at which 90% of doctors and 70% of nurses would recommend treatment,##REF##11804771##25## and most of the children were unwell with febrile illness as it affected their comfort, appetite, activity, and sleep.</p>", "<p>Fourthly, the success of blinding was assessed at the 48 hour nurse visit by asking parents to guess which drugs were active. Overall, the 153 parents who responded were not able to guess treatment, but the 83 who expressed a definite opinion did identify allocation more often than would be expected by chance. Although we carried out blinded taste tests and volunteers could not distinguish placebo from active drugs, some parents may have been better able to do so because they had more time to compare study drugs with known products in the home as well as observing their children’s responses to treatment. Although this could have influenced the parental recording of the discomfort outcome, we do not see how it could influence the outcome of time without fever.</p>", "<p>Finally, given the challenges of recruitment, our sample might not be representative of the general population. For example, we do not know if the possibility of receiving either or both drugs combined and the severity of the child’s illness influenced parents’ decisions to participate. If this was the case, we think these factors are more likely to be associated with differences in parental attitudes to illness than the children’s response to the drugs. The most common reason for ineligibility was insufficient fever, a factor we think is unlikely to be associated with any other physiological marker of response to drugs.</p>", "<title>Implications of this research</title>", "<p>It is good practice for parents, nurses, and doctors who have made the decision to treat young, unwell children with fever, to use the minimum number of drugs possible.##UREF##1##5## Although other studies have shown that paracetamol is superior to placebo,##REF##1941390##6##\n##REF##2663318##7##\n##REF##4878632##8## our study suggests that those wanting to achieve faster and more prolonged fever relief in the first four hours should use ibuprofen in preference to paracetamol. Similarly, where symptoms are expected to last at least 24 hours (probably most children with more severe symptoms at the onset of illness), those wanting to maximise the time without fever should probably start with ibuprofen but also consider paracetamol plus ibuprofen. Pragmatically, although our trial design did not specifically address this, we speculate that if a child remains unwell after a first dose of ibuprofen, subsequent alternation of paracetamol and ibuprofen for 24 hours would be more effective than either drug alone. This speculation is supported by a recent study showing that paracetamol was more effective than placebo when added to ibuprofen.##REF##16515705##14## The decision to start with ibuprofen or paracetamol plus ibuprofen, however, should also be influenced by an assessment of the benefits (an additional 2.5 hours without fever) compared with the risk of unintentionally exceeding the maximum recommended dose owing to the additional complexity of using two drugs. This risk is not theoretical. Even in the context of this supervised trial, between 6% and 13% of parents exceeded the maximum number of recommended doses in the first 24 hours.</p>", "<p>The pragmatism of the intervention changed with time, moving from efficacy in the first four hours to effectiveness in the second 24 hours. By 48 hours, considerably fewer study drugs were being given and this could partly explain the observed lack of effects on discomfort at this time. In the community, paracetamol and ibuprofen are usually dosed by age, and we recognise that calculating doses by weight means the results may inform primary and secondary care practice more than practice at home. We decided against a dose by age regimen, however, for two reasons. Firstly, given the recommendation of the children’s national service framework to dose by weight##UREF##7##26## and the dose by weight presentations in the British national formulary for children,##UREF##4##18## we believe that in the future more medicines for children will be given by weight. Secondly, we wanted to ensure that heavier children for their age received a therapeutic dose and to avoid exceeding the normal recommended dose for children who were light for their age. Comparing dose by weight with dose by age shows that children can receive as much as 50% more##UREF##8##27## or 50% less paracetamol and 100% more ibuprofen.</p>", "<p>Medicine bottles in the United States contain dosing advice by both age and weight and although healthcare professionals can clearly calculate dose by weight, we think two steps are needed before parents can routinely use weight to determine dose in other countries. Firstly, studies should investigate the safety implications of any differences between estimates of children’s weights measured by parents using domestic scales (or recently recorded weights in parent held children’s health records) and those measured by professionals using paediatric scales. Secondly, suppliers of antipyretics could consider routinely including dose by weight tables. Given that the complexity of using two drugs over a 24 hour period is more likely to lead to inadvertently exceeding the maximum recommended dose, we also believe that multiple blank charts should be supplied for parents to record when medicines have been given and how much.</p>", "<p>Recent case reports have highlighted the concern about renal toxicity in dehydrated children given ibuprofen.##REF##17293366##28##\n##REF##14977711##29## Although this serious effect is rare, we excluded children with dehydration from our trial and believe that ibuprofen should not routinely be given to children with, or at risk of, dehydration. Good evidence shows, however, that ibuprofen is as safe as paracetamol for children with asthma, where there is no evidence of sensitivity to non-steroidal anti-inflammatory drugs.##REF##11826230##30##</p>", "<p>We agree with the guidelines for fever from the National Institute for Health and Clinical Excellence (NICE) that antipyretics should be used only when children have fever associated with other symptoms,##UREF##1##5## although further research is needed to establish the effectiveness of antipyretics for the relief of these symptoms. However, we believe that the guidance on the use of two drugs combined need not be so cautious now that there is good evidence of superiority for both drugs over one drug for increasing time without fever over 24 hours.</p>", "<title>Conclusion</title>", "<p>Doctors, nurses, pharmacists, and parents wanting to use medicines to treat young, unwell children with fever should be advised to use ibuprofen first and to consider the relative benefits and risks of using paracetamol plus ibuprofen over a 24 hour period. There is no evidence from the accompanying cost effectiveness evaluation to contradict these findings.##UREF##3##17##</p>" ]
[ "<title>Conclusion</title>", "<p>Doctors, nurses, pharmacists, and parents wanting to use medicines to treat young, unwell children with fever should be advised to use ibuprofen first and to consider the relative benefits and risks of using paracetamol plus ibuprofen over a 24 hour period. There is no evidence from the accompanying cost effectiveness evaluation to contradict these findings.##UREF##3##17##</p>" ]
[ "<p><bold>Objective</bold> To investigate whether paracetamol (acetaminophen) plus ibuprofen are superior to either drug alone for increasing time without fever and the relief of fever associated discomfort in febrile children managed at home.</p>", "<p><bold>Design</bold> Individually randomised, blinded, three arm trial.</p>", "<p><bold>Setting</bold> Primary care and households in England.</p>", "<p><bold>Participants</bold> Children aged between 6 months and 6 years with axillary temperatures of at least 37.8°C and up to 41.0°C.</p>", "<p><bold>Intervention</bold> Advice on physical measures to reduce temperature and the provision of, and advice to give, paracetamol plus ibuprofen, paracetamol alone, or ibuprofen alone.</p>", "<p><bold>Main outcome measures</bold> Primary outcomes were the time without fever (&lt;37.2°C) in the first four hours after the first dose was given and the proportion of children reported as being normal on the discomfort scale at 48 hours. Secondary outcomes were time to first occurrence of normal temperature (fever clearance), time without fever over 24 hours, fever associated symptoms, and adverse effects.</p>", "<p><bold>Results</bold> On an intention to treat basis, paracetamol plus ibuprofen were superior to paracetamol for less time with fever in the first four hours (adjusted difference 55 minutes, 95% confidence interval 33 to 77; P&lt;0.001) and may have been as good as ibuprofen (16 minutes, −7 to 39; P=0.2). For less time with fever over 24 hours, paracetamol plus ibuprofen were superior to paracetamol (4.4 hours, 2.4 to 6.3; P&lt;0.001) and to ibuprofen (2.5 hours, 0.6 to 4.4; P=0.008). Combined therapy cleared fever 23 minutes (2 to 45; P=0.025) faster than paracetamol alone but no faster than ibuprofen alone (−3 minutes, 18 to −24; P=0.8). No benefit was found for discomfort or other symptoms, although power was low for these outcomes. Adverse effects did not differ between groups.</p>", "<p><bold>Conclusion</bold> Parents, nurses, pharmacists, and doctors wanting to use medicines to supplement physical measures to maximise the time that children spend without fever should use ibuprofen first and consider the relative benefits and risks of using paracetamol plus ibuprofen over 24 hours.</p>", "<p><bold>Trial registration</bold> Current Controlled Trials ISRCTN26362730.</p>" ]
[]
[ "<p><bold>Cite this as:</bold>\n<italic>BMJ</italic> 2008;337:a1302</p>" ]
[ "<fig id=\"fig1\" position=\"float\"><caption><p><bold>Fig 1</bold> Use of study drugs during first 24 hours. Shaded areas represent time that drug was to be given</p></caption></fig>", "<fig id=\"fig2\" position=\"float\"><caption><p><bold>Fig 2</bold> Participant flow through trial</p></caption></fig>", "<fig id=\"fig3\" position=\"float\"><caption><p><bold>Fig 3</bold> Mean temperature over first 24 hours after randomisation, by treatment group. *All children had temperatures greater than 37.2°C at baseline eligibility assessment, as measured by standard digital axillary thermometry. Temperature measured using a data logger was less than 37.2°C for 19 children because of delays between digital thermometry measure and drug dosing and differences between digital and data logger thermometry methods</p></caption></fig>" ]
[ "<table-wrap id=\"tbl1\" position=\"float\"><label>Table 1</label><caption><p> Baseline characteristics of children with fever randomised to three treatment groups. Values are numbers (percentages) of children unless stated otherwise</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Characteristic</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol (n=52)</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Ibuprofen (n=52)</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol plus ibuprofen (n=52)</th></tr></thead><tbody><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Boy</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">26 (50)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">37 (71)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">25 (48)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Girl</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">26 (50)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">15 (29)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">27 (52)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Mean (SD) weight (kg)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">13.0 (4.2)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">13.4 (3.9)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">12.6 (3.3)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"justify\" valign=\"top\">Mean (SD) age (months)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">28.7 (17.7)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">28.1 (17.4)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">25.1 (13.4)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Age (months)*:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 6-17</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (38)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">18 (35)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">19 (37)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 18-71</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">32 (62)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">34 (65)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">33 (63)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Mean (SD) baseline temperature (°C)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38.6 (0.6)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38.6 (0.6)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38.6 (0.6)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Temperature (°C)*:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> &lt;39</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">37 (71)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">37 (71)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">39 (75)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 39-41</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">15 (29)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">15 (29)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">13 (25)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Fever duration (hours)*:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> ≤24</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">18 (35)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">19 (37)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">19 (37)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> &gt;24</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">34 (65)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">33 (63)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">33 (63)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Antibiotic use*:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Yes</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">14 (27)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">15 (29)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">17 (33)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> No</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38 (73)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">37 (71)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">35 (67)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Paracetamol use 4-6 hours before randomisation:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Yes</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (38)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">17 (33)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (38)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> No</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">32 (62)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">35 (67)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">32 (62)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Ibuprofen use 6-8 hours before randomisation:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Yes</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4 (8)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2 (4)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3 (6)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> No</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">48 (92)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">50 (96)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">49 (94)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Discomfort*:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3 (6)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5 (9)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5 (9)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Not quite normal</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">31 (60)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">27 (52)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">30 (58)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"justify\" valign=\"top\"> Some pain or distress</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">18 (34)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">18 (35)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">14 (27)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"justify\" valign=\"top\"> Crying or very distressed</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0 (0)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2 (4)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3 (6)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Activity:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3 (6)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4 (8)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4 (8)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Quiet for longer than usual</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">12 (23)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">18 (35)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">23 (45)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Hardly moving about</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">31 (60)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">19 (36)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">19 (36)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Not moving about willingly</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6 (11)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">11 (21)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6 (11)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Appetite:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5 (10)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3 (6)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4 (8)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Eating less than normal</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">12 (23)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">14 (27)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">10 (19)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Eating much less than normal</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">35 (67)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">33 (63)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">36 (69)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Vomiting or refusing food or drink</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0 (0)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2 (4)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2 (4)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Sleep:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8 (15)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3 (6)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4 (8)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> More than usual</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (38)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">21 (40)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (38)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> More disturbed than usual</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">9 (17)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">15 (29)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">10 (19)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> A lot more disturbed than usual</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">15 (29)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">13 (25)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">18 (35)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Recruitment method:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Local</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">17 (33)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">18 (35)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">10 (19)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Remote</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">27 (52)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">26 (50)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">31 (60)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Community</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8 (15)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8 (15)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">11 (21)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Ethnicity:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> White</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">47 (90)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">47 (90)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">44 (85)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Other</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5 (10)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5 (10)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8 (15)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Diagnosis:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Otitis media</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">7 (14)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">11 (20)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8 (14)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Respiratory tract infection</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">12 (23)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">15 (28)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">17 (33)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Non-specific viral illness</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">21 (40)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (37)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">16 (31)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Other</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">12 (23)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8 (15)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">11 (22)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Previous febrile convulsion:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Yes</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2 (4)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1 (2)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2 (4)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> No</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">50 (96)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">51(9)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">50 (96)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Asthma:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Yes</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">9 (17)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4 (8)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6 (12)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> No</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">43 (83)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">48 (92)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">46 (88)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tbl2\" position=\"float\"><label>Table 2</label><caption><p> Descriptive statistics of outcomes (time without fever and no discomfort) at selected times. Values are numbers (percentages) unless stated otherwise</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Outcomes</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol (n=52)</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Ibuprofen (n=52)</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol plus ibuprofen (n=52)</th></tr></thead><tbody><tr><td colspan=\"4\" rowspan=\"1\" align=\"left\" valign=\"top\" content-type=\"TableSubHead\"><bold>Primary outcomes</bold></td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Mean (SD) time without fever in first 4 hours (minutes)*</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">116.2 (65.0)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">156.0 (57.6)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">171.1 (40.8)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">No discomfort at 48 hours†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">34 (65)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">37 (71)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">36 (69)</td></tr><tr><td colspan=\"4\" rowspan=\"1\" align=\"left\" valign=\"top\" content-type=\"TableSubHead\"><bold>Secondary outcomes:</bold></td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Outcomes at 24 hours:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Mean (SD) time until first fever clearance (minutes)‡</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">71.0 (69.1)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">42.2 (33.5)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">45.5 (34.3)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Mean (SD) time without fever in first 24 hours (minutes)*</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">940.3 (362.9)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1055.2 (329.7)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1217.4 (237.6)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> No discomfort†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">22 (44)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">36 (69)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">29 (56)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal activity†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (40)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (58)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">23 (48)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal appetite†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">10 (21)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">14 (27)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">14 (29)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal sleep†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">17 (37)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">13 (50)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (37)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Outcomes at 48 hours:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Mean (SD) temperature (°C)§</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">36.4 (0.89)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">36.4 (0.85)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">36.6 (1.01)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal activity†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">31 (60)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">37 (73)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">28 (54)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal appetite†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">21 (41)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">22 (44)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">21(41)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal sleep†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">27 (52)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">31 (61)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">25 (48)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Outcomes at day 5:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Mean (SD) temperature (°C)**</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">36.2 (0.93)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">36.1 (0.78)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">36.0 (0.66)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> No discomfort†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">43 (88)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38 (81)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38 (76)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal activity†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">44 (90)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">39 (85)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">37 (73)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal appetite†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">29 (58)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">29 (59)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">32 (62)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Normal sleep†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">31 (62)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">25 (50)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">27 (53)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tbl3\" position=\"float\"><label>Table 3</label><caption><p> Regression models for time without fever over first four hours (240 minutes) and no discomfort at 48 hours, adjusting for minimisation</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"1\" rowspan=\"2\" align=\"left\" valign=\"bottom\">Outcomes</th><th colspan=\"2\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Primary comparisons</th><th colspan=\"1\" rowspan=\"2\" align=\"center\" valign=\"bottom\">Secondary comparison: ibuprofen <italic>v</italic> paracetamol</th></tr><tr><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol plus ibuprofen <italic>v</italic> paracetamol</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol plus ibuprofen <italic>v</italic> ibuprofen</th></tr></thead><tbody><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Time without fever in first 4 hours*:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Adjusted difference (minutes)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">55.3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">16.2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">39.0</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 95% confidence interval</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">33.1 to 77.5†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">−7.0 to 39.4†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">15.9 to 61.0‡</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> P value</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">&lt;0.001†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.2†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">&lt;0.001‡</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">No discomfort at 48 hours§:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Adjusted odds ratio</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1.33</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.89</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1.50</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 95% confidence interval</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.49 to 3.56†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.32 to 2.43†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.53 to 4.26‡</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> P value</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.7†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">&gt;0.8†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">&gt;0.5‡</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tbl4\" position=\"float\"><label>Table 4</label><caption><p> Regression models for time without fever up to 24 hours, adjusting for minimisation</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"1\" rowspan=\"2\" align=\"left\" valign=\"bottom\">Outcomes</th><th colspan=\"2\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Primary comparisons</th><th colspan=\"1\" rowspan=\"2\" align=\"center\" valign=\"bottom\">Secondary comparison: ibuprofen <italic>v</italic> paracetamol</th></tr><tr><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol plus ibuprofen <italic>v</italic> paracetamol</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol plus ibuprofen <italic>v</italic> ibuprofen</th></tr></thead><tbody><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Time until first fever clearance*:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Adjusted difference (minutes)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">−23.5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3.0</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">−26.3</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 95% confidence interval</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">−44.8 to −2.2†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">−18.3 to 24.4†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">−48.3 to −4.3‡</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> P value</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.025†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">&gt;0.8†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.015‡</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Time without fever in first 24 hours§:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> Adjusted difference (hours)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4.4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2.5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1.9</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 95% confidence interval</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2.4 to 6.3†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.6 to 4.4†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">−0.2 to 4.0‡</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> P value</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">&lt;0.001†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.008†</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.076‡</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tbl5\" position=\"float\"><label>Table 5</label><caption><p> Five most common adverse effects. Values are numbers of children</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Adverse effect</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol (n=52)</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Ibuprofen (n=52)</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol plus ibuprofen (n=52)</th></tr></thead><tbody><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Diarrhoea</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">10</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">9</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">12</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Vomiting</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Rash</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Cough</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Cold to touch</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tbl6\" position=\"float\"><label>Table 6</label><caption><p> Number of doses of paracetamol alone or ibuprofen alone over 24 and 48 hours. Values are numbers (percentages) of children</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"bottom\">Drug use (dose No)</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol (n=52)</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Ibuprofen (n=52)</th><th colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">Paracetamol plus ibuprofen (n=52)</th></tr></thead><tbody><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Paracetamol or placebo in 24 hours:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">49 (94)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">51 (98)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">48 (92)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">44 (85)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">47 (90)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">34 (65)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">24 (46)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">22 (42)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6 (12)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3 (6)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4 (8)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Paracetamol or placebo in 48 hours:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">49 (94)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">51 (98)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">50 (96)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">49 (94)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">49 (94)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">42 (81)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">39 (75)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38 (73)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">35 (67)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">26 (50)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">24 (46)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 6</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">20 (38)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">11 (21)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">15 (29)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 7</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8 (15)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6 (12)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6 (12)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 8</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3 (6)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1 (2)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1 (2)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 9</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">—</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1 (2)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">—</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Ibuprofen or placebo in 24 hours:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">51 (98)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">48 (92)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">51 (98)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38 (73)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">39 (75)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">37 (71)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">7 (13)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6 (12)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">7 (13)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">—</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">—</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2 (4)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\">Ibuprofen or placebo in 48 hours:</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">52 (100)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">51 (98)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">49 (94)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">51 (98)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">45 (87)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">45 (87)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">46 (88)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">32 (62)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">34 (65)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">29 (56)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">18 (35)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5 (10)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">18 (35)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 6</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">7 (13)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4 (8)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">10 (19)</td></tr><tr><td colspan=\"1\" rowspan=\"1\" align=\"left\" valign=\"top\"> 7</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">—</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1 (2)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3 (6)</td></tr></tbody></table></table-wrap>" ]
[]
[ "<boxed-text position=\"float\" content-type=\"style3\"><sec><title>What is already known on this topic</title><list list-type=\"simple\"><list-item><p>Paracetamol plus ibuprofen are being increasingly used at home and in primary and secondary care for the relief of fever and its associated symptoms</p></list-item><list-item><p>Five previous trials of combined therapy mostly tested single doses for children in secondary care and reached conflicting conclusions</p></list-item></list></sec><sec><title>What this study adds</title><list list-type=\"simple\"><list-item><p>In the first four hours, temperature is reduced faster and for longer in children given ibuprofen than in those given paracetamol</p></list-item><list-item><p>In the first 24 hours, children given both drugs spent 4.4 hours less time with fever than those given paracetamol and 2.5 hours less time with fever than those given ibuprofen.</p></list-item><list-item><p>Parents and healthcare professionals should consider ibuprofen first and the relative benefits and risks of using combined therapy over 24 hours</p></list-item></list></sec></boxed-text>" ]
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[ "<table-wrap-foot><p>*Minimisation criterion (baseline temperature included as continuous variable in all models; baseline discomfort minimised distinguishing the top and bottom two levels because of anticipated frequencies but included as four level categorical variable in all models).</p></table-wrap-foot>", "<table-wrap-foot><p>*Time spent with temperature less than 37.2°C in first four hours after first dose of drug, using number of valid 30 second interval points from data logger; unknown for zero, one, and two children in three groups, respectively, by four hours, and zero, two, and two, respectively, by 24 hours. Time without fever over first four hours was 48 minutes for paracetamol, 65 minutes for ibuprofen, and 71 minutes for paracetamol plus ibuprofen and for time without fever in first 24 hours was 65 minutes for paracetamol, 73 minutes for ibuprofen, and 84 minutes for paracetamol plus ibuprofen.</p><p>†Children reported at relevant time to be “normal” (see table 1); denominators may vary owing to missing data (in most cases fewer than four children).</p><p>‡Time from baseline until temperature first falls below 37.2°C; unknown for five children (zero, two, and three, respectively) and right censored at 240 minutes for three children.</p><p>§Measured by research nurse; unknown for one, five, and two children, respectively.</p><p>**Measured by parent; unknown for four, seven, and three children, respectively.</p></table-wrap-foot>", "<table-wrap-foot><p>*Weighted by number of time points in first four hours contributing valid data on temperature. Positive differences indicate additional minutes below 37.2°C for first named treatment group compared with comparator.</p><p>†Primary comparisons after applying Dunnett’s correction (approximate P values obtained using extrapolation from limited published values<sup>21</sup>; uncorrected P values were &lt;0.001 and 0.11 for time without fever, 0.53 and 0.79 for discomfort).</p><p>‡Secondary comparison after applying Tukey’s correction (P values obtained using interpolation from extensive published values<sup>21</sup>; &lt;0.001 for temperature, 0.37 for discomfort).</p><p>§Odds of being well compared with not being well.</p></table-wrap-foot>", "<table-wrap-foot><p>*Negative differences indicate that first named treatment group has faster fever clearance time than comparator group.</p><p>†Primary comparisons after applying Dunnett’s correction (uncorrected P values were 0.016 and 0.75 for fever clearance, &lt;0.001 and 0.005 for time without fever).</p><p>‡Secondary comparison after applying Tukey’s correction (uncorrected P values were 0.006 for fever clearance, 0.033 for time without fever).</p><p>§Weighted by number of time points in first 24 hours contributing valid data on temperature; positive differences indicate additional hours with temperature less than 37.2°C for first named treatment group than for comparator.</p></table-wrap-foot>", "<fn-group><fn><p>We thank Avon, Gloucestershire, and Wiltshire NHS Direct; the Bristol general practitioner practices; the south Bristol walk-in centre; the emergency department of the Bristol Royal Hospital for Children; the children and parents who participated; the South West Medicines for Children Local Research Network; the research nurse team W Horseman, J Farrimond, R Powell, S Shatford, P Richards; the South West Medicines for Children Local Research Network nurse V Payne; W Patterson (trial coordinator); S Doohan and S Burke (project administrators); K Schroeder, M Weiss, and A Emond (co-applicants); Sara Whitburn (proof and background reading); K Pitcher (data entry and quality); the trial steering committee (AL Kinmonth, C Butler, J Peacock, M Blythe, and P Denyer); and the data monitoring and safety committee (R Bragonier, S Kerry, and J Chudleigh).</p></fn><fn fn-type=\"participating-researchers\"><p>Contributors: ADH, AAM, MF, and TJP conceived the study and wrote the protocol. The research nurse team collected the data under the supervision of NMR, CC, and SH. AAM, CC, and TJP cleaned and analysed the data. CC, ADH, and TJP initially drafted the paper, with subsequent contributions from all authors. ADH is the guarantor.</p></fn><fn fn-type=\"financial-disclosure\"><p>Funding: National Institute for Health Research health technology assessment programme (project No 03/09/01). The final study design, data collection and analysis, interpretation of results, and paper writing was the sole responsibility of the authors. For the duration of the trial, ADH held a postdoctoral award from the National Coordinating Centre for Research Capacity Development, Department of Health. The views and opinions expressed in this paper do not necessarily reflect those of the funding bodies. The active drugs and placebos were purchased from Pfizer and DHP Investigational Medicinal Products, respectively. Neither had any other role in the design, conduct, analysis, or reporting of the trial.</p></fn><fn fn-type=\"conflict\"><p>Competing interests: None declared.</p></fn><fn><p>Ethical approval: Bath research ethics committee (reference No 04/Q2001/197).</p></fn><fn><p>Provenance and peer review: Not commissioned; externally peer reviewed.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"haya551291.f1\"/>", "<graphic xlink:href=\"haya551291.f2\"/>", "<graphic xlink:href=\"haya551291.f3\"/>" ]
[]
[{"label": ["4"], "mixed-citation": ["Meremikwu M, Oyo-Ita A. Physical methods for treating fever in children. [Review] [38 refs]. "], "source": ["Cochrane Database Syst Rev"], "year": ["2003"]}, {"label": ["5"], "mixed-citation": ["National Institute for Health and Clinical Excellence. "], "source": ["Feverish illness in children. Assessment and initial management in children younger than 5 years"], "year": ["2007"]}, {"label": ["15"], "mixed-citation": ["Kramer LC, Richards PA, Thompson AM, Harper DP, Fairchok MP. Alternating antipyretics: antipyretic efficacy of acetaminophen versus acetaminophen alternated with ibuprofen in children. "], "source": ["Clin Pediatr (Phila)"], "year": ["2008"]}, {"label": ["17"], "mixed-citation": ["Hollinghurst S, Redmond N, Costelloe C, Montgomery AA, Fletcher M, Peters TJ, et al. Paracetamol plus ibuprofen for the treatment of fever in children (PITCH): economic evaluation of the randomised controlled trial. "], "source": ["BMJ"], "year": ["2008"]}, {"label": ["18"], "mixed-citation": ["British Medical Association, Royal Pharmaceutical Society of Great Britain, Royal College of Paediatrics and Child Health. "], "source": ["BNF for children"], "year": ["2007"]}, {"label": ["21"], "mixed-citation": ["European Parliament. European clinical trial directive 2001/20/EC. 2001."]}, {"label": ["22"], "mixed-citation": ["Zar J. "], "source": ["Biostatistical analysis"], "year": ["1994"]}, {"label": ["26"], "mixed-citation": ["Department of Health. "], "source": ["National service framework for children, young people and maternity services"], "year": ["2004"]}, {"label": ["27"], "mixed-citation": ["Bua J, L\u2019Erario I, Barbi E, Marchetti F. When off-label is a good practice: the example of paracetamol and salbutamol. "], "source": ["Arch Dis Child"], "year": ["2008"], "volume": ["93"], "fpage": ["546"]}]
{ "acronym": [], "definition": [] }
30
CC BY
no
2022-01-12 21:41:46
BMJ. 2008 Sep 2; 337:a1302
oa_package/ce/66/PMC2528896.tar.gz
PMC2528934
18781206
[ "<title>Introduction</title>", "<p>Road traffic injuries (RTIs) are an important cause of morbidity and mortality, and are projected to become the sixth leading cause of death and third leading cause of disability adjusted life years (DALYs) lost globally by the year 2020 ##REF##9167458##[1]##. Poorer nations are disproportionately affected by RTIs and account for approximately 85% of RTI deaths and 90% of RTI disability ##UREF##0##[2]##. In poorer countries of Latin America, RTIs are already the sixth leading cause of death and third leading cause of morbidity for all ages ##UREF##1##[3]##. While well-designed research, successful interventions, and legislative priority has led to a substantial decrease in the burden of RTIs in wealthier regions, the rates of RTIs in many poorer nations are increasing ##REF##16679167##[4]##.</p>", "<p>Children and pedestrians are especially vulnerable to traffic injuries, particularly in developing countries ##REF##12003888##[5]##, ##REF##14697797##[6]##, ##REF##16449050##[7]##, ##REF##9217700##[8]##, ##UREF##2##[9]##. In the low to middle income countries of the Americas, RTIs are the number one cause of death and morbidity for children aged 5–14, and a leading cause of death for children aged 0–4 ##UREF##1##[3]##. Additionally, the RTI fatality rate for children of poorer countries is as much as six times that of children from high income countries ##REF##12003888##[5]##. Pedestrians are involved most frequently in RTIs in the developing world, and represent up to 54% of those injured in Latin American studies ##REF##9217700##[8]##, ##REF##12772483##[10]##, ##REF##11325362##[11]##.</p>", "<p>Prevention of child pedestrian RTIs has focused on modifying both personal (education initiatives) and, more effectively, environmental (traffic calming) risk factors ##REF##2585230##[12]##, ##REF##12003885##[13]##, ##REF##12772494##[14]##, ##REF##2389759##[15]##, ##REF##8557453##[16]##, ##REF##9262499##[17]##, ##REF##8363665##[18]##. Environmental risk factors themselves, however, have been less rigorously studied ##UREF##3##[19]##. The personal risk factors encountered in the literature include age, gender, household overcrowding, poverty, single parent homes, and low levels of education in caregivers, while environmental risk factors include high traffic volumes, high vehicle speeds, presence of sidewalks, and density of curb side parking ##REF##2585230##[12]##, ##REF##2389759##[15]##, ##REF##8557453##[16]##, ##REF##9262499##[17]##, ##UREF##3##[19]##, ##REF##9788088##[20]##, ##REF##7833733##[21]##, ##REF##4034298##[22]##, ##REF##7742404##[23]##, ##REF##11937618##[24]##, ##REF##2751039##[25]##. The overwhelming majority of these studies were conducted in developed countries ##REF##16679167##[4]##, ##REF##16046946##[26]##, and the results are commonly relied upon when importing or creating intervention strategies for the developing world. The assumption, however, that developed world practices translate into effective prevention measures in poorer countries may be erroneous as they may not be affordable, may require disproportionate technologies, and may miss important risk factors unique to developing world settings ##REF##12772494##[14]##, ##UREF##4##[27]##, ##UREF##5##[28]##.</p>", "<p>The aim of this study was to assess personal and environmental risk factors for child pedestrian RTIs in the urban, developing world setting of Lima, Peru. Our intention is to aid the design of new RTI interventions or the translation of existing ones from high income nations to poorer ones based on locally relevant risk factors.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Study Design and Setting</title>", "<p>This analysis is a sub-study of a large, community based cross sectional study of childhood injuries in San Juan de Miraflores (SJM), a poor, urban district of Lima, Peru. It includes results from the cross sectional study and two nested case control studies exploring personal and environmental risk factors for child pedestrian RTIs. Studies were conducted between January 2005 and July 2006.</p>", "<title>Participants</title>", "<title>Cross sectional and personal risk factor case control studies</title>", "<p>In the cross sectional study, six health promoters with high school graduate level education administered door to door surveys in 12 SJM zones, divided along existing neighbourhood borders. Staff began randomly and proceeded until each zone was completed. Households with a consenting adult and at least one resident child aged ≤18 were eligible.</p>", "<p>In the personal risk factor case control study, health promoters administered follow up surveys to cases of child pedestrian RTIs and randomly selected age and gender matched controls from the original study. Subjects were recruited to a goal of 100 cases and 200 controls. Cases were children who incurred a RTI during pedestrian activity in SJM from the year 2000 onward. RTIs occurring in parking lots, driveways, or while the vehicle was reversing were excluded. Two controls per case were selected by random assortment of all potential controls, and then random number generation to identify the first and subsequent controls. Controls were included if they could be age (within one year) and gender matched to a case, and there was no family history of pedestrian RTI. There was no compensation for participation.</p>", "<title>Environmental case control study</title>", "<p>The environmental case control study used the same case-control sets as in the personal risk factor study. Goal recruitment was 40 case and 80 control environments. Case sites of RTIs were included if the environment in which the RTI occurred had not changed since the time of the injury (as reported by the guardian of the injured child), and the RTI occurred between the hours of 6:00 and 20:00 (to minimize personal safety risk to staff as the district in which the study took place was quite dangerous). Control environments were selected by considering the origin and destination of the case child prior to injury, and the distance (number of blocks) from the origin that the RTI occurred. As an example, if a case was injured travelling from his local market to home, three blocks from the market, we then identified which market the control subject normally visited, and the route they would take from the market to home. The control site was then three blocks from their market, on their route home.</p>", "<title>Data Collection</title>", "<p>All study surveys were extensively pilot tested, and completed surveys were reviewed by the principal investigator (JD).</p>", "<title>Cross sectional and personal risk factor case control studies</title>", "<p>In the cross sectional study we administered a semi-structured survey including the core data sets recommended by the WHO ##UREF##6##[29]##. Children aged ≥12 years and present at the time of the survey were interviewed in the presence of a guardian. If the child was not present or was &lt;12 years of age, a guardian was interviewed. The personal risk factor study required that the injured child and guardian be present at the time of interview and collected demographic data and school information in relation to the year in which the injury (or case injury for controls) took place and precise RTI time and location data for cases.</p>", "<title>Environmental case control study</title>", "<p>In the environmental case control study, structured, 1.5 hour assessments were performed. Briefly, we evaluated: pedestrian movement, including volume, use of cross walks, and street vendor density; vehicle movement, including speed, vehicle specific volumes (cars, trucks, public transportation, motorcycle taxis), and traffic code infractions; pedestrian infrastructure, including sidewalks, crosswalks, and crossing lights; vehicle infrastructure, including road conditions, traffic lights, speed bumps, lane demarcation, and curb side parking.</p>", "<p>Environmental assessments were performed on the same day of the week and at the same time of day as when the case injury occurred. Case and control assessments were done simultaneously by two health promoters per site. Measurements were made only in the direction the vehicle was travelling prior to causing the case injury, on a section of road extending 150 meters from the injury site. All measurements were made by direct observation. Vehicle speed was recorded during a dedicated 30 minute period using digital timers to measure the time to traverse 150 meters.</p>", "<title>Definitions</title>", "<p>We defined <italic>pedestrian activity</italic> as walking, running, or standing, but not cycling or skating. A <italic>road traffic injury (RTI)</italic> was any unintentional injury inflicted by a motorized vehicle. A <italic>serious RTI</italic> necessitated a healthcare consultation, i.e. visit to a hospital or health post. Of the RTIs identified in the cross sectional study, only serious RTIs were included in the case control studies. We considered <italic>poverty</italic> to be <italic>present</italic> if one or more of the following criteria were fulfilled: economic dependence (defined in Peru as ≥3 household occupants per wage earner), absence of indoor plumbing, dirt floors, and children in the home aged 6–12 not attending school. We considered <italic>overcrowding</italic> to be ≥4 people per room, excluding the kitchen, bathroom, and hallways. <italic>Education in the head of household</italic> and <italic>maternal education</italic> were <italic>low</italic> if primary schooling was incomplete. <italic>Years of residence</italic> refers to the total number of years in which the family has lived in the home where the interview took place. <italic>Speeds and volumes</italic> (i.e. pedestrian, vehicle) were considered <italic>high</italic> if they were in the highest tertile of recorded measurements. <italic>Avenues</italic>, <italic>streets</italic>, and <italic>roads</italic>, were defined according to city planning maps. <italic>Street vendors</italic> were sidewalk or street merchants without fixed locals.</p>", "<title>Statistics</title>", "<p>Sample size in the personal risk factor study was calculated with an assumed exposure in the control population of 50%, lowest detectable OR of 2.15, alpha equal to 0.05 and a power of 80 percent. Sample size in the environmental risk factor study was limited by time and funding considerations. Statistical analysis was performed using SPSS software (SPSS ®, SPSS Inc, ver. 11.5, Chicago, Illinois) and StatsDirect statistical software (StatsDirect Ltd ®, ver. 2.7.0, UK). Descriptive analyses determined the proportion and percentages of occurrences for binary and categorical variables. Conditional logistic regression methods were used to generate unadjusted matched odds ratios (OR) with 95% confidence intervals (CI) for each exposure variable and adjusted multivariate models for both personal and environmental risk factors. The best fitting multivariate models were built beginning with two variables with historic association to the outcome based on the review of the literature (poverty and maternal education for the personal risk factor study, and vehicle speed and volume for the environmental risk factor study), then adding subsequent variables if they improved the model. Improved models were those in which the −2 log likelihood ratio was greater than a critical value derived from the chi-square distribution table based on degrees of freedom at an alpha level of 0.05. Variables evaluated in the multivariate analysis but eliminated by statistical criteria are listed in ##SUPPL##0##Appendix S1##. All statistical tests were two-sided and p-values &lt;0.05 were considered statistically significant. Interactions between selected factors were tested however none were found to be significant.</p>", "<title>Ethics</title>", "<p>The study protocols, informed consent and assent forms, and data collection instruments were approved by the human research ethics committee of Asociación Benéfica PRISMA (FWA 00001219). All interviewed children signed written assent forms and one guardian per household provided written consent for the cross sectional and case control portions of the study.</p>" ]
[ "<title>Results</title>", "<title>Descriptive Analysis</title>", "<p>Participant recruitment and attrition are highlighted in ##FIG##0##Figure 1##. In the cross sectional study, 21811 households were approached, of which 8039 were eligible for participation. Of these, 63% consented and were surveyed and the final analysis included 5061 households and 10210 children (median two per household; characteristics described in ##TAB##0##Table 1##). Between 2000 and 2005, this population sustained 141 pedestrian RTIs, of which 117 (83%) were serious. No surveyed household reported child pedestrian RTI deaths during this period. In the personal risk factor study, there were four instances in which cases were clustered within households (two cases per household). Of these four homes, 50% (2·0) were poor, none had a low level of maternal education, there was a median of four resident children (range 2·0 to 5·0), and the median time of family residence was 11·5 years (range 2·0 to 15·0).</p>", "<p>Characteristics of serious pedestrian injuries are presented in ##TAB##1##Table 2##. In the 1–4 and 5–9 year old groups, RTIs most commonly occurred during trips to or from the market (50% and 40%, respectively) and playing in the street (27% and 28%, respectively). In the 10–14 year old group, RTIs most commonly involved trips to or from school (28%), the market (23%) or a relative's home (23%). Younger children (aged 1–9) were more commonly injured while crossing in non designated areas (70% of RTIs in this age group).</p>", "<p>Of the 120 evaluated sites, only one had a traffic light, two had stop signs, and there were no posted speed limits. Tertiles derived from aggregate case and control data for pedestrian volume and vehicle speed and volume were used to define strata in the risk factor analyses. The middle pedestrian volume tertile was 101 to 201 pedestrians/hr, the middle vehicle speed tertile was 33.9 to 44.6 km/hr, and the middle vehicle volume tertile was 24 to 249 vehicles/hr. The median number of vehicles per hour at the case sites was 244·0 (range 0·0 to 4503·0), composed of motorcycle taxis (29%), public transportation (buses and vans- 28%), taxis (26%), and cars (15%). At control sites, the median number of vehicles per hour was 66·0 (range 0·0 to 1212·0), with a similar profile composed of motorcycle taxi (33%), public transportation (25%), taxi (25%), and cars (13%). The median vehicle velocity at case and control sites was 40·7 km/hr (range 18·2 to 60·5) and 39·8 km/hr (range 20·4 to 83·1), respectively.</p>", "<title>Risk Factor Analyses</title>", "<p>Univariate associations between case and control characteristics and pedestrian RTI are presented in ##TAB##2##Table 3##. More hours per day in school was protective over pedestrian RTI, while larger streets, commercial or mixed commercial zones, and high vehicle volumes were associated with increased odds of pedestrian injury.</p>", "<p>The final multivariate conditional logistic regression models for personal and environmental risk factors are shown in ##TAB##3##Table 4##. More hours per day spent in school and years of family residency in the same home were protective against child pedestrian RTIs, while a greater number of children in the home, a greater number of street vendors, the absence of lane demarcations, and high vehicle volume and speed increased the odds of pedestrian injury.</p>" ]
[ "<title>Discussion</title>", "<p>Effective intervention design, or translation of existing interventions, for child pedestrian injury prevention to the world's poorer countries requires identification of locally relevant modifiable risk factors. This study describes the context in which child pedestrian RTIs occurred in an urban district of a major Latin American city, and identified both personal and environmental risk factors. Briefly, and in order of increasing strength of association, we found that more years of family residence in the same location and longer length of the school day were protective, while a greater number of children living in the home, a greater number of street vendors, higher vehicle speeds, absent lane demarcation, and higher vehicle volumes increased the risk of child pedestrian RTI. Our study of personal risk factors is among few such case control studies in Latin America, and, to the best of our knowledge, the environmental case control component is the first reported from the developing world.</p>", "<p>Personal risk factors for child pedestrian RTIs have been described extensively, but few are derived from case control studies in the developing world. We identified some similar associations, including familiarity with the local environment ##REF##9788088##[20]## (reflected by years of family residence) and the number of children living in the home ##REF##2751039##[25]##. However, other previously identified risk factors, such as poverty ##REF##2389759##[15]##, ##REF##9262499##[17]##, ##REF##9788088##[20]##, ##REF##4034298##[22]##, household crowding ##REF##2585230##[12]##, ##REF##9262499##[17]##, ##REF##9788088##[20]##, ##REF##4034298##[22]##, and low maternal education ##REF##9788088##[20]##, ##REF##2751039##[25]## were not significant predictors within our population. SJM is a low income zone and the relative lack of economic variability may have weakened the effect of poverty. Overcrowding in this community may be compensated by the traditional Peruvian family dynamic in which multiple adult relatives live in the same or in neighbouring homes, increasing the number of caretakers per child. A similar effect may abrogate the anticipated importance of low maternal education. The protective effect of increased time spent in school has not been described previously. We found that each additional hour of school conferred a 48% decrease in the odds of a pedestrian RTI. This is unlikely a function of the added educational benefit of longer schooling, but rather a change in exposures. Children that are in school less may make more frequent trips to the market or spend more time playing in the street, the two most common exposures prior to RTI in this community. They may also be performing these activities at riskier times of day, when traffic patterns are more conducive to pedestrian RTI.</p>", "<p>Environmental case control studies, designed to identify environmental risk factors amenable to modification, are rarely performed in pedestrian RTI investigations. One reason is the challenge inherent in selecting appropriate control sites. Prior studies have first chosen a control child and then the control site in relation to this child's home based upon the distance and/or direction that the case RTI occurred in relation to the case home ##REF##8557453##[16]##, ##REF##7833733##[21]##. This method, however, fails to consider two important variables - the points of origin and destination of the case child prior to injury. Our innovative technique for control selection used distance as in previous studies, but with relation to the points of origin and destination of the case child prior to the injury. Our approach takes account of what children are doing when they are injured and tries to answer the question of why only one of the three children doing the same thing in different places should suffer an injury. This was a challenging undertaking and case selection was limited to those for whom the journey's start and end points could be clearly defined (e.g. home to school) so that adequate definition of the comparable control journey was feasible. We found strong associations between child pedestrian RTIs and higher traffic volume and higher vehicle speed, consistent with other studies from wealthier countries ##REF##2389759##[15]##, ##REF##8557453##[16]##, ##REF##7833733##[21]##, ##REF##7742404##[23]##. Similar to studies by Roberts and Mueller, our final model incorporates vehicle speed as a categorical, rather than continuous, variable ##REF##2389759##[15]##, ##REF##7833733##[21]##. The risk of injury to a child may be similar on a street where the average vehicle speed is 25 km/hr (lowest tertile) and 35 km/hr (middle tertile), while the risk increases once the average vehicle speed reaches 45 km/hr (highest tertile). The risk of injury, however, on a street where the average vehicle speed is 55 km/hr may not be significantly different from that of the street where the average speed is 45 km/hr, as both these values are above the critical speed limit. The increased risk of pedestrian RTI associated with a greater number of street vendors and absent lane demarcation has not previously been described. Street vendors may create hazardous conditions by obstructing portions of the street, diverting traffic, concealing oncoming vehicles from view, or distracting pedestrians and drivers. Absent lane demarcations likely contribute to disorderly traffic flow, perhaps leading to unpredictable traffic patterns and making it more difficult for children to judge safe times to cross the street. These two factors are particularly common in the developing world and may be important considerations when designing child RTI interventions there.</p>", "<p>The strengths of this research include the large scale community based descriptive analysis and concurrent identification of both personal and environmental risk factors in the same community. The data are derived from and thus applicable to a developing world setting, addressing well recognized gaps in our understanding of child pedestrian RTIs in the world's highest risk regions. Finally, our novel approach to control environment selection makes an important contribution to the field of RTI research design. The low overall sampling rate of the cross sectional survey from which the cases and controls were generated should be considered when assessing the generalizability of the study. However, at the same time, the number of households and children surveyed was large. The difficult reality of conducting household surveys in a developing world setting is that childcare is often relegated to older siblings who cannot provide consent to participate and there exists a distrust of strangers grounded in the very tangible threat of home invasion and kidnappings. As only homes with at least one resident child were eligible for study inclusion, cases where an only child had been fatally injured in a RTI would have been missed, and, while likely a rare occurrence, this is a potential source of selection bias. Also, both case control studies involved injuries occurring up to five years prior to surveying, thus recall bias and changing roadside environments are important considerations. It is unlikely, however, that recall of socioeconomic and demographic factors would be biased significantly by having suffered an RTI. To minimize confounding by changing street environments, case families were asked if environmental modifications were made since the time injury. Affirmative or equivocal responses required selection of a new case site. Additionally, the sample size for the environmental risk factor study was small, thus increasing the probability of a type II error, and measurements were done by direct observation, therefore reported measures, while internally consistent and validated, should be considered approximations. Finally, children are only at risk for pedestrian RTIs when they are exposed to traffic while walking, therefore exposure factors (such time spent in school, walking to school, playing in the street, etc) are considered risk factors in our analysis. As the data is derived from case control studies, relationships between risk factors and outcomes represent associations and causality cannot be inferred.</p>", "<p>Our findings have important public health implications. Most injuries affected children aged 5–9 years, and occurred while unaccompanied, going to or from market, playing in the street, and crossing in non designated areas suggesting room for targeted behavioural interventions as well as the need for local traffic calming measures. Taxis and motorcycle taxis were the most frequent vehicles responsible, thus law enforcement could have a particularly helpful role. In particular, motorcycle taxis are a common mode of transport in Lima, but drivers are often under-aged, vehicles are often filled beyond capacity, and they frequently violate traffic codes. The protective effect of time spent at school suggests the viability of longer school days or increased access to after school programs as a means of preventing child pedestrian injuries. Finally, our environmental analysis provides evidence to support measures to reduce traffic volumes and speeds, limit the number of street vendors on a given stretch of road, and improve lane demarcation.</p>", "<p>It is clear that as disparities between high and low income countries continue to grow with regard to child pedestrian injuries, our goal should be to develop prevention strategies targeting risk factors relevant to a developing world context. This study advances that process by identifying both personal and environmental risk factors for child pedestrian RTIs in a major Latin America city.</p>" ]
[]
[ "<p>Conceived and designed the experiments: JD RG DB DAJM. Performed the experiments: JD MT DAJM. Analyzed the data: JD. Contributed reagents/materials/analysis tools: JD RG DB DAJM. Wrote the paper: JD.</p>", "<title>Background</title>", "<p>Child pedestrian road traffic injuries (RTIs) are an important cause of death and disability in poorer nations, however RTI prevention strategies in those countries largely draw upon studies conducted in wealthier countries. This research investigated personal and environmental risk factors for child pedestrian RTIs relevant to an urban, developing world setting.</p>", "<title>Methods</title>", "<p>This is a case control study of personal and environmental risk factors for child pedestrian RTIs in San Juan de Miraflores, Lima, Perú. The analysis of personal risk factors included 100 cases of serious pedestrian RTIs and 200 age and gender matched controls. Demographic, socioeconomic, and injury data were collected. The environmental risk factor study evaluated vehicle and pedestrian movement and infrastructure at the sites in which 40 of the above case RTIs occurred and 80 control sites.</p>", "<title>Findings</title>", "<p>After adjustment, factors associated with increased risk of child pedestrian RTIs included high vehicle volume (OR 7·88, 95%CI 1·97–31·52), absent lane demarcations (OR 6·59, 95% CI 1·65–26·26), high vehicle speed (OR 5·35, 95%CI 1·55–18·54), high street vendor density (OR 1·25, 95%CI 1·01–1·55), and more children living in the home (OR 1·25, 95%CI 1·00–1·56). Protective factors included more hours/day spent in school (OR 0·52, 95%CI 0·33–0·82) and years of family residence in the same home (OR 0·97, 95%CI 0·95–0·99).</p>", "<title>Conclusion</title>", "<p>Reducing traffic volumes and speeds, limiting the number of street vendors on a given stretch of road, and improving lane demarcation should be evaluated as components of child pedestrian RTI interventions in poorer countries.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>The authors wish to thank Dr. Steve Cohen and Dr. Mkaya Mwamburi for their statistical help. We would also like to thank Lilia Cabrera, Gladys Oña, Mercedes Mendoza, Carmen Loza, Jessica Pena, Karina Sànchez, Alejandrina Sànchez, Maria Luisa Navarette, Sonia Lopez, Eleana Sànchez, Fanny Garcìa, Ruth Limassca, Jeny Rodrìguez, Miriam Huayta, Esther Soto, Beatriz Castro, Virginia Huancarè – the field workers and research nurses involved – for their dedication and commitment to the field work. We would like to thank Ian Roberts for helpful preliminary discussions on methodology and design.</p>" ]
[ "<fig id=\"pone-0003166-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003166.g001</object-id><label>Figure 1</label><caption><title>In the cross sectional study, 24% (5832) and 36% (7940) of homes approached were not surveyed because no adult was home after two attempts or there were no resident children, respectively.</title><p>Of the remaining 8039 households, 2973 (37%) refused to participate and five households (12 children) were excluded due to incomplete data. In the personal risk factor case control study, health promoters made two visits to homes in which no one was present, after which time the case or control was replaced. In 37 instances no adult was present on the repeat visit. Of the 40 cases selected for the environmental analysis, 93% were injured within the two years preceding the study. *Traffic volumes were too high to allow for accurate measurement in 2 case environments, thus they and their corresponding control environments were removed and replaced. RF = Risk Factors.</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003166-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003166.t001</object-id><label>Table 1</label><caption><title>Cross sectional study population characteristics (n = 10210).</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Percent</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gender-male</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5269</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">51·6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Poverty- present</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5635</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55·2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Education of head of household-low<xref ref-type=\"table-fn\" rid=\"nt101\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1951</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19·1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Overcrowding-yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">785</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7·7</td></tr><tr><td colspan=\"3\" align=\"left\" rowspan=\"1\">Ages (years)<xref ref-type=\"table-fn\" rid=\"nt102\">†</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">461</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4·5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1–4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2297</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22·5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5–9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2864</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28·1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10–14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2790</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27·3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">15–18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1797</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17·6</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003166-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003166.t002</object-id><label>Table 2</label><caption><title>RTI Characteristics (Cross Sectional Study).</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics of serious RTI</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Percent</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">RTI requiring hospital/clinic visit<xref ref-type=\"table-fn\" rid=\"nt103\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">117</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;12 hours</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">86</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">76·1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;12 hours</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23·9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median days of hospitalization (n = 27)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td colspan=\"3\" align=\"left\" rowspan=\"1\">\n<bold>Age at time of serious RTI (years)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1–4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18·8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5–9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">47</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40·2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10–14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33·3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">15–18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7·7</td></tr><tr><td colspan=\"3\" align=\"left\" rowspan=\"1\">\n<bold>Activity</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median distance from home (blocks)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2·5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median distance from point of origin (blocks)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td colspan=\"3\" align=\"left\" rowspan=\"1\">RTI occurred during:</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Trip to/from store/market</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35·0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Trip to/from school</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15·4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Trip to/from relative's home</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12·0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Play</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20·5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Other<xref ref-type=\"table-fn\" rid=\"nt104\">†</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17·1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Alone or with another minor</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">82</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">70·1</td></tr><tr><td colspan=\"3\" align=\"left\" rowspan=\"1\">\n<bold>Time of day</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Morning</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28·2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Afternoon or evening</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71·8</td></tr><tr><td colspan=\"3\" align=\"left\" rowspan=\"1\">\n<bold>Location</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Road or street</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39·3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Avenue</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">59·8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Highway</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·9</td></tr><tr><td colspan=\"3\" align=\"left\" rowspan=\"1\">\n<bold>Agent</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Taxi or mototaxi</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41·0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Private auto</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23·1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Public transportation (bus, minibus, or van)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22·2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Other (truck, motorcycle)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13·7</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003166-t003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003166.t003</object-id><label>Table 3</label><caption><title>Characteristics of case and control participants and sites in the risk factor analyses<xref ref-type=\"table-fn\" rid=\"nt105\">*</xref>.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cases (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Controls (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Matched OR (95% CI)</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Personal risk factor analysis</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gender- male</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">67 (67·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">134 (67·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age- mean±SD</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11·5±4·4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11·9±4·1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Poverty- present</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">47 (47·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">103 (51·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·86 (0·52–1·41)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal education- low</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10 (10·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (4·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·86 (0·73–4·75)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Overcrowding- yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12 (12·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12 (6·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2·00 (0·90–4·45)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of resident children- median (range)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2·0 (1·0–9·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2·0 (1·0–8·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·18 (0·99–1·42)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Years of family residence- median (range)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10·5 (0·1–46·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (0·2–50·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·98 (0·96–1·00)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hours/day in school- mean±SD<xref ref-type=\"table-fn\" rid=\"nt106\">†</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>4·8±0·96</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>5·1±0·77</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0·56 (0·37–0·83)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Attend school mostly in the afternoon- yes<xref ref-type=\"table-fn\" rid=\"nt106\">†</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27 (29·3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">42 (21·8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·37 (0·79–2·40)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Walks to school- yes<xref ref-type=\"table-fn\" rid=\"nt106\">†</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">83 (89·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">164 (85·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·36 (0·64–2·91)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Allowed to play in street- yes<xref ref-type=\"table-fn\" rid=\"nt106\">†</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40 (40·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">67 (33·7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·38 (0·82–2·32)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Family has car- yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13 (13·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">38 (19·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·66 (0·35–1·27)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Home with yard- yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">66 (66·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">128 (64·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·09 (0·66–1·79)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Blocks from home to nearest park- median (range)<xref ref-type=\"table-fn\" rid=\"nt106\">†</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·0 (0·0–12·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·0 (0·0–10·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·98 (0·83–1·15)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Environmental risk factor analysis</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Time of day- morning</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (17·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (17·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Location- avenue</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>28 (70·0)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>35 (43·8)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>3·90 (1·51–10·09)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Zone- commercial or mixed commercial</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>26 (65·0)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>35 (43·8)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>3·28 (1·24–8·65)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pedestrian volume- high<xref ref-type=\"table-fn\" rid=\"nt107\">‡</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (40·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">24 (30·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·80 (0·79–4·09)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vehicle volume- high<xref ref-type=\"table-fn\" rid=\"nt107\">‡</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>20 (50·0)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>20 (25·0)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>6·49 (1·83–23·0)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vehicle Speed- high<xref ref-type=\"table-fn\" rid=\"nt107\">‡</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18 (45·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22 (27·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2·10 (0·95–4·63)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of Street vendors- median (range)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0–25)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0–21)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·21 (0·99–1·48)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Absent Lane Demarcation<xref ref-type=\"table-fn\" rid=\"nt106\">†</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29 (74·4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50 (62·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·71 (0·73–4·02)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Dirt road- yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (7·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13 (16·3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·44 (0·12–1·59)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Speed bump- yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (5·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (11·3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·44 (0·10–2·06)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gated community- yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (5·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (7·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·67 (0·13–3·30)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pedestrian crosswalk present- yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (5·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 (10·0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·50 (0·11–2·35)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Sidewalk present- yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27 (67·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62 (77·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·63 (0·28–1·42)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;50% curbside parking</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (10·3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (6·3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2·38 (0·38–14·97)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Park/play area nearby- yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17 (42·5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35 (43·8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·96 (0·47–1·94)</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003166-t004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003166.t004</object-id><label>Table 4</label><caption><title>Multivariate associations between personal and environmental risk factors and pedestrian RTI<xref ref-type=\"table-fn\" rid=\"nt108\">*</xref>.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Risk Factor</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Matched OR (95%CI)</td></tr></thead><tbody><tr><td colspan=\"2\" align=\"left\" rowspan=\"1\">\n<bold>Personal</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Poverty</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0·58 (0·31–1·08)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low Maternal Education Level</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·83 (0·65–5·17)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of resident children</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>1·25 (1·00–1·56)</bold>\n<xref ref-type=\"table-fn\" rid=\"nt109\">†</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Years of residence</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0·97 (0·95–0·99)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hours/day in school<xref ref-type=\"table-fn\" rid=\"nt110\">‡</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0·52 (0·33–0·82)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Attend school mostly in the afternoon<xref ref-type=\"table-fn\" rid=\"nt110\">‡</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1·31 (0·70–2·46)</td></tr><tr><td colspan=\"2\" align=\"left\" rowspan=\"1\">\n<bold>Environmental</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vehicle volume- high<xref ref-type=\"table-fn\" rid=\"nt111\">§</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>7·88 (1·97–31·52)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vehicle Speed- high<xref ref-type=\"table-fn\" rid=\"nt111\">§</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>5·35 (1·55–18·54)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of Street vendors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>1·25 (1·01–1·55)</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Absent Lane Demarcation<xref ref-type=\"table-fn\" rid=\"nt112\">∥</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>6·59 (1·65–26·26)</bold>\n</td></tr></tbody></table></alternatives></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003166.s001\"><label>Appendix S1</label><caption><p>(0.02 MB DOC)</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"nt101\"><label>*</label><p>9 missing values.</p></fn><fn id=\"nt102\"><label>†</label><p>1 missing value.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt103\"><label>*</label><p>4 missing values for duration of hospital visit; percentages shown are of the 113 with known duration.</p></fn><fn id=\"nt104\"><label>†</label><p>Includes to/from friends home, bus stop, restaurant, parent's work, park, movies, and unspecified purpose.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt105\"><label>*</label><p>Matched OR calculated with conditional logistic regression methods. OR denotes odd ratio, and CI confidence interval; NA- OR not calculated because cases/controls were matched by these variables.</p></fn><fn id=\"nt106\"><label>†</label><p>For hours/day in school and attends school mostly in pm, and walks to school-7 cases and 7 controls were not attending school at the time of injury; For allowed to play in street- 1 missing control value; For blocks from home to nearest park- 4 missing control values; For absent lane demarcation- 1 missing case value.</p></fn><fn id=\"nt107\"><label>‡</label><p>High designation corresponds to the highest tertile of all values recorded at all 120 sites (vehicle volume≥250 vehicles/hr; vehicle speed≥44·7 km.hr, pedestrian volume≥201pedestrians/hr).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt108\"><label>*</label><p>OR denotes odd ratio, and CI confidence interval; Personal and environment al regressions were performed separately.</p></fn><fn id=\"nt109\"><label>†</label><p>p = 0·048.</p></fn><fn id=\"nt110\"><label>‡</label><p>7 cases and 7 controls were not attending school at the time of injury.</p></fn><fn id=\"nt111\"><label>§</label><p>High designation corresponds to the highest tertile of all values recorded at all 120 sites (vehicle volume≥250 vehicles/hr; vehicle speed≥44·7 km.hr).</p></fn><fn id=\"nt112\"><label>∥</label><p>1 missing case value.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>The study was funded in part by the Tutorial in Tropical Health at JHU/Peru Overseas Sites Grant (5T35AI007646) and the Fogarty International Center/ Ellison Medical Foundation-Overseas Fellowships in Global Health and Clinical Research. DAJM is supported by the Wellcome Trust. The authors' work was independent of the funders.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"pone.0003166.g001\"/>", "<graphic id=\"pone-0003166-t001-1\" xlink:href=\"pone.0003166.t001\"/>", "<graphic id=\"pone-0003166-t002-2\" xlink:href=\"pone.0003166.t002\"/>", "<graphic id=\"pone-0003166-t003-3\" xlink:href=\"pone.0003166.t003\"/>", "<graphic id=\"pone-0003166-t004-4\" xlink:href=\"pone.0003166.t004\"/>" ]
[ "<media xlink:href=\"pone.0003166.s001.doc\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["2"], "element-citation": ["\n"], "surname": ["Peden"], "given-names": ["M"], "year": ["2004"], "source": ["The world report on road traffic injury prevention"], "publisher-loc": ["Geneva"], "publisher-name": ["World Health Organization"]}, {"label": ["3"], "element-citation": ["\n"], "collab": ["World Health Organization"], "year": ["1999"], "source": ["Injury, a leading cause of the global burden of disease"], "publisher-loc": ["Geneva"], "publisher-name": ["WHO"]}, {"label": ["9"], "element-citation": ["\n"], "surname": ["Roberts"], "given-names": ["I"], "year": ["1995"], "article-title": ["Injuries to child pedestrians."], "source": ["BMJ"], "volume": ["310"], "fpage": ["413"], "lpage": ["414"]}, {"label": ["19"], "element-citation": ["\n"], "surname": ["Peden"], "given-names": ["M"], "year": ["2001"], "source": ["Proceedings of WHO meeting to develop a 5- year strategy for road traffic injury prevention"], "publisher-loc": ["Geneva"], "publisher-name": ["World Health Organization"]}, {"label": ["27"], "element-citation": ["\n"], "surname": ["Perel", "McGuire", "Eapen", "Ferraro"], "given-names": ["P", "M", "K", "A"], "year": ["2004"], "article-title": ["Research on preventing road traffic injuries in developing countries is needed."], "source": ["BMJ"], "volume": ["328"], "fpage": ["895"]}, {"label": ["28"], "element-citation": ["\n"], "surname": ["Bartlett"], "given-names": ["SN"], "year": ["2002"], "article-title": ["The problem of children's injuries in low-income countries: a review."], "source": ["Health Policy Plan"], "volume": ["17"], "fpage": ["1"], "lpage": ["13"]}, {"label": ["29"], "element-citation": ["\n"], "collab": ["World Health Organization"], "year": ["2004"], "source": ["Guidelines for conducting community surveys on injuries and violence"], "publisher-loc": ["Geneva"]}]
{ "acronym": [], "definition": [] }
29
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 10; 3(9):e3166
oa_package/b0/a4/PMC2528934.tar.gz
PMC2528935
18797502
[ "<title>Introduction</title>", "<p>Researchers have long noted that men tend to have lower index∶ring finger ratios than women ##UREF##0##[1]##, ##REF##20982098##[2]##, ##REF##14943709##[3]##. Anecdotal reports of subtle differences in such measures of hand shape between mathematicians and engineers, as compared with artists and litterateurs, for example, date back at least a century ##UREF##1##[4]##. This 2D∶4D ratio is lower, more masculine-typical, in both men and women of greater physical fitness, or who participate at more elite levels of athletic competition ##REF##11182575##[5]##–##REF##17420996##[10]##. 2D∶4D has been proposed to correlate with health and personality traits because of the common organizational influence of prenatal testosterone across these traits ##UREF##2##[11]##–##UREF##4##[14]##. Although studies linking prenatal environment to variation in digit ratio do imply a role for fetal testosterone, a direct cause-and-effect relation is far from proven. For example, girls with congenital adrenal hyperplasia have lower, more male-like digit ratios ##REF##12441204##[15]##, ##REF##12488105##[16]##. Congenital adrenal hyperplasia involves disruption of the glucocorticoid synthesis pathway in the adrenal glands and leads to an absence of circulating glucocorticoids, triggering an over-production of androstenedione, which in turn results in abnormally high adrenal androgen production. The ratio of testosterone to estradiol in amniocentesis samples collected in the second trimester correlated with the child's 2D∶4D ratio at two years of age in a sample of 18 boys and 15 girls ##REF##15113628##[17]##. Female dizygotic twins having male co-twins have more masculine left hand 2D∶4D than those with female co-twins ##REF##16143332##[18]##. Similarly, mice that gestate next to males have larger ratios than those gestating next to females ##REF##18080736##[19]##, suggesting an effect of fetal testosterone in the opposite direction. Behavioral studies correlating 2D∶4D in humans and mice also suggest similar, but opposite effects: humans with lower 2D∶4D show greater propensity for aggression ##REF##15555497##[20]##–##UREF##6##[22]##, whereas inbred mouse strains with larger 2D∶4D were more likely to bite while being handled ##REF##16011578##[23]##. Lower digit ratios are associated with greater physical fitness or athletic competitiveness in men and women ##REF##11182575##[5]##–##REF##17420996##[10]##, but mouse strains with higher digit ratios tend (p = 0.058) to show higher daily activity levels (measured by the total number of beam breaks per day in an individual live-in cage) ##REF##16011578##[23]##.</p>", "<p>The overwhelming trend in human digit ratio studies shows more masculinized 2D∶4D to be associated with more masculinized behavior within each sex, so the studies linking sporting achievement and digit ratio make intuitive sense. However, the idea that 2D∶4D simply reflects the organizational effects of testosterone cannot be the whole story. The emphasis on prenatal testosterone as the major determinant of digit ratio runs counter to the fact that variation in digit ratio among human ethnic groups is far larger than differences between the sexes ##REF##12959899##[24]##–##REF##16688484##[26]##. Similarly, lab mice show large inter-strain differences in 2D∶4D ##REF##16011578##[23]##, but evidence for a sex difference in lab mice is mixed: two small studies show a higher 2D∶4D in females ##REF##12115273##[27]##, ##REF##12581609##28##, while a larger study finds no difference ##REF##16011578##[23]##. Whether digit ratio in mice is sexually dimorphic, and whether variation in rodent digit ratio can serve as a reliable proxy for prenatal environment in behavioral studies, is a matter of debate ##REF##16594983##[29]##, ##UREF##8##[30]##. Some ##UREF##2##[e.g. 11]## have argued that the digit ratio effect is driven by regulation of HOX genes by circulating androgens. Evidence of digit ratio effects from other taxa have been ambiguous. In lizards, sex differences have been reported in opposing directions for different species within the same study ##UREF##9##[31]##. Studies of <italic>Anolis carolinensis</italic> have reported significant effects of sex as well as significant differences between laboratories/populations ##UREF##10##[32]##, but another smaller study reports no significant difference ##UREF##11##[33]##. Similar patterns of results have been reported in birds, where sex differences found in zebra finches ##UREF##12##[34]## were not replicated by a larger study ##UREF##13##[35]##, and in pheasants where sex differences in 2D∶4D were found in one population ##REF##17136513##[36]##, but not another ##REF##15878573##[37]##. Given the highly conserved nature of HOX genes, and their pleiotropic effect on morphological development of both gonads and fingers, one ought to expect a highly robust sex effect on digit ratio across taxa ##REF##12581609##[28]##.</p>", "<p>We used an experimental evolution approach ##UREF##14##[38]## to investigate the link between behavior and digit ratios in laboratory house mice. We compared mice from lines that have been selectively bred for high rates of voluntary exercise with their unselected control lines. Mice from the selected (High Runner, HR) lines exhibit many differences relative to control lines, including higher voluntary wheel-running speeds, home-cage activity, maximal aerobic capacity, and predatory aggression, but no difference in maternal or intermale aggression ##REF##9670598##[39]##–##REF##18329645##[42]##. Importantly, the HR lines also show 2-fold elevated baseline circulating corticosterone, but no apparent difference in circulating testosterone ##REF##14609543##[40]##, ##REF##18329645##[42]##, ##UREF##15##[43]##. We hypothesized that, if digit ratio reflects the organizational effects of testosterone, then we would find a consistent sex difference in the mice. The digit-ratio-reflects-testosterone hypothesis predicts no difference between HR and control lines, given the absence of an effect of the selective-breeding regime on testosterone levels. We found a sex difference, higher 2D∶4D in females than males, statistically significant in the selected lines only, and an effect of selective breeding, higher 2D∶4D ratios in HR lines, in both sexes. The latter effect seems to contradict human studies, where masculinized digit ratios are associated with greater physical fitness or sporting achievement.</p>" ]
[ "<title>Materials and Methods</title>", "<p>The selective breeding methodology is described in ##REF##9670598##[39]##. To achieve a large sample size (total N = 1,013), mice were sampled from generations 45–48. Digits were measured by RHY (blind to generation, sex, line, and linetype), following the same procedures as in our previous work ##REF##16504142##[13]##, ##UREF##4##[14]##. Briefly, hind paws preserved in 4% paraformaldehyde were straightened and fixed palm-up to an adhesive backing, then photographed via stereoscope. Digit length was measured from the mid-point of the basal crease to digit tip (##FIG##0##Fig. 1##). Each paw was photographed and measured twice while the paw was left in the same position. The mean value of the two measures (which were highly correlated, Pearson's r = 0.9 for both left and right paws), was used for analysis. To calculate the reliability of the 2D∶4D measures, we re-imaged a random sample of 50 mice several months after the original images were taken, then calculated the intraclass correlation coefficient following ##UREF##16##[47]##. Left paws: ICC = 0.66 (95%CI: 0.472&lt;ICC&lt;0.792), F(49,50) = 4.89, P≪0.001), Right paws: ICC = 0.544 (95%CI: 0.317&lt;ICC&lt;0.713), F(49,50) = 3.39, P≪0.001).</p>", "<p>We used REML estimation in SAS Procedure Mixed (SAS Institute, Cary, NC) to perform mixed-model ANOVA. Main fixed effects were linetype (HR vs. C), sex, and the sex-by-linetype interaction (generation was included as a nuisance variable). Random effects were family nested within line, replicate lines nested within linetype, and the sex-by-line(linetype) interaction term (the number of families in each of the eight lines was 32, 39, 27, 34, 28, 33, 28 &amp; 28 for lines 1 through 8 respectively; note lines designated 1, 2, 4 &amp; 5 were control lines, while 3, 6, 7 &amp; 8 were selected lines). We also separated HR and C lines for two-way ANOVAs of sex and line, both treated as fixed, with family as random nested within line (see ##FIG##1##Fig. 2##). Two-tailed P values are presented.</p>" ]
[ "<title>Results</title>", "<p>We compared hind limb 2D∶4D, measured from the mid-point of the basal crease to digit tip (##FIG##0##Fig. 1##), of the four HR lines (male∶female N = 66∶79, 74∶89, 69∶62, 77∶69) with those of the four Control (C) lines (N = 48∶61, 49∶54, 58∶58, 53∶47).</p>", "<p>Overall, 2D∶4D was higher in females than males, and in HR lines than Control lines (##FIG##1##Fig. 2##). On the right limb, differences were significant for sex (F<sub>(1,6)</sub> = 12.88, p = 0.0115) and linetype (F<sub>(1,6)</sub> = 11.76, p = 0.0140), without significant interaction between these main effects (F<sub>(1,6)</sub> = 2.99, p = 0.1345) (for the random effects, χ<sup>2</sup> = 5.10, d.f. = 3, P = 0.0779). Results were similar on the left limb, but did not reach statistical significance (sex F<sub>(1,6)</sub> = 4.88, p = 0.0691; linetype F<sub>(1,6)</sub> = 4.71, p = 0.0730; interaction F<sub>(1,6)</sub> = 0.19, p = 0.6803) (for the random effects, χ<sup>2</sup> = 37.11, d.f. = 3, P&lt;0.0001). The finding of significant effects only on the right side fits the general pattern of previous research on human beings ##UREF##2##[11]##, ##UREF##3##[12]##.</p>", "<p>Although the interaction between sex and linetype was not statistically significant, ##FIG##1##Figure 2## suggests that the significant sex effect on the right paw is driven by the HR lines. Indeed, separate analyses of the Control and HR lines showed no sex difference for either limb in Control lines (right F<sub>(1,91)</sub> = 1.63, p = 0.2047; left F<sub>(1,91)</sub> = 1.76, p = 0.1879), whereas HR lines showed a sex difference in 2D∶4D on both rear limbs (right F<sub>(1,81)</sub> = 14.56, p = 0.0003; left F<sub>(1,81)</sub> = 5.74, p = 0.0189).</p>" ]
[ "<title>Discussion</title>", "<p>Selective breeding for the behavioral trait of high voluntary exercise (wheel running) raised the 2D∶4D digit ratio in these mice, suggesting that the selection regime “feminized” them, as females in this study had larger 2D∶4D ratios than males. The direction of the sex effect, larger in females than in males, agrees with those studies that have found sex differences in mice ##REF##12115273##[27]##, ##REF##12581609##[28]##. Similarly, the effect of selective breeding parallels the trend among inbred strains where higher total daily activity correlates positively with digit ratio ##REF##16011578##[23]##. Therefore, the present results seem to contradict the human studies, where masculinized digit ratios are associated with greater physical fitness or sporting achievement.</p>", "<p>Several possible explanations exist for this discrepancy, the first being that the HR lines are indeed the more feminized, but that high voluntary wheel running in mice is not a homologous trait to the traits assayed in the human studies. Female mice run more than males ##REF##18329645##[42]##. In human studies, the physical fitness measures used can be broadly classified as either level of competition in hierarchically organized sports ##REF##11182575##[5]##–##REF##17008344##[7]## or objective performance scores on athletic tasks ##REF##16403410##[8]##–##REF##17420996##[10]##. In a study of an otherwise unselected sample of 77 women and 102 men (mostly university students) ##REF##16403410##[8]##, the number of hours of exercise a week was measured, as a potentially confounding variable, and correlated significantly, and positively, with digit ratio (females, left hand: r = 0.27, P = 0.017; males, right hand: r = 0.38, P = 0.0001; note that these sex-by-hand combinations were the only ones correlating with physical fitness in the original study). This suggests that voluntary exercise in humans, unlike physical fitness, is associated with less masculinized 2D∶4D. Here, we found no such relationship in mice. In any case, the literature linking masculinized 2D∶4D with greater physical fitness in not unequivocal; for example, lower 2D∶4D is associated with earlier age of first myocardial infarction in men ##REF##12581609##[28]##.</p>", "<p>A second possible explanation is that higher exercise rates are in fact more “masculinized,” but that lower digit ratios do not indicate greater masculinization in these mice. The present data do not provide unequivocal support for larger 2D∶4D in females. The unselected control lines, like those in ##REF##16011578##[23]##, do not show statistically significant sexual dimorphism. Other evidence, such as intrauterine position ##REF##18080736##[19]##, suggests an effect in the opposite direction, higher in more masculinized individuals. On balance, the lack of any sex effect in that direction suggests strongly that if digit ratio indicates masculinization, then the high-running lines are the more feminized, and the possibility that lower digit ratios are more masculinized is a poor candidate explanation for our results.</p>", "<p>A third possible explanation for our results is that propensity for voluntary physical exercise and 2D∶4D are not linked to masculinization or, if they are, not through prenatal androgen exposure. Although 2D∶4D was higher in high-running lines of mice, they do not have higher testosterone, at least as adults ##REF##14609543##[40]##. However, the sex and selection regime effects on 2D∶4D match those for adult baseline circulating corticosterone: females have higher corticosterone levels than males, and HR lines have higher levels than Controls ##REF##18329645##[42]##, ##UREF##15##[43]##. Similarly, females have higher 2D∶4D than males, and HR have higher 2D∶4D than Controls. Although increased circulating glucocorticoids are generally thought to depress testosterone, some evidence suggests that they are positively correlated in the human fetus ##REF##15724043##[44]##. We are unaware of any published work directly examining the effects of glucocorticoids on digit ratio. However, experimental manipulations of hormones other than testosterone have been demonstrated to influence digit ratio. Male, but not female, ring necked-pheasants show decreased right limb 2D∶4D in response to increased egg estradiol ##REF##17136513##[36]##, whereas they show an increase in left limb 2D∶3D–not 2D∶4D–in females, but not males, in response to increased egg testosterone ##REF##15878573##[37]##. Given the many factors that have the ability to affect digit ratio, it is clearly more complicated than a simple testosterone-driven manliness metric. The significant additive genetic effects on human digit ratios imply a possible common effect of genes on both 2D∶4D and behavioral traits, aside from any common hormonal influence ##REF##15724043##[44]##, ##REF##16611491##[45]##. Our results cast some doubt on 2D∶4D serving as an indicator of prenatal testosterone exposure, per se. Nonetheless, our results support the idea that relative digit length reflects some endocrinology-related aspect of development. Further investigation of the interacting effects of genetic variation, perinatal hormone levels, and such environmental influences as prenatal maternal stress and perinatal maternal care will be required before the full story is understood.</p>" ]
[]
[ "<p>Conceived and designed the experiments: PLH TGJ. Performed the experiments: RHYY JLM RMH. Analyzed the data: TGJ. Contributed reagents/materials/analysis tools: TGJ. Wrote the paper: PLH TGJ.</p>", "<p>The ratio of the length of the second digit (index finger) divided by the fourth digit (ring finger) tends to be lower in men than in women. This 2D∶4D digit ratio is often used as a proxy for prenatal androgen exposure in studies of human health and behavior. For example, 2D∶4D ratio is lower (i.e. more “masculinized”) in both men and women of greater physical fitness and/or sporting ability. Lab mice have also shown variation in 2D∶4D as a function of uterine environment, and mouse digit ratios seem also to correlate with behavioral traits, including daily activity levels. Selective breeding for increased rates of voluntary exercise (wheel running) in four lines of mice has caused correlated increases in aerobic exercise capacity, circulating corticosterone level, and predatory aggression. Here, we show that this selection regime has also increased 2D∶4D. This apparent “feminization” in mice is opposite to the relationship seen between 2D∶4D and physical fitness in human beings. The present results are difficult to reconcile with the notion that 2D∶4D is an effective proxy for prenatal androgen exposure; instead, it may more accurately reflect effects of glucocorticoids, or other factors that regulate any of many genes.</p>" ]
[]
[ "<p>We thank Andy Iwaniuk for inspiration, Doug Wong-Wylie for stereoscope and camera use, Nalin Fernando for photographic assistance and Wolfgang Forstmeier and an anonymous reviewer for helpful comments on an earlier version of this manuscript.</p>" ]
[ "<fig id=\"pone-0003216-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003216.g001</object-id><label>Figure 1</label><caption><title>Measurement of digit lengths.</title><p>Right rear palm of male mouse from a line bred for high running, illustrating digit lengths, measured from the mid-point of the basal crease to digit tip. The 2D∶4D ratio was 573.6∶585.0 pixels, yielding a 2D∶4D of 0.9805.</p></caption></fig>", "<fig id=\"pone-0003216-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003216.g002</object-id><label>Figure 2</label><caption><title>Digit ratios by sex and selection line.</title><p>2D∶4D finger length ratios by sex and selection regime for the eight lines of mice. Control are lines 1, 2, 4 and 5 (lab designations), High Runner selected lines are 3, 6, 7 and 8), open bars indicate females, and shaded bars males. Values are least square means and associated standard errors from models (separately for Control and Selected lines) including sex, line, sex-by-line interaction, and generation as fixed effects (SAS Procedure Mixed: family nested within line and the sex-by-family(line) interaction were random effects). See text for P values and for results of analyses comparing the four Control with the four High Runner lines.</p></caption></fig>" ]
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[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>Supported by Natural Sciences and Engineering Research Council (NSERC Canada) PGS-M scholarship to R.H.Y., NSERC Discovery Grant (249685) to P.L.H., and National Science Foundation grant (IOB-0543429) to T.G. J.L.M was supported by the University of California Office of the President Dissertation Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"pone.0003216.g001\"/>", "<graphic xlink:href=\"pone.0003216.g002\"/>" ]
[]
[{"label": ["1"], "element-citation": ["\n"], "surname": ["George"], "given-names": ["R"], "year": ["1930"], "article-title": ["Human finger types."], "source": ["Anat Rec"], "volume": ["46"], "fpage": ["199"], "lpage": ["204"]}, {"label": ["4"], "element-citation": ["\n"], "surname": ["Baker"], "given-names": ["F"], "year": ["1888"], "article-title": ["Anthropological notes on the human hand."], "source": ["Am Anthropol"], "volume": ["1"], "fpage": ["51"], "lpage": ["76"]}, {"label": ["11"], "element-citation": ["\n"], "surname": ["Manning"], "given-names": ["JT"], "year": ["2002"], "source": ["Digit ratio: a pointer to fertility behavior and health"], "publisher-loc": ["New Brunswick, NJ"], "publisher-name": ["Rutgers University Press"]}, {"label": ["12"], "element-citation": ["\n"], "surname": ["Putz", "Gaulin", "Sporter", "McBurney"], "given-names": ["DA", "SJC", "RJ", "DH"], "year": ["2004"], "article-title": ["Sex hormones and finger length What does 2D\u22364D indicate?"], 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["42"], "fpage": ["755"], "lpage": ["764"]}, {"label": ["25"], "element-citation": ["\n"], "surname": ["Manning", "Barley", "Walton", "Lewis-Jones", "Trivers", "Singh"], "given-names": ["JT", "L", "J", "DI", "RL", "D"], "year": ["2000"], "article-title": ["The 2nd\u22364th digit ratio sexual dimorphism population differences and reproductive success: evidence for sexually antagonistic genes?"], "source": ["Evol Human Behav"], "volume": ["21"], "fpage": ["163"], "lpage": ["183"]}, {"label": ["30"], "element-citation": ["\n"], "surname": ["Hurd", "Wahlsten"], "given-names": ["PL", "D"], "year": ["2006"], "article-title": ["No sex difference in mouse digit ratio: reply to Voracek."], "source": ["Genes Brain Behav"], "volume": ["5"], "fpage": ["300"], "lpage": ["302"]}, {"label": ["31"], "element-citation": ["\n"], "surname": ["Rubolini", "Pupin", "Sacchi", "Gentilli", "Zuffi"], "given-names": ["D", "F", "R", "A", "MAL"], "year": ["2006"], "article-title": ["Sexual dimorphism in digit length ratios in two lizard species."], "source": ["Anat Rec"], "volume": ["288A"], "fpage": ["491"], "lpage": ["497"]}, {"label": ["32"], "element-citation": ["\n"], "surname": ["Chang", "Doughty", "Wade", "Lovern"], "given-names": ["JL", "S", "J", "MB"], "year": ["2006"], "article-title": ["Sexual dimorphism in the second-to-fourth digit length ratio in green anoles, "], "italic": ["Anoles carolinensis"], "source": ["Can J Zool"], "volume": ["84"], "fpage": ["1489"], "lpage": ["1494"]}, {"label": ["33"], "element-citation": ["\n"], "surname": ["Lombardo", "Thorpe"], "given-names": ["MP", "PA"], "year": ["2008"], "article-title": ["Digit ratios in green Anolis lizards ("], "italic": ["Anolis carolinensis"], "source": ["Anat Rec"], "volume": ["291"], "fpage": ["433"], "lpage": ["440"]}, {"label": ["34"], "element-citation": ["\n"], "surname": ["Burley", "Foster"], "given-names": ["NT", "VS"], "year": ["2004"], "article-title": ["Digit ratio varies with sex, egg order, and strength of mate preference in zebra finches."], "source": ["Proc R Soc Lond B"], "volume": ["271"], "fpage": ["239"], "lpage": ["244"]}, {"label": ["35"], "element-citation": ["\n"], "surname": ["Forstmeier"], "given-names": ["W"], "year": ["2005"], "article-title": ["Quantitative genetics and behavioural correlates of digit ratio in the zebra finch."], "source": ["Proc R Soc Lond B"], "volume": ["272"], "fpage": ["2641"], "lpage": ["2649"]}, {"label": ["38"], "element-citation": ["\n"], "surname": ["Garland", "Rose"], "given-names": ["T", "MR"], "suffix": ["Jr"], "year": ["2009"], "source": ["Experimental evolution: concepts, methods, and applications of selection experiments"], "publisher-loc": ["Berkeley CA"], "publisher-name": ["University of California Press"], "comment": ["In press"]}, {"label": ["43"], "element-citation": ["\n"], "surname": ["Malsich", "Saltzman", "Gomes", "Rezende", "Jeske"], "given-names": ["JL", "W", "FR", "EL", "DR"], "year": ["2007"], "article-title": ["Baseline and 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{ "acronym": [], "definition": [] }
47
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 17; 3(9):e3216
oa_package/67/90/PMC2528935.tar.gz
PMC2528936
18795096
[ "<title>Introduction</title>", "<p>Innate immunity uses gene-encoded antimicrobial peptides to form a first line of host defense against noxious microorganisms ##REF##11807545##[1]##, ##REF##15703760##[2]##. A large amount of antimicrobial peptides have been identified from animals, plants and microorganisms. Several families of antimicrobial peptides including cathelicidin, liver-expressed antimicrobial peptide (LEAP) or hepcidin, histatin, and defensin have been identified from mammalians ##REF##12960280##[3]##–##REF##15025852##[7]##. Defensins and hepcidins are characterized by the presence of multiple disulfide bridges, whereas histatins and most of cathelicidins are linear molecules without disulfide bridges.</p>", "<p>After the first discovery of cathelicidin (Bac5) from bovine neutrophils, a large amount of cathelicidins have been identified from other mammalians ##REF##16716248##[8]##–##REF##8416958##[13]##. As other antimicrobial peptide families, structurally divergent cathelicidins have been found, even in a single mammalian species. For example, there are at least seven cathelicidins in cattle, horse, pig, sheep, and goat ##REF##16716248##[8]##. Some exceptions are in human, rhesus monkey, mouse, rat, and guinea pig, only a single cathelicidin was found ##REF##16716248##[8]##, ##REF##11238224##[14]##–##REF##9278433##[18]##.</p>", "<p>Cathelicidin antimicrobial peptides are released from their corresponding inactive precursors by proteolytic cleavage ##REF##16716248##[8]##. The cathilicidin family of proteins is characterized by the presence of a highly conserved anionic cathelin domain ##REF##12960280##[3]##, ##REF##16716248##[8]##, ##REF##10931440##[19]##. Cathelin is an inhibitor of the cysteine proteinase cathepsin L ##REF##2792375##[20]##. In the precursors of cathelicidins, the highly conserved cathelin domains composed of about 100 amino acid residues is flanked by a signal peptide fragment (approximately 30 residues long) on its N-terminus, and by a structurally divergent cationic antimicrobial peptide region on its C-terminus ##REF##16716248##[8]##. Upon activation, most of cathelicidin precursors proteolytically cleaved to release the cathelin domain and the C-terminal mature antimicrobial peptides. Some intact cathelicidin precursors are also found in the biological fluids where cathelicidin expressed ##REF##12960280##[3]##, ##REF##12226737##[21]##. Elastase seems to be the most common peptidase to release mature cathelicidins ##REF##1425666##[22]##, ##REF##9038306##[23]##. In human hCAP18, however, protease-3 cleaves the proprotein ##REF##11389039##[24]##. Mature cathelicidins can be further degraded by some serine proteases because multiple cationic amino acid residues (Arg or Lys) are in the sequences of cathelicidins ##REF##12759353##[25]##. In addition, hCAP18 could be degraded by aspartyl protease (gastricsin) at vaginal pH. Some hydrolytic fragments of cathelicidin were found to possess increased antimicrobial abilities ##UREF##0##[26]##.</p>", "<p>Recently, several cathelicidins have been identified from some non-mammalian vertebrates including hagfish ##REF##15019197##[27]##, rainbow trout ##REF##16041021##[28]##, ##REF##16377685##[29]##, atlantic salmon ##REF##16377685##[29]##, and chicken ##REF##16326712##[30]##, ##REF##17827276##[31]##. As the oldest jawless craniates, hagfish lacks adaptive immunity ##REF##16716248##[8]##, ##UREF##1##[32]##. The presence of cathelicidins in hagfish may indicate that cathelicidin genes appeared early in phylogenesis ##REF##16716248##[8]##. Cathelicidins have been found from most of vertebrates including fish, bird, mammalian, whereas no cathelicidins have been found from amphibians and reptiles. In this wok, a cathelicidin from snake was identified and characterized.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Materials</title>", "<p>\n<italic>B. fasciatus</italic> crude venom and venomous glands were collected from Guang Xi Province, China. The SMART™ PCR cDNA synthesis kit was purchased from Clontech, USA. Chromatography media Sephadex G-50 and CM-Sephadex C-25 were obtained from Amersham Bioscience, Sweden. Trifluoroacetic acid (TFA, HPLC grade) was from Perkin-Elmer. Acetonitrile (ACN, HPLC grade) was bought from Fisher Chemicals. 2,2,2-trifluroethanol-d3 98% (TFE-d3), sodium dodecyl-d<sub>25</sub> sulfate (SDS-d25) 98.7%, trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP)-d<sub>4</sub> 98% and D<sub>2</sub>O 99% were purchased from Cambridge Isotope Laboratories. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) C4 column (30 cm×0.46 cm) was from Agilent. The pMD18-T vector was from Takara, Dalian, China. All other reagents were of analytical or sequencing grade. The animals used for the experiments were treated according to the protocols evaluated and approved by the ethical committee of Kunming Institute of Zoology.</p>", "<title>Isolation of cathelicidin-BF</title>", "<p>The purification procedure was according to our previously report ##REF##18164783##[33]##, ##REF##17383773##[34]##. 0.4 g <italic>B. fasciatus</italic> crude venom was first fractionated using gel filtration chromatography Sephadex G-50 column (26 cm×100 cm), equilibrated with 50 mM Tris–HCl, 50 mM NaCl (pH 7.8). The elution was performed with the same buffer and monitored at UV absorption of 280 nm. The peak having antimicrobial activity was collected and further dialyzed against PBS (pH 6.0). The dialyzed product was next subjected to the cation-exchange CM-Sephadex C-25 column (1.6 cm×30 cm). The elution was achieved with a linear NaCl gradient, at a flow rate of 1 ml/min. The peak with antimicrobial activity was collected and finally purified by reverse phase high performance liquid chromatography (C4), equilibrated with 0.1% (v/v) TFA/water. The elution was performed with a liner gradient of acetonitrile at a flow rate of 0.7 ml/min.</p>", "<title>Primary structural analysis</title>", "<p>The amino acid sequence of the N-terminus was determined by the automated Edman degradation using an Applied Biosystems pulsed liquid-phase sequencer, model 491. Electrospray ionization mass spectrometry (ESI-MS) was used to determine the molecular weight by a Finnigan LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA) in positive-ion mode. The sample solutions (50%H2O/50%ACN) were infused into the mass spectrometer via a Harvard syringe pump (Holliston, MA, USA). The spray voltage was set to +4.5 kV. Spectra were acquired by summing 30 scans.</p>", "<title>CD and NMR spectroscopy</title>", "<p>Circular dichroism (CD) spectra were recorded at 298 K on a JASCO J-810 spectrometer (Jasco, Japan). Samples were prepared by dissolving the peptide powder to a concentration of 90 µM in TFE/H<sub>2</sub>O mixtures or in SDS micelles of different concentrations. The spectra were measured between 190 and 250 nm using 0.1 cm path-length cell with 1 nm bandwidth, 1 sec response time, and a scan speed of 100 nm/min. Three consecutive scans per sample were performed, added and averaged followed by subtraction of the signal of the solvent. The secondary structure elements of the peptides were estimated according to the Yang formula ##REF##9762080##[35]##.</p>", "<p>Samples for nuclear magnetic resonance (NMR) measurements contained 4 mM cantheicidin-BF in TFE-d3/H<sub>2</sub>O (9∶1, v/v) at pH 6.5, or in 300 mM SDS-d25 at pH 6.5. All NMR spectra were recorded at 298 K on a Varian Unity INOVA 600 MHz spectrometer equipped with three RF channels and a triple resonance z-axis pulsed-field gradient probe. The 2D <sup>1</sup>H-<sup>1</sup>H TOCSY spectra were acquired with a mixing time of 75 ms, while <sup>1</sup>H-<sup>1</sup>H NOESY spectra were acquired with mixing times of 200 and 300 ms. The watergate approach was employed for water suppression. Data were collected with 256 and 1024 complex data points in t1 and t2 dimensions, respectively. Signals were averaged over 64 transients. All NMR spectra were processed and analyzed using the NMRPipe/NMRDraw software and the Sparky program ##REF##8520220##[36]##, ##UREF##2##[37]##. Linear prediction in the t1 dimension was used before the Fourier transformation. Assignments of the proton resonances were achieved using both TOCSY and NOESY spectra. The <sup>1</sup>H chemical shifts were referenced to TSP. The secondary structure was predicted using the H<sub>α</sub> Chemical Shift Index approach ##REF##8589602##[38]##.</p>", "<title>SMART cDNA synthesis</title>", "<p>Total RNA was extracted using TRIzol (Life Technologies, Ltd.) from the venomous glands of <italic>B. fasciatus</italic>. cDNA was synthesized by SMART™ techniques by using a SMART™ PCR cDNA synthesis kit (Clontech, Palo Alto, CA). The first strand was synthesized by using cDNA 3′ SMART CDS Primer II A, <named-content content-type=\"gene\">5′-AAGCAGTGGTATCAACGCAGAGTACT</named-content> (30) N-1N-3′ (N = A, C, G or T; N-1 = A, G or C), and SMART II An oligonucleotide, <named-content content-type=\"gene\">5′-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3′</named-content>. The second strand was amplified using Advantage polymerase by 5′ PCR primer II A, <named-content content-type=\"gene\">5′-AAGCAGTGGTATCAACGCAGAGT- 3′</named-content>.</p>", "<title>Screening of cDNA encoding cathelicidin-BF</title>", "<p>The cDNA synthesized by SMART™ techniques was used as template for PCR to screen the cDNAs encoding serine protease inhibitor. Two oligonucleotide primers, BFS<sub>1</sub>\n<named-content content-type=\"gene\">5′-AA(A/G)TT(T/C)TT(T/C)AG(A/G)AA(A/G)(C/T)T(A/T/C/G)AA(A/G)AA (A/G)-3′</named-content>, in the reverse direction, a specific primer designed according to the amino acid sequence determined by Edman degradation and primer II A as mentioned in “<italic>SMART cDNA synthesis</italic>” in the sense direction were used in PCR reactions. The DNA polymerase was Advantage polymerase from Clontech (Palo Alto, CA) The PCR conditions were: 2 min at 94°C, followed by 30 cycles of 10 sec at 92°C, 30 sec at 50°C, 40 sec at 72°C. Finally, the PCR products were cloned into pGEM®-T Easy vector (Promega, Madison, WI). DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377.</p>", "<title>Expression profile of tissues</title>", "<p>Reverse transcription-polymerase chain reaction (RT-PCR) was carried out to analyze gene expression of cathelicidin-BF in <italic>B. fasciatus</italic>. Total RNA extraction from different tissues and first-strand cDNA synthesis were the same as described above. The primers were, forward primer, 5′-cathelicidin: <named-content content-type=\"gene\">5′-ATGGAAGGGTTCTTCTGGA AGACC-3′</named-content>, and reverse primer, 3′-cathelicidin: <named-content content-type=\"gene\">5′-CAAATTAGAAGGGGATGGAG ACC-3′</named-content>. PCR conditions were: 95°C (3 min), and 30 cycles of 95°C (30 s), 56°C (30 s), 72°C (3 min) followed by a 15 min extension period at 72°C. The control PCR was performed using the specific primers (forward primer, actin-s <named-content content-type=\"gene\">5′-GGGTGTGATGGT TGGCATGG-3′</named-content>, and reverse primer, actin-as <named-content content-type=\"gene\">5′-TGGCTGGAAGAGGGCTTCTG-3′</named-content>) for snake actin, using the same conditions as above.</p>", "<title>Alignment and phyogenetic comparison of cathelicidins</title>", "<p>Cathelicidin sequences were obtained from the protein database at the National Center for Biotechnology Information. The phylogenetic tree is constructed by neighbor-joining analysis, using the ClustalW program (version 1.8).</p>", "<title>Antimicrobial testing</title>", "<p>Antimicrobial activities of cathelicidin-BF and cathelicidin-BF15 (VKRFKKFFRKLKKSV) were tested according to our previous methods ##REF##17384140##[39]##–##REF##17764786##[42]##. Ampicillin, benzylpenicillin (Amresco) and Imipenem and Cilastatin Sodium for Injection (ICS, Merck) were used as positive controls. The details were provided in the ##SUPPL##0##Materials and Methods S1##.</p>", "<title>Bacteria killing kinetics</title>", "<p>\n<italic>In vitro</italic> bacteria killing kinetics of cathelicidin-BF, ICS (its minimal inhibitory concentration (MIC) for <italic>Escherichia coli</italic> 08A866 is 0.15 µg/ml), and HDW (an antimicrobial peptide from the frog of <italic>Rana nigrovittata</italic>, with a amino acid sequence of FIGPVLKIATSILPTAICKIFKKC, its MIC for <italic>E. coli</italic> 08A866 is 18.7 µg/ml), respectively, were determined according to the methods described by Mygind <italic>et al</italic>\n##REF##16222292##[43]##. The details were provided in the ##SUPPL##0##Materials and Methods S1##.</p>", "<title>Hemolysis, cytotoxicity, serum stability</title>", "<p>Hemolytic activity was checked by incubating the tested samples with human red blood cells to determine hemoglobin releasing ability by measuring the absorbance at 540 nm, using 1% Triton X-100 as a positive control. Cytotoxicity and serum stability were measured according the methods described by Mygind <italic>et al</italic>\n##REF##16222292##[43]##. The details were provided in the ##SUPPL##0##Materials and Methods S1##.</p>", "<title>Synthetic Peptides</title>", "<p>All of the peptides used for the bioactivity assays and NMR analysis in this paper were synthesized by the peptide synthesizer (433A, Applied Biosystems) in AC SCIENTIFIC (Xi An) INC. (Xi An, China) and analyzed by HPLC and MALDI-TOF mass spectrometry to confirm that the purity was higher than 95%. All peptides were dissolved in water.</p>" ]
[ "<title>Results</title>", "<title>Isolation of cathelicidin-BF from the snake venoms of <italic>B. fasciatus</italic>\n</title>", "<p>The crude snake venom was separated into four fractions by Sephadex G-50 gel filtration as our previous report (##SUPPL##1##Figure S1a##) (##SUPPL##1##Fig. S1a##) ##REF##18164783##[33]##, ##REF##17383773##[34]##. The fraction III, containing antimicrobial activity was further subject to CM-Sephadex C-25 cation-exchange column, and nine sub-fractions were collected (##SUPPL##1##Figure S1b##). The fraction VI with both trypsin-inhibitory and antimicrobial activities was further purified using RP-HPLC. The peak with antimicrobial activity is marked with an arrow in ##SUPPL##1##Figure S1c##. The purified antimicrobial peptide was named cathelicidin-BF. The molecular mass and purity of purified cathelicidin-BF was further analyzed by a ESI mass spectrometry, giving a [M+7H]<sup>7+</sup>, [M+7H]<sup>6+</sup>, [M+7H]<sup>5+</sup>and [M+7H]<sup>4+</sup>of 521.1, 607.6, 729.1 and 991.5 (##SUPPL##2##Figure S2##), indicating that purified cathelicidin-BF has a molecular weight of 3637.5–3638.5.</p>", "<title>Structure characterization of cathelicidin-BF</title>", "<p>Purified cathelicidin-BF was subjected to amino acid sequence analysis using automated Edman degradation. Its amino acid sequence is KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF. Cathelicidin-BF is composed of 30 amino acid residues including 12 basic residues (9 Lys and 2 Arg), 5 phenylalanines, and only one acidic amino acid residue (Glu). It is a lysine-rich and phenylalanine-rich peptide. Analysis using the ExPASy MW/pI tool (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.expasy.ch/tools/pi_tool.html\">http://www.expasy.ch/tools/pi_tool.html</ext-link>) showed that cathelicidin-BF had the predicted pI (isoelectric point) of 11.79 and a predicted molecular weight of 3637.5 that matched well with the observed mass by ESI mass spectrometry (##SUPPL##2##Figure S2##). By BLAST search, no similar sequence was found in GenBank.</p>", "<p>Several positive clones, which contained an insert of 750 bp were identified and isolated from <italic>B. fasciatus</italic> venomous gland cDNA library. The complete nucleotide sequence of cDNA (GenBank accession EU753183) and deduced amino acid sequence of cathelicidin-BF precursor are shown in ##FIG##0##Figure 1##. Unexpectedly, the cathelicidin-BF precursor displays the maximal similarity (47%) with predicted myeloid cathelicidin 3 from <italic>Ornithorhynchus anatinus</italic> (GenBank accession XP_001512130) by BLAST search. The protein precursor is composed of 191 amino acid (aa) residues, including a predicted signal peptide, a conserved cathelin domain and a mature cathelicidin-BF. Noticeably, four cysteines that are conserved in the cathelin domain of all mammalian cathelicidins are also invariantly spaced in cathelicidin-BF precursor, suggesting that the snake cathelicidin-BF precursor is a real mammalian cathelicidin.</p>", "<p>The amino acid sequence of cathelicidin-BF determined by Edman degradation is identical with the amino acid sequence deduced from the cDNA sequence. There is a possible cleavage site (Valine<sup>157</sup>) for elastase at the N-terminus of the mature cathelicidin-BF (##FIG##0##Figure 1##). Based on the possible cleavage site, a 34-aa peptide should be released from the precursor, but the purified cathelicidin-BF is only composed of 30 aa. Different from other cathelicidins, there is an acidic doman between the cathelin doman and the antimicrobial peptide in the cathelicidin-BF precursor (##FIG##1##Figure 2##).</p>", "<p>Evolution analysis revealed that all vertebrate cathelicidins formed three distinct clusters with fish cathelicidins located in a separated clade from others. Supported by a bootstrap value of 77%, cathelicidin-BF was clustered with platypus CATH-3 (##FIG##2##Figure 3##). Although platypus is a mammal, it also has reptilian features, For instance, it lays eggs and it is venomous ##UREF##3##[44]##. The close evolution relationship of cathelicidin-BF found in the venoms of <italic>B. fasciatu</italic>s with platypus cathelicidins may provide further proof for platypus's reptilian features.</p>", "<title>Secondary structures detected by CD and NMR</title>", "<p>The secondary structure elements in different solvent environments were detected by CD spectroscopy (##SUPPL##3##Figure S3##, ##SUPPL##7##Table S1##). In H<sub>2</sub>O, the CD spectrum of cathelicidin-BF showed a strong negative band at 200 nm, indicative of a random-coil conformation. Interestingly, in TFE/H<sub>2</sub>O mixtures, the CD spectra showed double minima at 208 and 222 nm, indicating a highly α-helical conformation. The signals at 208 and 222 nm were intensified gradually by increasing concentrations of TFE, which indicated that the helicity of the peptide was increased in more hydrophobic or membrane-mimetic environments. The CD spectra of the peptide in SDS micelles also showed a typical α-helix pattern and the content of the α-helix structure increased with the increasing SDS concentration.</p>", "<p>NMR spectra recorded on the peptide in SDS micelles were of low quality and can not be used for structural analysis, might due to aggregation of the peptide in negative charged SDS micelles. Therefore, the helical structure of the peptide in TFE/H<sub>2</sub>O mixture was investigated using NMR spectroscopy. ##SUPPL##8##Table S2## lists the nearly complete assignments of the proton chemical shifts of cantheicidin-BF in TFE/H<sub>2</sub>O mixture (9∶1, v/v). Comparison of the HN-HN region of NOESY spectrum recorded on cantheicidin-BF in H<sub>2</sub>O with that in TFE/H<sub>2</sub>O (9∶1, v/v) illustrates that, the peptide adopts a stable secondary structure in TFE/H<sub>2</sub>O mixture (##SUPPL##4##Figure S4##). The H<sub>α</sub> CSI prediction is indicative of a helical structure in the N-terminal region comprising residues F2–F18 (##SUPPL##5##Figure S5##), although a well-defined three-dimensional structure of the peptide in TFE/H<sub>2</sub>O mixture has not been obtained yet, mainly due to the deficiency of enough unambiguous conformational restraints for the exact structural analysis.</p>", "<p>The amphipathic helical conformation is well known to be a crucial factor for many antimicrobial peptides to interact with membranes ##REF##14561728##[45]##, ##REF##16170803##[46]##. On basis of the rough structural analysis described above, it is indicated that the N-terminal region of cantheicidin-BF adopts a typical amphipathic α-helical conformation (##SUPPL##6##Figure S6##) as many other cantheicidins.</p>", "<title>Expression profile of tissues</title>", "<p>Using actin as control, expression pattern of cathelicidin-BF was investigated by RT-PCR. Tissue distribution of cathelicidin-BF expression in snake tissues were illustrated in ##FIG##3##Fig. 4##. All the selected tissues including stomach, trachea, skin, muscle, heart, kidney, lung, brain, intestine, spleen, liver, ovary and venomous gland can express this protein.</p>", "<title>Antimicrobial activities</title>", "<p>As listed in ##TAB##0##Table 1##, cathelicidin-BF and its analogue, cathelicidin-BF15 showed strong antimicrobial activities against tested microorganisms. Of the 40 tested microorganism strains, cathelicidin-BF exerted potent antimicrobial ability against most of Gram-negative bacteria (either standard strains or clinically isolated drug-resistance strains). For most of <italic>E. coli</italic>, the MICs are lower than 2.3 µg/ml, while ampicillin, benzylpenicillin and ICS are effective only to standard strain with a MIC of 18.7, 37.5 and 0.15 µg/ml respectively. The lowest MIC for <italic>K. pneumoniae</italic> is 0.3 µg/ml, while ampicillin, benzylpenicillin and ICS are effective only to standard strain with a MIC of 150, 18.7 and 9.4 µg/ml respectively. In contrast, most of <italic>S. aureus</italic> are not so sensitive for cathelicidin-BF, only one strain could be killed by cathelicidin-BF with a low MIC (4.7 µg/ml). Another Gram-positive bacteria genus, <italic>Bacillus</italic> also seems to be sensitive for cathelicidin-BF and cathelicidin-BF15. A dangerous clinically isolated strain, <italic>Salmonella typhi</italic> could also be killed by cathelicidin-BF and cathelicidin-BF15 with a low MIC (1.2 µg/ml). Cathelicidin-BF and cathelicidin-BF15 are the same effective to some fungi as bacteria, for example, <italic>C. albicans</italic> ATCC2002 (with a MIC of 4.7 µg/ml).and <italic>P. pastoris</italic> (with a MIC of 0.3 µg/ml). Cathelicidin-BF exerted obvious antimicrobial activity against some saprophytic fungus such as <italic>A. terreus</italic> GIM3.34 (with a MIC of 18.7 µg/ml), <italic>A. niculans</italic> (with a MIC of 4.7 µg/ml), and <italic>C. globosum</italic> (with a MIC of 37.5 µg/ml). All the tested classic antibiotics including Ampicillin, Benzylpenicillin and ICS had no effect on these funguses. Several other cathelicidin-BF analogues, KF1–11 (KFFRKLKKSVK), KF12–19 (KRAKEFFK) and KF20–30 (KPRVIGVSIPF) had no any antimicrobial activity.</p>", "<p>The antimicrobial activity of cathelicidin-BF in different solutions was also investigated as listed in ##TAB##1##Table 2##. In 150 mM phosphate buffer solution (PBS) and 150 mM NaCl solution, cathelicidin-BF had stronger antimicrobial activities that in water. It suggested that salts could increase cathelicidin-BF's antimicrobial ability.</p>", "<title>Bacteria killing kinetics</title>", "<p>Using the antibiotics ICS as a positive control, antibacterial properties of the snake cathelicidin-BF were tested by the colony counting assay. As listed in ##TAB##2##Table 3## and ##TAB##3##Table 4##, cathelicidin-BF could rapidly exert its antibacterial activities. It just took less than 1 minute to kill all the <italic>E. coli</italic> at the concentration of 1, 5 or 10 times of MIC. The antibacterial activity was proved to be lethal for <italic>E. coli</italic>. <italic>E. coli</italic> was not capable of resuming growth on agar plates after a 6-h treatment with concentrations above the corresponding MICs. In contrast, the antibiotics, ICS could not clean the bacteria within 6 h at the concentration of 1 or 5 times of MIC. Only 10 times MIC of ICS could clean all the <italic>E. coli</italic> within 6 h. Furthermore, <italic>E. coli</italic> treated by 1 time MIC of ICS was capable of resuming growth during 6 h.</p>", "<p>In order to compare properties with other antimicrobial peptide, the frog antimicrobial peptide HDW was used as a control. Their bacteria killing kinetics during 30 min was listed in ##TAB##3##Table 4##. Although HDW had a rapid bacteria killing ability, cathelicidin-BF is faster to clean <italic>E. coli</italic> than HDW. Cathelicidin-BF just took less than 1 minute to clean <italic>E. coli</italic>, while HDW took several minutes.</p>", "<title>Hemolysis, cytotoxicity, serum stability</title>", "<p>Cathelicidin-BF had little hemolytic activity on human red blood cells even with peptide concentrations up to 400 µg/ml. At the same concentration, cathelicidin-BF was neither cytotoxic for mouse macrophage (RAW264.7) nor for human liver tumor cell (HepG<sub>2</sub>) (data not shown). Thus, it showed considerable selectivity for microorganisms over mammalian cells <italic>in vitro</italic>.</p>", "<p>Serum stability was checked by incubating 100 µg/ml cathelicidin-BF and cathelicidin-BF15 with 90% fresh normal human serum at 37°C for 0, 1, 2, 3, 6, 10 and 24 hours. For cathelicidin-BF, antimicrobial activities against <italic>E. coli</italic> 08A866 could not be detected after 3 h-incubation, while cathelicidin-BF15 could keep its antimicrobial activity up to 10 h in 90% fresh normal human serum. Cathelicidin-BF15 seems to be more stable than cathelicidin-BF in serum.</p>" ]
[ "<title>Discussion</title>", "<p>Antimicrobial peptides (AMPs) and their precursor molecules form a central part of biological immunity. For the species which lack adaptive immunity, AMPs play a key role to defense microorganism infection. For their capacity to rapidly inactive infectious agents and to probably inhibit the emergence of drug resistance, AMPs have attracted considerable attention, especially for the treatment of antibiotic-resistant pathogens. The most two important AMP families, defensin and cathelicidin have been found in mammalians, birds and fish. Coincidently, both defensin and cathelicidin have not been found in both reptiles and amphibians although a beta-defensin-like protein with unusual disulfide connectivity (C1–C6/C2–C5/C3–C4), which is different from other the vertebrate beta-defensins, has been identified from a marine turtle ##REF##18341335##[47]##, ##REF##16700051##[48]##. Several hundreds of gene-encoded AMPs have been found from amphibians ##REF##2229048##[12]##, ##REF##17384140##[39]##–##REF##17764786##[42]##, ##REF##14726199##[49]##. Only a few peptides or proteins from reptiles have been found to exert antimicrobial activities, and most of them are phospholipases A<sub>2</sub> or its derivatives ##REF##17383773##[34]##, ##REF##15793109##[50]##–##REF##17044815##[52]##, and L-amino acid oxidase ##REF##1796476##[53]##.</p>", "<p>In the attempt to find AMPs from the snake venoms of <italic>B. fasciatus</italic>, which is a rich source of biological peptides or proteins with therapeutic potential, an AMP, cathelicidin-BF has been isolated and characterized. By screening the cDNA, cathelicidin-BF was unexpectedly found to be a C-terminus of a cathelicidin. The cathelicidin-BF precursor is composed of 191 amino acid residues with conserved cathelin domain that was flanked by signal peptide and by mature cathelicidin-BF. A conserved cleavage site (Valine<sup>157</sup>) for elastase in the processing and maturation of bovine, porcine and chicken cathelicidins ##REF##16326712##[30]## is also existed in the cathelicidin-BF precursor, suggesting that the snake cathelicidin is possibly processed by elastase-like proteases. Based on the hypothesis, cathelicidin-BF precursor should release a 34-aa peptide fragment (KRFKKFFRKLKKSVKKRAKEFFKKPRVIGVSIPF), which has a 4-aa (KRFK) extension at the N-terminus of the 30-aa cathelicidin-BF (KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF). Two reasons may explain the length difference between the predicted 34-aa C-terminal peptide and the purified 30-aa cathelicidin-BF in this case: 1, the predicted cleavage site is right, and the purified 30-aa cathelicidin-BF is from the further processing of the 34-aa peptide; 2, Valine<sup>157</sup> in the cathelicidin-BF precursor is not the exact protease cleavage site. In fact, some cathelicidins is not cleaved by elastase to release C-terminal active peptide fragments as mentioned in this “introduction”. An obvious feather of cathelicidin-BF is that there is a high density (40%) of basic amino acid residues in its sequence. As some other cathelicidins ##REF##16326712##[30]##, there are multiple aromatic amino acid residues in cathelicidin-BF's sequence (5 Phenylalanines). An atypical feature of cathelicidin-BF precursor is that an acidic domain insertion (EEGEQKQEEGNEEEKEVEEEEQEED EKD) is located between the cathelin domain and the mature cathelicidin-BF. This region potentially could affect preproprotein net charge, stability, activity or processing. The similar acidic regions are also found in amphibian antimicrobial precursors, which are located between the signal peptide domains and the mature antimicrobial domains ##REF##17384140##[39]##–##REF##17764786##[42]##. Amphibian acidic regions act as a role to neutralize the positive charge of the mature antimicrobial domains and to avoid possible toxicity of the precursor proteins.</p>", "<p>The data of antimicrobial testing indicated that cathelicidin-BF is clearly among the most potent cathlicidins discovered to date. Among the 40 strains of tested microorganisms, 15 strains could be killed by cathelicidin-BF at &lt;0.6 µM. For a variety of microorganisms, cathelicidin-BF had better antimicrobial ability than ampicillin, benzylpenicillin and ICS. Cathelicidin-BF's obvious ability to kill some saprophytic fungus such as <italic>A. terreus</italic>, <italic>A. niculans</italic> and <italic>C. globosum</italic> is also very interesting. It may be used as agricultural antibiotics against plant or food pathogenic microorganisms. To our knowledge, this is the first report of cathelicidin's antimicrobial activities against saprophytic fungus. In addition, cathelicidin-BF had very rapid microbe-killing efficacy. Cathelicidin-BF could kill <italic>E. coli</italic> within one minute at the dose of one time MIC. All the results suggest that cathelicidin-BF is an excellent candidate for clinical or agricultural antibiotics.</p>" ]
[]
[ "<p>Conceived and designed the experiments: DL RL. Performed the experiments: YW JH XL HY RL JW AW. Analyzed the data: YW JH XL HY RL JW AW DL RL. Contributed reagents/materials/analysis tools: DL RL. Wrote the paper: YW JH XL DL RL.</p>", "<title>Background</title>", "<p>Cathelicidins are a family of antimicrobial peptides acting as multifunctional effector molecules of innate immunity, which are firstly found in mammalians. Recently, several cathelicidins have also been found from chickens and fishes. No cathelicidins from other non-mammalian vertebrates have been reported.</p>", "<title>Principal Findings</title>", "<p>In this work, a cathelicidin-like antimicrobial peptide named cathelicidin-BF has been purified from the snake venoms of <italic>Bungarus fasciatus</italic> and its cDNA sequence was cloned from the cDNA library, which confirm the presence of cathelicidin in reptiles. As other cathelicidins, the precursor of cathelicidin-BF has cathelin-like domain at the N terminus and carry the mature cathelicidin-BF at the C terminus, but it has an atypical acidic fragment insertion between the cathelin-like domain and the C-terminus. The acidic fragment is similar to acidic domains of amphibian antimicrobial precursors. Phylogenetic analysis revealed that the snake cathelicidin had the nearest evolution relationship with platypus cathelicidin. The secondary structure of cathelicidin-BF investigated by CD and NMR spectroscopy in the presence of the helicogenic solvent TFE is an amphipathic α-helical conformation as many other cathelicidins. The antimicrobial activities of cathelicidin BF against forty strains of microorganisms were tested. Cathelicidin-BF efficiently killed bacteria and some fungal species including clinically isolated drug-resistance microorganisms. It was especially active against Gram-negative bacteria. Furthermore, it could exert antimicrobial activity against some saprophytic fungus. No hemolytic and cytotoxic activity was observed at the dose of up to 400 µg/ml. Cathelicidin-BF could exist stably in the mice plasma for at least 2.5 hours.</p>", "<title>Conclusion</title>", "<p>Discovery of snake cathelicidin with atypical structural and functional characterization offers new insights on the evolution of cathelicidins. Potent, broad spectrum, salt-independent antimicrobial activities make cathelicidin-BF an excellent candidate for clinical or agricultural antibiotics.</p>" ]
[ "<title>Supporting Information</title>" ]
[]
[ "<fig id=\"pone-0003217-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003217.g001</object-id><label>Figure 1</label><caption><title>The cDNA sequence encoding cathelicidin-BF and the predicted precursor amino acid sequence.</title><p>The amino sequence of purified cathelicidin-BF is <italic>boxed</italic>. The stop codon is indicated by a <italic>star</italic> (*). The potential polyadentlation signal (<named-content content-type=\"gene\">AATAAA</named-content>) is <italic>underlined</italic>.</p></caption></fig>", "<fig id=\"pone-0003217-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003217.g002</object-id><label>Figure 2</label><caption><title>Multiple sequence alignment of snake cathelicidin with other representative cathelicidins.</title><p>Cathelicidin-BF precursor is aligned with porcine, bovine, human, chicken and hagfish cathelicdins. <italic>Dashes</italic> are inserted to optimize the alignment, and conserved residues are <italic>shaded</italic>. Two intramolecular disulfide bonds in the cathelin pro-sequence are shown. Mature cathelicinds are <italic>underlined</italic>, and their net charge (<italic>in parenthesis</italic>) and length are also indicated. The acidic fragment insertion in cathelicidin-BF is <italic>boxed</italic>.</p></caption></fig>", "<fig id=\"pone-0003217-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003217.g003</object-id><label>Figure 3</label><caption><title>Phylogenetic analysis of cathelicidins.</title><p>Phylogenetic dendrogram obtained by neighbour-joining analysis based on the proportion difference (p-distance) of aligned amino acid sites of the full-length peptide sequences. Only bootstrap values &gt;50% (expressed as percentages of 1000 resamplings) are shown at branching points. Snake cathelicidin-BF is boxed.</p></caption></fig>", "<fig id=\"pone-0003217-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003217.g004</object-id><label>Figure 4</label><caption><title>RT-PCR analysis of cathelicidin gene expression pattern in various snake tissues using gene-specific primers with actin as a control.</title></caption></fig>" ]
[ "<table-wrap id=\"pone-0003217-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003217.t001</object-id><label>Table 1</label><caption><title>antimicrobial activity comparison of cathelicidin-BF with antibiotics.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Microorganism strains</td><td colspan=\"6\" align=\"left\" rowspan=\"1\">MIC(ug/ml)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">BF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">BF-15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Amp</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ben</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ICS</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HDW</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Bacillus subtilis</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.02</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.004</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Bacillus pumilus</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.015</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Bacillus cereus</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">150</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">150</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Pseudomonas aeruginosa</italic> ATCC27853</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">150</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P. aeruginosa</italic> (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P.aeruginosa</italic> 08031205(IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P.aeruginosa</italic> 08031014(IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Escherichia coli</italic>ATCC25922</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>E. coli</italic> 08A852 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>E. coli</italic> 08A866(IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>E. coli</italic> 08031017 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>E. coli</italic> 08032813 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>E. coli</italic> 08040726 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>E. coli</italic> 08040722 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Staphylococcus aureus</italic> ATCC2592</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>S. aureus</italic> ATCC25923</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;400</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>S. aureus</italic> 08A865 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;400</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>S. aureus</italic> 08A875 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>S. aureus</italic> 08031002 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>S. aureus</italic> 08031013 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>S. aureus</italic> 08032706 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>S. aureus</italic> 08032712 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>S. aureus</italic> 08032810 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Acinetobacter calcoaceticus</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Sphingobacterium siyangense</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Sacharibacillus kuerlensis</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Serratia marcescens</italic> SA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;400</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Serratia marcescens</italic> MA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;400</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Pseudomonas luteola</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Salmonella typhi</italic> (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klebsiella pneumoniae</italic> (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>K. pneumoniae</italic> 08031012 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>K. pneumoniae</italic> 08040202 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>K. pneumoniae</italic> 08040724 (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Enterococcus faecium</italic> (IS, DR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">150</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Aspergillus terreus</italic> GIM3.34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Aspergillus niculans</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Chaetomium globosum</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Candida albicans</italic> ATCC2002</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Pichia pastoris</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003217-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003217.t002</object-id><label>Table 2</label><caption><title>Antimicrobial activity of cathelicidin BF in different solutions.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Microorganism</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">MIC</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">150 mM NaCl</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">150 mM PBS</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>E. coli</italic>ATCC25922</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P. aeruginosa</italic> ATCC27853</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>S. aureus</italic> ATCC2592</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>C. albicans</italic> ATCC2002</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.4</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003217-t003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003217.t003</object-id><label>Table 3</label><caption><title>Bacterial killing kinetics of cathelicidin-BF against <italic>E. coli</italic>.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td colspan=\"6\" align=\"left\" rowspan=\"1\">Amount of bacteria co-cultured with different samples for different time (CFU)</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Time (h)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td colspan=\"6\" align=\"left\" rowspan=\"1\">Samples</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">BFx1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">BFx5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">BFx10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ICSx1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">224</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1082</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ICSx5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ICSx5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.9% salt water</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">234</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2341</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14109</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003217-t004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003217.t004</object-id><label>Table 4</label><caption><title>Bacterial killing kinetics of cathelicidin-BF and HDW against <italic>E. coli</italic>.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td colspan=\"6\" align=\"left\" rowspan=\"1\">Amount of bacteria co-cultured with different samples for different time (CFU)</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Time (min)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30</td></tr><tr><td colspan=\"6\" align=\"left\" rowspan=\"1\">Samples</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">BFx1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">BFx5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">BFx10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HDWx1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">49</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HDWx10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">38</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.9% salt water</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">69</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">56</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">66.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">68.7</td></tr></tbody></table></alternatives></table-wrap>" ]
[]
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[]
[]
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[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003217.s001\"><label>Materials and Methods S1</label><caption><p>Detail materials and methods</p><p>(0.03 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003217.s002\"><label>Figure S1</label><caption><p>Purification of the cathelicidin from snake venom. (a) Gel filtration chromatography. Sephadex G-50 column (2.6 cm×100 cm), equilibrated and developed with 50 mM Tris-HCl plus 50 mM NaCl (pH 7.8) at a flow rate of 0.3 ml/min, fractions were collected. (b) Cation-exchange chromatography. CM-Sephadex C-25 column (16 cm×40 cm) elution was achieved with a liner NaCl gradient, at a flow rate of 1 ml/min. (c) RP-HPLC chromatography. C4 reverse phase column, equilibrated with 0.1% (v/v) TFA/water, elution was performed with an acetonitrile liner gradient at a flow rate of 0.7 ml/min. The purified peptide with antimicrobial activity is indicated by an arrow.</p><p>(0.17 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003217.s003\"><label>Figure S2</label><caption><p>Electrospray ionization mass spectrometry analysis of the RP-HPLC peak containing antimicrobial activity.</p><p>(0.11 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003217.s004\"><label>Figure S3</label><caption><p>Circular dichroism spectra recorded on cathelicidin-BF in different solvent environments. (A) a∼e : in SDS micelles of 0, 30, 60, 90, 120 mM; (B) a∼e: in TFE/H<sub>2</sub>O mixtures of 1∶9, 3∶7, 5∶5, 7∶3, 9∶1 (v/v).</p><p>(0.13 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003217.s005\"><label>Figure S4</label><caption><p>HN-HN regions of 2D <sup>1</sup>H-<sup>1</sup>H NOESY spectra recorded on cathelicidin-BF in H<sub>2</sub>O (left) and in TFE/H<sub>2</sub>O mixture (9∶1, v/v) (right).</p><p>(0.12 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003217.s006\"><label>Figure S5</label><caption><p>Hα CSI prediction for the cathelicidin-BF peptide in TFE/H<sub>2</sub>O mixture (9∶1, v/v). h: helix; c: coil.</p><p>(0.16 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003217.s007\"><label>Figure S6</label><caption><p>The opposing position of the hydrophilic and hydrophobic side chains can be seen in this end-on representation of the α-helix in the N-terminal region of cathelicidin-BF.</p><p>(0.18 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003217.s008\"><label>Table S1</label><caption><p>Contents of helical structures of cathelicidin in TFE/H<sub>2</sub>O mixtures or in SDS micelles measured by CD.</p><p>(0.03 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003217.s009\"><label>Table S2</label><caption><p>\n<sup>1</sup>H chemical shifts of cathelicidin-BF in TFE/H<sub>2</sub>O mixture (9∶1, v/v) at 298 K</p><p>(0.07 MB DOC)</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"nt101\"><label/><p>MIC: minimal inhibitory concentration. These concentrations represent mean values of three independent experiments performed in duplicates. BF: cathlicidin-BF, BF-15: cathlicidin-BF15, Amp: ampicillin, Ben: benzylpenicillin, ICS: Imipenem and Cilastatin Sodium for Injection, ND: no detectable activity, -: no assay, IS: clinically isolated strain, DR: drug resistance for ampicillin and benzylpenicillin.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt102\"><label/><p>MIC: minimal inhibitory concentration. These concentrations represent mean values of three independent experiments performed in duplicates.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt103\"><label/><p>BF: cathlicidin-BF, CFU: colony forming unit, ICS: Imipenem and Cilastatin Sodium for Injection, ×1, ×5 and ×10: 1, 5 and 10 times</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt104\"><label/><p>BF: cathlicidin-BF, CFU: colony forming unit, ×1, ×5 and ×10: 1, 5 and 10 times, HDW: A amphibian antimicrobial peptide from <italic>Rana nigrovittata</italic>.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>This work was supported by grants of 2007AA02Z192 and 2007AA100602 from the Ministry of Science and Technology of the People's Republic of China, and KSCX2-YW-G-024 and KSCX2-YW-R-20 from Chinese Academy of Sciences.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"pone.0003217.g001\"/>", "<graphic xlink:href=\"pone.0003217.g002\"/>", "<graphic xlink:href=\"pone.0003217.g003\"/>", "<graphic xlink:href=\"pone.0003217.g004\"/>", "<graphic id=\"pone-0003217-t001-1\" xlink:href=\"pone.0003217.t001\"/>", "<graphic id=\"pone-0003217-t002-2\" xlink:href=\"pone.0003217.t002\"/>", "<graphic id=\"pone-0003217-t003-3\" xlink:href=\"pone.0003217.t003\"/>", "<graphic id=\"pone-0003217-t004-4\" xlink:href=\"pone.0003217.t004\"/>" ]
[ "<media xlink:href=\"pone.0003217.s001.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003217.s002.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003217.s003.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003217.s004.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003217.s005.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003217.s006.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003217.s007.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003217.s008.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003217.s009.doc\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["26"], "element-citation": ["\n"], "surname": ["Murakami", "Lopez-Garcia", "Braff", "Dorschner", "Gallo"], "given-names": ["M", "B", "MH", "RA", "RL"], "year": ["2004"], "article-title": ["Postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense."], "source": ["J Immunol"], "volume": ["172"], "fpage": ["070"], "lpage": ["3077"]}, {"label": ["32"], "element-citation": ["\n"], "surname": ["Raison", "dos Remedios", "Jorgensen", "Lomholt", "Weber", "Malte"], "given-names": ["RL", "NJ", "JM", "JP", "RE", "H"], "year": ["1998"], "article-title": ["The hagfish immune system,"], "source": ["The Hagfish Immune System"], "publisher-name": ["Chapman and Hall"], "fpage": ["334"], "lpage": ["344"]}, {"label": ["37"], "element-citation": ["\n"], "surname": ["Goddard", "Kneller"], "given-names": ["TD", "DG"], "article-title": ["SPARKY 3,"], "publisher-loc": ["San Francisco"], "publisher-name": ["University of California"]}, {"label": ["44"], "element-citation": ["\n"], "surname": ["Whittington", "Papenfuss", "Bansal", "Torres", "Wong"], "given-names": ["CM", "AT", "P", "AM", "ES"], "year": ["2008"], "article-title": ["Defensins and the convergent evolution of platypus and reptile venom genes."], "source": ["Genome Res [Epub ahead of print]"]}]
{ "acronym": [], "definition": [] }
53
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 16; 3(9):e3217
oa_package/56/0e/PMC2528936.tar.gz
PMC2528937
18795097
[ "<title>Introduction</title>", "<p>Hepatocyte Growth Factor (HGF), also known as Scatter Factor (SF), is a pleiotropic cytokine of mesenchymal origin that mediates a characteristic array of biological activities including cell proliferation, survival, motility and morphogenesis ##UREF##0##[1]##–##REF##7775566##[3]##. Its high affinity receptor, the tyrosine kinase Met, is expressed by a wide range of tissues including epithelial, endothelial, hematopoietic, neuronal and muscular cells ##REF##8268192##[4]##, ##REF##14685170##[5]##. Embryonic muscle precursor cells express Met and migrate following HGF gradients during embryo development ##REF##7822413##[6]##–##REF##10193678##[10]##. Genetic impairment of HGF-Met signaling in mice leads to abnormal muscle development in the limbs, thorax and tongue ##REF##7854452##[11]##–##REF##7651534##[13]##, and newborns -which are ataxic and have breathing problems- die a few hours later because they cannot suck mother's milk ##REF##8898205##[14]##. In the adult, the HGF-Met pathway is involved in muscle regeneration following injury. Muscle satellite cells, which reside in the stroma of muscular tissues and express both HGF and Met ##REF##10639617##[15]##, represent a pool of muscle precursors that are activated and stimulated to divide when muscle regeneration or adaptive growth is needed ##REF##10644525##[16]##, ##REF##9473336##[17]##. Autocrine HGF-Met stimulation plays a key role in mediating activation and early division of satellite cells, but is shut off in a second phase in order to allow the cells to exit the cell cycle and to enter the differentiation process ##REF##9166406##[18]##, ##REF##9551084##[19]##. HGF stimulation of cultured satellite cells promotes cell proliferation and inhibits myogenic differentiation ##REF##10825239##[20]##.</p>", "<p>Magic Factor-1 (Met-Activating Genetically Improved Chimeric Factor-1 or Magic-F1) is an HGF-derived, engineered protein that contains two Met-binding domains repeated in tandem. It has a reduced affinity for Met and, in contrast to HGF, it elicits activation of the AKT but not the ERK signaling pathway. As a result of its partial ability to activate Met signaling, Magic-F1 is not mitogenic but conserves the ability to protect cells against apoptosis. We have analyzed the effects of Magic-F1 on muscular cells both <italic>in vitro</italic> and in mice. We show that Magic-F1 protects myogenic precursors against apoptosis and thus enhances the differentiation process, which is naturally accompanied by cell death. This pro-differentiative effect is observed both in cultured myogenic cell systems and in two different <italic>in vivo</italic> models. Remarkably, constitutive or transient expression of Magic-F1 in a mouse model of muscular dystrophy partially rescues the dystrophic phenotype and allows animals to perform better in a classic tread mill functional test. These features make Magic-F1 a novel, potential molecular tool to counteract muscle wasting in major muscular diseases including cachexia and muscular dystrophy.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Magic-F1 Factor engineering and purification</title>", "<p>Magic-F1 is an engineered factor containing two HGF NK2 domains joint by a linker. The exact amino acidic sequence of Magic-F1 corresponds to: residues 1–285 of human HGF (Gene Bank # M73239); a linker with the sequence (GGGGS)<sub>3</sub>; residues 30–285 of human HGF; a poly-histidine tag with the sequence DDDKHHHHHH. Factors were produced in a CHO cell line (ATTC, Rockville, Maryland). Purification was performed by dual-step affinity chromatography using a heparin-Sepharose column and a Ni<sup>2+</sup>-chelate column (Amersham Pharmacia, Uppsala, Sweden). Activated human recombinant HGF was purchased from R&amp;D Systems (Minneapolis, Minnesota) while Metron Factor-1 (Metr. in ##SUPPL##0##Figure S1##) recombinant protein ##REF##11981563##[53]## was produced at Dompé Pharmaceutical Company S.p.A. (L'Aquila, Italy).</p>", "<title>ELISA binding assay</title>", "<p>HGF and FcMET (a chimera consisting of the extracellular domain of MET fused to the Fc region of a human IgG<sub>1</sub>) were purchased from R&amp;D Systems. Binding of Magic-F1 and HGF to Fc-Met was measured by ELISA using the receptor in solid phase and the ligands in liquid phase. A fixed concentration (100 ng/well) of Fc-Met was adsorbed to 96-well ELISA plates and incubated with increasing concentrations of ligands. Binding was revealed using biotinylated anti-HGF antibodies (R&amp;D). Binding data were analyzed and fit using Prism software (Graph Pad Software, San Diego, California).</p>", "<title>Immunoreagents</title>", "<p>The antibodies used in this study were obtained as follows: anti-human HGF for both Western blotting and immunoprecipitation, Santa Cruz Biotechnology (Santa Cruz, California); anti-human Met for Western blotting, Santa Cruz; anti-human Met for immunoprecipitation, as described ##REF##14685170##[5]##; anti-mouse Met, Santa Cruz; anti-AKT and anti-phospho-AKT, New England Biolabs (Beverly, Massachusetts); anti-MAPK (p42–44/ERK) and anti-phospho-MAPK, Promega (Madison, Winsconsin); anti-laminin polyclonal rabbit antibodies, Sigma (St. Louis, Missouri); anti-desmin rabbit polyclonal antibody, Sigma; MF20 and embryonic myosin monoclonal antibody, Developmental Studies Hybridoma Bank (Iowa City, Iowa).</p>", "<title>Receptor activation and signal transduction</title>", "<p>For receptor activation analysis, quiescent cells plated on collagen-coated 100 mm plates (Becton Dickinson, Franklin Lakes, New Jersey) were stimulated with 5 nM HGF or Magic-F1 for 30 min at 37°C and then lysed as described ##REF##1334458##[54]##. Lysates were immunoprecipitated with anti-Met and analyzed by Western blotting using anti-phosphotyrosine antibodies. For signal-transduction analysis, cells were stimulated as above for different times and then lysed. For MAPK and AKT activation, lysates were directly analyzed by Western blotting using antibodies specific for the activated forms of the signaling molecules. Quantification of enhanced chemiluminescence signal was performed using a STORM apparatus and Image Quant software (Molecular Dynamics, Amersham Biosciences, Sunnyvale, California).</p>", "<title>Cell cultures and bioassays</title>", "<p>Mouse myogenic cell line C2C12 was maintained in DMEM supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10% FBS. C2C12 cells were induced to differentiate into myotubes by replacing 10% FBS with 2% horse serum (HS). Differentiation was completed in 7–8 days. All cultures were performed at 37°C in a humidified incubator with 5% CO<sub>2</sub> and 95% air. Satellite cells were prepared as previously described ##REF##17293855##[46]##. Briefly, muscle fragments were digested with 2% collagenase II (Invitrogen, Carlsbad, California) for 60 min at 37°C. Digested cells were discarded and fragments were incubated again with 0.05% trypsin (Invitrogen) for 15 min at 37°C with gentle agitation. After the incubation, isolated cells were collected and fragments were incubated again until the whole tissue was digested (usually three times). Isolated cells were pooled, centrifuged and resuspended in DMEM supplements with 20% pre-screened FCS, 1% gentamycin, and plated onto collagen coated dishes at a density of 10<sup>4</sup> cells×cm<sup>2</sup>. Contamination by non-myogenic cell was reduced by pre-plating the cell suspension onto plastic dishes where fibroblasts tend to adhere more rapidly. Differentiation was induced shifting the medium to DMEM supplemented with 2% horse serum. Cell morphology was examined daily with a phase-contrast microscope connected to an image analyzer. Cells were trypsinized daily and counted on a hemocytometer. Cell viability was determined by trypan blue dye exclusion assay. Cell cytotoxicity was performed using an XTT-based <italic>in vitro</italic> toxicology assay kit (Sigma) according to manufacturer's protocol. Incubation medium was collected after 3 hours and read spectrophotometrically at a wavelength of 450 nm. Background signals, obtained from plates without cells, were subtracted from sample readings. Apoptosis was quantified using an ApopTag Fluorescein In situ Apoptosis detection kit (Chemicon, Temecula, California) according to the manufacturer's protocol. Cell differentiation was carried out for 8 days. Cells were grown on 6 cm Petri dishes until sub-confluent, washed with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes and then permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. After incubation with PBS containing 10% normal serum, samples were incubated overnight at 4°C with anti-GFP at 1∶200 dilution, anti myosin heavy chain (MF20) antibody at 1∶2 dilution. After incubation, cells were washed three times in PBS and incubated with the appropriate FITC- or TRITC-conjugated secondary antibodies for 1 hour at room temperature. After washing in PBS, cells were analyzed under a fluorescent microscope and photographed. As a control for the immunofluorescence method, we omitted the primary antibody and no staining was detected under these conditions. Cell nuclei were counterstained with DAPI.</p>", "<title>Plasmids and DNA preparation</title>", "<p>Magic-F1 was cloned into pIRESneo (Clontech, Italy) for C2C12 transfection experiments whereas it was cloned into pcDNA3 (Invitrogen) containing the cytomegalovirus (CMV) promoter for electrotransfer experiments (pCMV-Magic-F1); a pCMV-bgal plasmid coding for beta-galactosidase and a pCMV-hHGF plasmid coding for human hepatocyte growth factor were also used. Plasmids were prepared by using standard procedures. All plasmid preparations was obtained using a GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit (Sigma) and contained a high percentage of supercoiled DNA (70–80%). No RNA was detectable by gel electrophoresis.</p>", "<title>DNA electro-transfer and animal handling</title>", "<p>Mouse experiments were performed in the San Raffaele Hospital SPF Animal Care Facilities according to international ethical guidelines (EEC Council Directive 86/609; NIH Guide for the Care and Use of Laboratory Animals, 1985). Authorization for animal experimentation was obtained from the Italian Ministry of Health. Gene transfer into skeletal muscle mediated by electric pulse was performed as previously reported ##REF##10200250##[33]##. Briefly, 20 µg of DNA in 10 µl of PBS was injected into the <italic>tibialis anterior</italic> or in the <italic>quadriceps</italic> muscle of anesthetized, 10 day-old C57Bl/6 mice (Iffa Credo, St. Germain sur l'Arbresle, France) with a Hamilton syringe. There were 10 muscles included in each experimental group. Five minutes after DNA injection trans-cutaneous electric pulses were applied by two stainless steel plate electrodes placed 3.8–4.3 mm apart, at each side of the leg. Electrical contact with the leg skin was ensured by shaving each leg and applying a conductive gel. Square-wave electric pulses (eight pulses; 200 V/cm; 20 ms per pulse; 1 Hz) were generated by a digital Stimulator (Panlab 3100, Biological Instruments, Varese, Italy).</p>", "<title>Muscular regeneration analysis</title>", "<p>Acute skeletal muscle damage was induced in male and female MLC1F/Magic-F1transgenic mice and control mice (7 animals/group) by i.m. injection of 10 nM cardiotoxin (Gentaur, Brussels, Belgium) in physiologic solution (0.9% w/v NaCl). Control mice were injected with physiologic solution alone. At 3, 7, and 14 days after drug injection, mice were sacrified and subjected to histological evaluation and morphometric analysis of <italic>tibialis anterior</italic>. After excision, muscles were sectioned (4–6 µm) and processed for immunofluorescence analysis using the primary antibodies listed above. All sections were washed three times in PBS and incubated with 10% donkey serum for 30 min at RT before the addition of the appropriate Alexa 488-, Alexa 594- or Alexa 647-conjugated donkey secondary antibodies. Alternatively, some sections were stained with hematoxylin and eosin and examined by an independent histopathologist not informed of sample identity to determine muscle fiber sizes using Scion Image software (Scion, Frederick, Maryland).</p>", "<title>Biochemical and molecular analysis</title>", "<p>Western blot analysis of cells or tissues was performed as described ##REF##12459784##[41]##, 55. Total RNA from control or treated cells was extracted using Trizol reagent (Invitrogen) and analysed by PCR after reverse transcription with random hexamers. RT-PCR analysis has been performed using the following primers:</p>", "<p>Bax</p>", "<p>Fw <named-content content-type=\"gene\">5′-TGTTTGCTGATGGCAACTTC-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-GATCAGCTCGGGCACTTTAG-3′</named-content>\n</p>", "<p>Bcl-2</p>", "<p>Fw <named-content content-type=\"gene\">5′-GGGATGCCTTTGTGGAACTA-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-CTCACTTGTGGCCCAGGTAT-3′</named-content>\n</p>", "<p>P53</p>", "<p>Fw <named-content content-type=\"gene\">5′-GGATGCCCGTGCTGCCGAGGAG-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-AGTGAAGGGAC TAGCATTGTC-3′</named-content>\n</p>", "<p>Magic-F1</p>", "<p>Fw <named-content content-type=\"gene\">5′-TTCAGAAGTTGAATGCATGACCTG-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-TCTTCTTTTCCTTTGTCCCTCTAG-3′</named-content>\n</p>", "<p>GAPDH</p>", "<p>Fw <named-content content-type=\"gene\">5′-TTCACCACCATGGAGAAGGC-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-GGCATGGACTGTGGTCATGA-3′</named-content>\n</p>", "<p>MyoD</p>", "<p>Fw <named-content content-type=\"gene\">5′-TGCACTTCCACCAACCCCAACCAGC-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-CCTGGACTCGCGCACCGCCTCACT-3′</named-content>\n</p>", "<p>Met</p>", "<p>Fw <named-content content-type=\"gene\">5′-AGAAATTCATCAGGCTGTGAAGCGCG-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-TTCCTCCGATCGCACACATTTGTCG-3′</named-content>\n</p>", "<p>PAX3</p>", "<p>Fw <named-content content-type=\"gene\">5′-AGGAGGCGGATCTAGAAAGGAAG-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-TGTGGAATAGACGTGGGCTGGTA-3′</named-content>\n</p>", "<p>Myf5</p>", "<p>Fw <named-content content-type=\"gene\">5′-GAGCTGCTGAGGGAACAGGTGGAGA-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-GTTCTTTCGGGACCAGACAGGGCTG-3′</named-content>\n</p>", "<p>IGF1</p>", "<p>Fw <named-content content-type=\"gene\">5′-CTGTGCCCCACTGAAGCCTA-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-GGACTTCTGAGTCTTGGGCATG-3′</named-content>\n</p>", "<p>Myostatin</p>", "<p>Fw <named-content content-type=\"gene\">5′-AGTGACGGCTCTTTGGAAGATG-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-AGTCAGACTCGGTAGGCATGGT-3′</named-content>\n</p>", "<p>Follistatin</p>", "<p>Fw <named-content content-type=\"gene\">5′-CTGTACAAGACCGAACTGAGC-3′</named-content>\n</p>", "<p>Rv <named-content content-type=\"gene\">5′-TCCACAGTCCACGTTCTCACA-3′</named-content>\n</p>", "<title>Generation of Magic-F1 and α-SG knock-out/Magic-F1 transgenic mice</title>", "<p>We constructed the transgene by inserting the Magic-F1 construct into the pMex plasmid containing the 1,500-bp fragment of the MLC promoter, an 840-bp fragment of SV40 poly(A), and a 900-bp fragment from the 3′ end of the MLC1f/3f gene, which acts as an enhancer ##REF##11175789##[34]##; provided by Dr. Antonio Musarò, University of Rome, Italy. We microinjected the transgene into the male pronucleus of fertilized eggs from FVB mice (Jackson Laboratories, Bar Harbor, Maine) that were implanted into pseudopregnant foster mothers. We identified positive transgenic mice by PCR. For PCR detection, sense and antisense primers specific respectively for the MLC1F promoter and the linker region of Magic-F1 were used. Transgenic founders were mated with wild-type FVB mice to generate F1 offspring. After obtaining MLC1F/Magic-F1 mice we mated them with α-SG knock-outs ##REF##9744877##[36]## to generate α-SG knock-out/Magic-F1 transgenic mice. The animals were housed in a temperature controlled (22°C) room with a 12∶12 hours light-dark cycle. All studies have been performed using Tg:MLC1F/Magic-F1 hemizygous mice, following the protocols approved by the Animal Care and Use Committee of the San Raffaele Institute (IACUC 264) and communicated to the Ministry of the Health and local authorities according to Italian law.</p>", "<title>Adenovirus preparation and administration</title>", "<p>pAd/CMV-Magic-F1/V5-DEST was engineered using the ViraPower Adenoviral Expression System from Invitrogen. The Adenoviral vector was linearized with <italic>Pac</italic>I restriction enzyme and transfected into 293A cells. Cells were grown in Iscove Medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin (Sigma). After complete detachment of cells, the supernatant was used to superinfect 293A cells. The purification of Adenoviral particles was performed with Vivapure AdenoPACK 100TM (Sartorius, Goettingen, Germany) starting from 200 ml of cell culture. Juvenile α-SG knock-out mice (8 weeks old) were anesthetized with an intraperitoneal injection of avertin (0.2 ml/10 g bodyweight of a 1.2% solution), hair was shaved from the skin and pAd-Magic-F1 suspension (2.5×10<sup>9</sup> pfu diluited in 30 µl of PBS containing 100 ng of VEGF) were injected i.m. with a 30-gauge needle in the center of <italic>gastrocnemius</italic>, <italic>quadriceps</italic> and <italic>tibialis anterior</italic>. To prevent an immune-mediated clearance of adeno-infected fibers, all mice were immunosuppressed with FK506 (5 mg/day/Kg, subcutaneously). The immunosuppressive treatment was started on the day before the Adenoviral injection and continued until mice were sacrificed.</p>", "<title>Treadmill analyses</title>", "<p>Treadmill analyses were carried out using a six-lane motorized treadmill (Exer 3/6 Treadmill; Columbus Instruments, Columbus, Ohio) supplied with shocker plates. The first trial was performed at low intensity and for short duration to accustom the mice to the exercise (5 m/min for 5 minutes, after which the speed was increased 1 m/min every 2 minutes until it reached 9 m/min). After the first trial, the treadmill was run at an inclination of 0° at 5 m/min for 5 minutes, after which the speed was increased 1 m/min every 1 minute. The test was stopped when the mouse remained on the shocker plate for more than 20 s without attempting to re-engage the treadmill, and the time to exhaustion was determined.</p>" ]
[ "<title>Results</title>", "<title>Engineering of Magic-F1, a bivalent Met ligand</title>", "<p>Mature HGF is a dimeric molecule consisting of a α- and a β-chain joint by a disulphide bridge ##REF##2531289##[21]##. The α-chain contains a leader peptide for secretion, an N-domain similar to the activation domain of plasminogen, and four kringle domains (K 1–4) typical of the blood clotting cascade proteases ##REF##1280830##[22]##. In functional terms, HGF is a bivalent molecule containing two distinct Met binding sites, one in the α-chain high affinity; ##REF##1321034##[23]## and one at in the β-chain low affinity; ##REF##9722511##[24]##. Isolated HGF domains containing only one receptor binding site (HGF NK1, HGF NK2, HGF α-chain, HGF β-chain) can bind to the Met receptor but do not activate it ##REF##1280830##[22]##–##REF##8662798##[25]##, thus suggesting that a bivalent molecule is necessary to achieve receptor activation. Consistent with this idea, some monovalent scatter factor subdomains (HGF NK1, HGF NK2) display a partial agonistic activity when they are stabilized in a dimeric form by extracellular matrix proteoglycans ##REF##8636243##[26]##. To generate new recombinant proteins capable of inducing specific patterns of biological responses, we engineered several artificial molecules containing different HGF domain in various combination. Magic-F1, the prototype of this series, contains the signal peptide plus the N-domain and the first two kringles repeated in tandem and joint by a linker (##FIG##0##Fig. 1A##). A poly-histidine tag was engineered at the C-terminal end to facilitate protein purification. Since the high affinity Met binding site lies within the N and K1 domains ##REF##1321034##[23]##, Magic-F1 is a bivalent ligand. Magic-F1 recombinant protein was produced using both transiently and stably transfected CHO cells, and was purified by affinity chromatography as described in the Experimental Protocol section (##FIG##0##Fig. 1B##). The affinity of Magic-F1 for Met was measured in a ELISA binding assay using a recombinant chimera between Met and the Fc portion of a human immunoglobulin Fc-Met; ##REF##1334493##[27]##. Fc-Met was absorbed in solid phase and exposed to increasing concentrations of Magic-F1 or HGF in liquid phase. Binding was revealed using biotinylated anti-HGF antibodies. This analysis revealed that Magic-F1 has an affinity for Met that is approximately 7–8 times lower than that of HGF (<italic>i.e.</italic> 0.8 nM; ##FIG##0##Fig. 1C##). These data are consistent with previous measurements that determined the affinity of different subdomains of HGF for Met ##REF##1321034##[23]##.</p>", "<title>Magic-F1 does not induce myoblast proliferation</title>", "<p>Since HGF has been shown to affect satellite cell proliferation and differentiation, the action of the Magic-F1 on these biological processes was investigated by different approaches. We first subjected the myogenic cell line C2C12 ##REF##6839359##[28]## to different biological and biochemical assays in the presence of recombinant Magic-F1. Myoblast proliferation was evaluated by culturing C2C12 cells with Magic-F1, HGF or no factor as control. While HGF induced myoblast proliferation in a dose-dependent manner, Magic-F1 did not affect proliferation even at high concentrations as well as NK2 (##FIG##0##Fig. 1E##). As phosphorylation of Met is necessary for the activation of the HGF signaling cascade ##UREF##0##[1]##, we tested whether Magic-F1 could induce Met receptor phosphorylation. Immunoprecipitation analysis of Met followed by Western blot analysis using anti-phosphotyrosine antibodies revealed that both HGF and Magic-F1 induce phosphorylation of Met in C2C12 cells (##FIG##0##Fig. 1D##), indicating that the inability of Magic-F1 to affect myoblasts proliferation is not due to defective receptor activation. Since HGF is able to promote cell proliferation through the ERK pathway and to prevent apoptosis through AKT signaling ##REF##11134526##[29]##, we next tested the ability of Magic-F1 to activate these two distinct pathways. While HGF induced phosphorylation of both MAPK and AKT. Magic-F1, differently form NK2, induced phosphorylation of AKT. Moreover, consistent with the idea that HGF and Magic-F1 compete for the same binding site on Met, Magic-F1 inhibited HGF-mediated MAPK phosphorylation (##FIG##0##Fig. 1F##).</p>", "<title>Magic-F1 promotes myoblast differentiation and survival</title>", "<p>Next, we generated several stable clones of C2C12 myoblasts expressing Magic-F1 (##FIG##1##Fig. 2A##). Surprisingly, C2C12 cells expressing Magic-F1 differentiated at a faster rate compared to controls. In fact, they started to express myosin heavy chain, a marker of terminal differentiation, only one day following switch to differentiation medium (##FIG##1##Fig. 2B##). Consistent with accelerated differentiation, the myogenic markers MyoD and Myf5 were up-regulated while the Pax3 protein was down-regulated (##FIG##1##Fig. 2D##). Moreover, Magic-F1 increased the expression of 30 out of 36 genes known to be upregulated during C2C12 differentiation ##REF##14688207##[30]##; ##SUPPL##1##Figure S2##. Magic-F1-expressing C2C12 cells fused into myotubes containing on average more nuclei than controls, while HGF did not affect myoblast fusion (##FIG##1##Fig. 2C##). Interestingly, in stable clones expressing Magic-F1, myostatin expression but not follistatin or IGF1 expression was down-regulated earlier compared to controls (##FIG##1##Fig. 2E##). This is in agreement with previous data showing promotion of myoblast differentiation and muscle hypertropy following myostatin ablation ##REF##9356471##[31]##, ##REF##9139826##[32]##. Finally, cells expressing Magic-F1 displayed a marked reduction in the expression of several pro-apoptotic genes, including Bad, Bax and p53 (##FIG##1##Fig. 2F##) suggesting that the anti-apoptotic properties documented for HGF ##REF##11134526##[29]## are conserved in Magic-F1. Thus, Magic-F1 is an engineered, HGF-derived protein that elicits a selective pattern of biological responses on myoblasts. Firstly, it is a partial agonist of Met that activates the AKT pathway but not the ERK pathway. Secondly, it conserves the anti-apoptotic activity of HGF but not its mitogenic properties. Thirdly, it significantly enhances the differentiation potential of myoblasts without affecting their proliferation. The latter property is likely to be due to its inability to activate the ERK pathway.</p>", "<title>Electro-enhanced Magic-F1 DNA transfer in vivo promotes muscle hypertrophy and protects myocites against apoptosis</title>", "<p>Efficient secretion of therapeutic proteins can be induced into skeletal muscle through electro-enhanced DNA transfer ##REF##10200250##[33]##. Using this technology, we tested the activity of Magic-F1 on mouse skeletal muscles <italic>in vivo</italic>. A plasmid encoding Magic-F1 was co-electroporated with a plasmid expressing β-galactosidase into the <italic>tibialis anterior</italic> and <italic>quadriceps</italic> muscles of juvenile mice (postnatal day 10) as described.</p>", "<p>A vector encoding HGF and an empty vector without insert were used as controls. Histological analysis using X-gal staining showed that β-galactosidase was widely expressed one week after intra-muscular DNA electrotransfer but rapidly declined afterwards (##FIG##2##Fig. 3A##). Expression of the foreign genes also reached its maximum one week post-transfer and lasted for up to three weeks, as determined by RT-PCR analysis (##FIG##2##Fig. 3B##). Morphometric analysis performed on the <italic>tibialis anterior</italic> and <italic>quadriceps</italic> (9 mice for each group and 300–460 fibres for each sample were analyzed) revealed a significant increase of the cross-sectional area of Magic-F1-electrotransferred muscles compared to the control muscles starting two weeks after electrotransfer (##FIG##2##Fig. 3C##) as well as an increase in fiber perimeter (not shown). Representative images of electroporated <italic>quadriceps</italic> stained with hematoxylin and eosin are shown in ##FIG##2##Fig. 3D##. Next, we evaluated whether Magic-F1 could protect muscle cells against apoptosis. To this end, we performed a TUNEL analysis of muscle sections one week after <italic>in vivo</italic> electrotransfer. This analysis indeed showed a decreased number of apoptotic nuclei (TUNEL positive) in muscles treated with either Magic-F1 or HGF (##FIG##2##Fig. 3E##). Taken together, the <italic>in vitro</italic> and <italic>in vivo</italic> data presented here suggest that Magic-F1 induces hypertrophy in the developing skeletal muscle by enhancing the differentiation and fusion ability of myogenic cells and by protecting them against apoptosis.</p>", "<title>Magic-F1 transgenic mice display hypertrophic fast-twitch fibers and improved running ability</title>", "<p>To further investigate the ability of Magic-F1 to promote muscle hypertrophy, we generated transgenic mice expressing Magic-F1 under the control of the skeletal muscle-specific regulatory elements of the rat myosin light chain <italic>MLC1F</italic> gene locus ##REF##11175789##[34]##; ##FIG##3##Fig. 4A##. MLC1F/Magic-F1 transgenic lines were identified by genotyping PCR with primers specific for the Magic-F1 coding sequence (##FIG##3##Fig. 4B##). Expression of the Magic-F1 transgene in adult mice was detected by RT-PCR in all muscles analyzed; on the contrary, no signal was detected in the liver of transgenic mice or in any organ of wild-type animals (##FIG##3##Fig. 4C##). Protein expression in fast transgenic muscles was also confirmed by Western blotting analysis (##FIG##3##Fig. 4D##). Embryonic and post-natal development of MLC1F/Magic-F1 transgenic animals occurred without overt differences compared to control mice. Skeletal muscle hypertrophy became apparent at around 5 weeks of age, consistent with the <italic>in vivo</italic> electrotransfer results. Morphometric analysis of the fast <italic>tibialis anterior</italic> muscles in transgenic mice showed a statistical significant increment of myofiber cross-sectional areas compared to age-matched wild-type controls (##FIG##3##Fig. 4E##, left histogram). Interestingly, morphometric analysis of slow-twitch <italic>soleus</italic> muscles unveiled no difference between transgenic and control animals (##FIG##3##Fig. 4E##, right histogram), even though transgene expression was detected in the <italic>soleus</italic> muscle (see ##FIG##3##Fig. 4C##). A treadmill test was performed in order to evaluate the effect of Magic-F1 on muscular performance. This <italic>in vivo</italic> motility assay revealed that MLC1F/Magic-F1 transgenic mice cover on average a longer distance in comparison to their wild-type counterparts (##FIG##3##Fig. 4F##), thus demonstrating that Magic-F1-induced muscle hypertrophy results in increased muscular performance.</p>", "<title>Magic-F1 transgenic mice display enhanced muscle regenerative capacity</title>", "<p>In muscular dystrophy disorders, fiber degeneration is only partially counterbalanced by regeneration of new fibers by satellite cells ##REF##15464452##[35]##. Hypertrophic factors represent a potential therapeutic approach against muscle wasting. We therefore analyzed the effect of Magic-F1 on muscle regeneration. Muscle damage was induced in the <italic>tibialis anterior</italic> muscles of adult MLC1F/Magic-F1 transgenic or wild-type mice by a single intramuscular injection of cardiotoxin. MLC1F/Magic-F1 transgenic animals responded to muscle crush by rapidly activating the regenerative program. Three days after cardiotoxin injection, an enhanced number of centrally-nucleated regenerating myofibers and an increased expression of the regeneration hallmark protein, embryonic myosin heavy chain (MyHC), was observed in damaged muscles of transgenic mice compared to those of age-matched wild-type animals (##FIG##4##Fig. 5A##). Furthermore, one week post-injury, muscle fibers of MLC1F/Magic-F1 transgenic mice were characterized by enhanced peripherycal localization of nuclei and by the down-regulation of embryonic MyHC, indicating successful completion of the regeneration program. In contrast, in wild-type animals, regeneration persisted for a few more days (##FIG##4##Fig. 5A##). Interestingly, also regenerating centrally-nucleated fibers in the MLC1F/Magic-F1 transgenic mice appeared to have a greater cross-sectional area in comparison to wild-type animals after 3 days of injury (##SUPPL##2##Figure S3##). Consistent with these observations, satellite cells collected from MLC1F/Magic-F1 transgenic showed enhanced differentiation potential <italic>in vitro</italic> compared to satellite cells from wild-type mice. Furthermore, satellite cells from MLC1F/Magic-F1 transgenic mice were more differentiation-prone as revealed by smaller clone size and accelerated appearance of differentiated myotubes (##FIG##4##Fig. 5B##). Moreover, cardiotoxin induced a rapid apoptotic response in injected areas, which appeared to be strongly reduced in MLC1F/Magic-F1 transgenic animals (##FIG##4##Fig. 5C and D##). Rapid and efficient muscle regeneration in transgenic muscles subjected to cardiotoxin treatment is also explained by earlier and increased expression of the muscle master genes <italic>MyoD</italic> and <italic>Myf5</italic> (##FIG##4##Fig. 5E##). This resulted in reduction of central nucleated fibers at 10 days following cardiotoxin treatment (##FIG##4##Fig. 5F##) and in greater cross-sectional area of regenerated transgenic fibers compared to wild-type animals (##SUPPL##2##Figure S3B##).</p>", "<title>Magic-F1 partially rescues the dystrophic phenotype of alpha-sarcoglycan knock-out mice</title>", "<p>The therapeutic potential of Magic-F1 was tested in alpha-sarcoglycan (α-SG) knock-out mice, which represent an established animal model of muscular dystrophy. Due to their genetic defect, these mice display persistent degeneration and regeneration areas in skeletal muscles ##REF##9744877##[36]##. To achieve Magic-F1 expression in these mice, we undertook two different approaches. Firstly, we crossed Magic-F1 transgenic mice with α-SG knock-out animals, thus generating α-SG knock-out mice expressing Magic-F1 in their muscles (##FIG##5##Fig. 6A##). Secondly, we engineered an adenoviral vector ##REF##9054512##[37]## expressing Magic-F1 and administered it by intramuscular injection to 45 day-old α-SG knock-out female mice under immunosuppressive conditions ##REF##8573612##[38]##. Morphological analysis of the <italic>tibialis anterior</italic> of α-SG knock-out/Magic-F1 transgenic mice revealed significant muscular hypertrophy compared to α-SG knock-out controls, which persisted until at least 6 months of age (##FIG##5##Fig. 6B##). Consisted with this, α-SG knock-out/Magic-F1 transgenic mice performed much better than control α-SG knock-out mice in a classic treadmill test (##FIG##5##Fig. 6C##). Adenovirus-mediated delivery of Magic-F1 also ameliorated the dystrophic phenotype of α-SG knock-out mice, although to a reduced extent compared to α-SG knock-out/Magic-F1 transgenic mice (##FIG##5##Fig. 6C##). This may be due to the lower expression levels of Magic-F1 achieved by adenoviral transduction (see Western blot analysis in ##FIG##5##Fig. 6A##). In any case, the values obtained were statistically significant compared to dystrophic animals treated with a control adenovirus (##FIG##5##Fig. 6C##).</p>" ]
[ "<title>Discussion</title>", "<p>Protein engineering allows creating recombinant factors displaying selective biological functions. This is particularly useful for pleiotropic factors eliciting several different biological responses like HGF. Magic-F1, an engineered protein derived from HGF, maintains the ability to protect cells against apoptosis and to promote myoblast differentiation, but is devoid of any mitogenic activity typical of its parental factor. This results in remarkable enhancement of skeletal muscle regeneration without induction of cell proliferation, a crucial feature for its potential therapeutic application. Notably, muscle hypertrophy was induced in normal and regenerating muscle both when Magic-F1 was present as a transgene and when it was delivered to post-natal muscles, as it would occur in a cell or gene therapy context.</p>", "<p>The potential relevance of inducing muscle hypertrophy to the treatment of muscle disorders in humans has been suggested by studies involving <italic>mdx</italic> mice, which carry a mutation in the dystrophin gene and therefore serve as a genetic model of Duchenne's muscular dystrophy ##REF##6583703##[39]##. For example, <italic>mdx</italic> mice lacking myostatin were found not only to be stronger and more muscular than their <italic>mdx</italic> counterparts with normal myostatin, but also to have reduced fibrosis and fat deposition, suggesting sustained muscle regeneration ##REF##12447939##[40]##. Furthermore, injection of neutralizing monoclonal antibodies directed against myostatin into either wild-type or <italic>mdx</italic> mice increases muscle mass and specific force, suggesting that myostatin plays an important role in regulating muscle growth in adult animals ##REF##12459784##[41]##. Magic-F1 is a molecule with a potential clinical application as it can induce muscle hypertrophy by both down-regulating myostatin and directly activating MyoD, Myf5 and several anti-apoptotic pathways. Interestingly, no side effects have been observed in skeletal muscles following electro-enhanced Magic-F1 DNA transfer or in transgenic mice expressing the Magic-F1 under the control of a muscle-specific promoter.</p>", "<p>Our data showing the inability of Magic-F1 to induce the ERK pathway together with an inhibitory interference with HGF-induced ERK activation are particular relevant to a potential therapeutic use of this engineered factor. In fact, several tissues other than myocytes and satellite cells express the Met receptor, including epithelial cells of kidney, liver, lung, skin, breast and the whole gastrointestinal tract, as well as neurons, endothelial cells and hematopoietic precursors ##UREF##0##[1]##, ##REF##14685170##[5]##. Furthermore, Met overexpression is a very frequent event in human cancer ##REF##15949770##[42]##. This raises the concern that stimulating the proliferation of Met-expressing cells may lead to tumor formation or progression ##REF##18506889##[43]##. In this regard, the lack of any mitogenic activity makes Magic-F1 a potentially safe cytokine for cell therapy.</p>", "<p>Because of its potent and selective effect on myoblast survival and differentiation, Magic-F1 promoted muscular hypertrophy in all mouse models analyzed. This biological activity, revealed by <italic>in vitro</italic> experiments, was extensively confirmed by the analysis of muscles treated by electro-enhanced DNA transfer or derived from transgenic mice expressing Magic-F1 under the control of a muscle-specific promoter. Interestingly, a statistically significant increase of myofiber cross-sectional areas was observed in the <italic>tibialis anterior</italic> muscles but not in slow-twitch <italic>soleus</italic> muscles. This can be attributed to the specificity of the promoter, active in fast twitch fibers, and to the fact that the amount of circulating Magic-F1 (escaping from fast-twitch muscles) is not enough to induce muscle hypertrophy. Moreover, following cardiotoxin treatment, regenerating centrally-nucleated fibers in the MLC1F/Magic-F1 transgenic mice appeared to have a greater cross-sectional area compared to wild-type animals. This can be explained by the enhanced differentiation potential of satellite cells, which indeed displayed an earlier differentiation program <italic>in vitro</italic> compared to cells isolated from wild-type mice. We previously reported the presence of myogenic precursors, named mesoangioblasts, in the skeletal muscles of mice ##REF##12855815##[44]##, dogs ##REF##17108972##[45]## and humans ##REF##17293855##[46]##. These cells could also be positively affected by Magic-F1 and we cannot exclude their participation in the regeneration of skeletal muscle tissues. On the other hand, the rapid apoptotic response in cardiotoxin-treated muscles is strongly reduced in MLC1F/Magic-F1 transgenic mice. This results in a more evident muscular hypertrophy of transgenic muscles.</p>", "<p>Several authors have reported that HGF inhibits muscle differentiation both <italic>in vitro</italic> and <italic>in vivo</italic>\n##REF##9551084##[19]##, ##REF##10825239##[20]##. Recently, it has been reported that HGF gene therapy improves LV remodeling and dysfunction post-infarction through promotion of cardiomyocyte hypertrophy, and that HGF plays a role in the induction of stem cell commitment to the cardiomyocyte lineage ##REF##15682436##[47]##–##REF##12695295##[49]##. Magic-F1 exhibits biological effects in the renewal of skeletal muscles tissues similar though not identical to those observed for HGF in cardiac tissue regeneration. Further studies are necessary to elucidate the different potential effects of HGF in this context and –in this sense– supplementary studies on Magic-F1 signal transduction could provide useful information.</p>", "<p>Successful adenovirus-mediated gene delivery under immunosuppressive conditions in adult muscles was previously demonstrated ##REF##8854096##[50]##, ##REF##9614557##[51]##. In the present study, we transduced muscle fibers of juvenile α-SG knock-out mice with adenoviral vectors carrying Magic-F1 cDNA. All injected mice showed a physiological benefit and performed much better compared to mock-treated dystrophic animals in treadmill tests. As discussed, the less efficient rescue of the dystrophic phenotype by adenovirus-mediated Magic-F1 delivery compared to the crossing with Magic-F1 transgenic mice is conceivably due to incomplete muscle transduction. Importantly, in those mice in which all dystrophic fibers were transduced, the treadmill test performance was similar to that covered by control, non-dystrophic animals (not shown).</p>", "<p>In conclusion, Magic-F1 is a soluble, engineered factor that displays marked anti-apoptotic and pro-differentiative clues on muscle precursors. Its ability to promote and enhance muscle regeneration makes it a potential candidate molecule for regenerative medicine, particularly for muscular dystrophy syndromes and other muscle degenerative disorders. Given the small size of its cDNA (approximately 1 kb), Magic-F1 may be used alone in a gene therapy setting or inserted as a second adjuvant gene in a vector already encoding a therapeutic gene, for example encoding a deacetylase inhibitor ##REF##16980968##[52]##. The lack of mitogenic activity allows a safe use of Magic-F1 as a therapeutic cytokine, promoting muscle regeneration without the potential risk of stimulating uncontrolled proliferation.</p>" ]
[]
[ "<p>Conceived and designed the experiments: MC SB MS. Performed the experiments: AF CO. Analyzed the data: LB MGCdA PMC GC. Contributed reagents/materials/analysis tools: RB FM MA VB CB YT PM. Wrote the paper: MS.</p>", "<title>Background</title>", "<p>Hepatocyte Growth Factor (HGF) is a pleiotropic cytokine of mesenchymal origin that mediates a characteristic array of biological activities including cell proliferation, survival, motility and morphogenesis. Its high affinity receptor, the tyrosine kinase Met, is expressed by a wide range of tissues and can be activated by either paracrine or autocrine stimulation. Adult myogenic precursor cells, the so called satellite cells, express both HGF and Met. Following muscle injury, autocrine HGF-Met stimulation plays a key role in promoting activation and early division of satellite cells, but is shut off in a second phase to allow myogenic differentiation. In culture, HGF stimulation promotes proliferation of muscle precursors thereby inhibiting their differentiation.</p>", "<title>Methodology/Principal Findings</title>", "<p>Magic-Factor 1 (Met-Activating Genetically Improved Chimeric Factor-1 or Magic-F1) is an HGF-derived, engineered protein that contains two Met-binding domains repeated in tandem. It has a reduced affinity for Met and, in contrast to HGF it elicits activation of the AKT but not the ERK signaling pathway. As a result, Magic-F1 is not mitogenic but conserves the ability to promote cell survival. Here we show that Magic-F1 protects myogenic precursors against apoptosis, thus increasing their fusion ability and enhancing muscular differentiation. Electrotransfer of Magic-F1 gene into adult mice promoted muscular hypertrophy and decreased myocyte apoptosis. Magic-F1 transgenic mice displayed constitutive muscular hypertrophy, improved running performance and accelerated muscle regeneration following injury. Crossing of Magic-F1 transgenic mice with α-sarcoglycan knock-out mice –a mouse model of muscular dystrophy– or adenovirus-mediated Magic-F1 gene delivery resulted in amelioration of the dystrophic phenotype as measured by both anatomical/histological analysis and functional tests.</p>", "<title>Conclusions/Significance</title>", "<p>Because of these features Magic-F1 represents a novel molecular tool to counteract muscle wasting in major muscular diseases such as cachexia or muscular dystrophy.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>We are grateful to Gaetano Clavenna and Sergio Dompé for continuous support, Sergio Ottolenghi and Gianpaolo Papaccio for helpful discussion, Cristina Barbieri, Antonio Citro, Flavio Ronzoni, Stefania Crippa and Chiara Ciuffreda for skilled technical assistance. We thank K.P. Campbell (Howard Hughes Medical Institute, University of Iowa) for providing α-SG-deficient mice, Christina Vochten and Luigi Vercesi for the professional secretarial service, and Paolo Luban for a kind donation.</p>" ]
[ "<fig id=\"pone-0003223-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003223.g001</object-id><label>Figure 1</label><caption><title>Magic-F1 elicits partial activation of the Met pathway in C2C12 myogenic cells.</title><p>(A) Schematic representation of the Magic-F1 molecule. The indicated restriction sites refers to the corresponding cDNA map. <italic>Bam</italic>H1 and <italic>Sal</italic>1 were destroyed and the DNA fragment was cloned into the <italic>Eco</italic>RV restriction site of the pIRES-neo plasmid. (B) Purification of Magic-F1 by metal-chelate affinity chromatography. Following elution, fractions (F1-6) were resolved by SDS-PAGE in non-reducing conditions along with bovine serum albumine (BSA) standards. Proteins were revealed by Coomassie staining. MW, molecular weight; kDa, kilo Dalton units (C) ELISA binding assay. A fixed amount (100 ng/well) of Fc-Met chimera was absorbed in solid phase and exposed to increasing concentrations of HGF or Magic-F1 in liquid phase. Binding was revealed using biotinylated anti-HGF antibodies. (D) Met phosphorylation analysis in C2C12 cells. Cells were stimulated with no factor (Ctrl), 5 nM Magic-F1 (M) or 5 nM HGF (HGF), and Met phosphorylation was determined using anti-phosphotyrosine antibodies (IB). The same blots were reprobed with anti-Met antibodies to normalize the amount of receptor immunoprecipitated (IP). (E) Growth curves of C2C12 cells treated with the indicated concentrations of Magic-F1, HGF or no factor. (F) Signal transduction analysis. Cells were stimulated with no factor (Ctrl), 5 nM HGF, 5 nM NK2, 5 nM Magic-F1 (M), or 5 nM Magic-F1 and 5 nM HGF (M+HGF). Cell lysates were analyzed by Western blotting using antibodies against ERK or AKT (total) as well as antibodies against the phosphorylated forms of these signal transducers (ph).</p></caption></fig>", "<fig id=\"pone-0003223-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003223.g002</object-id><label>Figure 2</label><caption><title>Morphological analysis of C2C12 cells expressing Magic-F1.</title><p>(A) Magic-F1 detection in the culture media of stably transfected clones by Western blot analysis. (B) Immunofluorescence analysis for myosin heavy chain expression on Magic-F1 expressing clones (right panels) or control clones (left panels). Cells were analyzed after 1 day (1 d), 3 days (3 d) and 5 days (5 d). Nuclei were stained with DAPI. (C) Fusion index of C2C12 cells stably transfected with Magic-F1, HGF or mock-transfected (Ctrl). Fusion index is the ratio between the number of myocites with two or more nuclei versus the total number of myocites. (D) RT-PCR analysis of myogenic transcription factors (Myf-5, Pax3 and MyoD) on stably transfected clones (M) or control cells (C). GAPDH is used as internal control. (E) RT-PCR analysis of myostatin, follistatin and IGF1 expression in proliferating (P) versus differentiating (D) C2C12 cells. C, control cells; M, cells expressing Magic-F1. Skeletal muscle tissue (sk Mus) and a mouse embryo at 10.5 days (E10.5) were also used as controls. GAPDH was used as an internal control. (F) RT-PCR analysis of pro-apoptotic genes Bad, Bax and p53 in C2C12 clones stably transfected with Magic-F1 (M) or control cells (Ctrl). GAPDH was used as an internal control.</p></caption></fig>", "<fig id=\"pone-0003223-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003223.g003</object-id><label>Figure 3</label><caption><title>In vivo electrotransfer-mediated delivery of Magic-F1 to muscles.</title><p>10-day-old mice were subjected to <italic>in vivo</italic> electroporation of a combination of two plasmids expressing beta-galactosidase (β-gal) and HGF or Magic-F1, respectively. (A) Percent of β-gal-positive fibers following X-gal staining 4 weeks after electroporation. Representative images of electroporated muscles 1 week (1 w) and 4 weeks (4 w) after electrotransfer are shown in the upper panel. (B) RT-PCR analysis of electroporated muscles expressing HGF or Magic-F1 (data refers to samples obtained from <italic>quadriceps</italic>). (C) Histograms of morphometric analysis performed on <italic>tibialis anterior</italic>. Nine mice per group were analyzed. For each mouse, 300–460 fibers were examined (<italic>p</italic>&lt;0.01). (D) H&amp;E staining of <italic>quadriceps</italic> electroporated muscles. Note the larger fibers formed after 4 weeks in the Magic-F1 group relative to the control (Ctrl) or HGF group. (E) Tunel analysis of <italic>quadriceps</italic> 1 week after <italic>in vivo</italic> electrotransfer.</p></caption></fig>", "<fig id=\"pone-0003223-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003223.g004</object-id><label>Figure 4</label><caption><title>Morphological and functional analysis of Magic-F1 transgenic mice.</title><p>(A) Transgenic construct used for two different microinjections into ES cells. (B) Representative example of tail genotyping by PCR. The M13newborn is positive for Magic-F1 integration. In two different microinjections we obtained 2 founders out of 14, that generated two different transgenic colonies. (C) RT-PCR analysis of Magic-F1 expression in transgenic (MLC1F/Magic-F1) and wild-type muscles (WT). No signal was detected in the liver of transgenic mice (Liv) or in any organ of wild-type animals. (D) Western blot analysis of Magic-F1 expression using anti-HGF antibodies or anti-GAPDH antibodies as control. A 60 kDa band appeared only in muscles from transgenic mice. (E) Morphometric analysis of <italic>tibialis anterior</italic> (left histogram) and <italic>soleus</italic> (right histogram) muscles. For each sample; 300–400 fibers were analyzed. (F) Distance performed by transgenic and control mice on a treadmill test. For more information, please refer to the <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref> section.</p></caption></fig>", "<fig id=\"pone-0003223-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003223.g005</object-id><label>Figure 5</label><caption><title>Magic-F1 promotes muscular regeneration.</title><p>(A) Immunofluorescence analysis of muscle fibers using antibodies against embryonic myosin heavy chain (red) or laminin (green) in the <italic>tibialis anterior</italic> of transgenic and wild-type mice. Nuclei were stained with DAPI. (B) Immunofluorescence analysis for desmin (middle panels, in green) and myosin heavy chain (lower panels, in red) of satellite cells isolated from <italic>tibialis anterior</italic> of Magic-F1 transgenic mice (M) and wild-type (WT) mice subjected to cardiotoxin treatment. Nuclei are stained with DAPI (in blue). The upper panels show a phase contrast image of satellite cell clones, 3 days after low density seeding. (C) TUNEL analysis of <italic>tibialis anterior</italic> after 3, 7 and 14 days after cardiotoxin treatment. (D) Quantification of apoptotic nuclei (ap nuclei) relative to the experiment described in C. Red line, transgenic mice; blue line, wild-type mice. (E) RT-PCR analysis of myogenic transcription factor expression (MyoD and Myf5) conducted on <italic>tibialis anterior</italic> from transgenic (M) or wild-type (WT) mice. (F) Representative images of <italic>tibialis anterior</italic> muscles stained with H&amp;E extracted from Magic-F1 transgenic mice and wild-type mice 10 days after cardiotoxin treatment. Note the larger size of fibers in the Magic-F1 group (M) compared to the control group (WT).</p></caption></fig>", "<fig id=\"pone-0003223-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003223.g006</object-id><label>Figure 6</label><caption><title>Magic-F1 increases muscle strength in α-SG knock-out mice.</title><p>(A) After performance of an exhaustion treadmill tests, mice were sacrificed and Magic-F1 expression was evaluated by RT-PCR (left panel, upper lanes) and Western blot analysis (right panel, upper lanes) on <italic>tibialis anterior</italic> muscles of the indicated mice. Lane 1, α-SG knock-out dystrophic mice injected with an adenovirus expressing Magic-F1; lane 2, Magic-F1 transgenic mice; lane 3, α-SG knock-out/Magic-F1 transgenic mice; lane 4, α-SG knock-out dystrophic mice injected with an empty adenovirus. GAPDH was used as internal control (bottom panel). (B) H&amp;E staining of <italic>tibialis anterior</italic> (left upper panel) and <italic>quadriceps</italic> (left lower panel) muscles from 3 month-old Magic-F1 mice, <italic>tibialis anterior</italic> of double transgenic α-SG knoc-out/Magic-F1 transgenic mice (upper middle panel), α-SG knock-out mice (right upper panel), α-SG knock-out mice injected with Ad-Magic-F1 (middle lower panel) or with Ad-Mock (right lower panel). Mice were sacrificed 14 days after the exhaustion treadmill tests. The bar is a 50 µm marker. (C) Exhaustion treadmill tests carried on wild-type mice (blue label), α-SG knock-out/Magic-F1 transgenic mice (red label), α-SG knock-out mice injected with Ad-Magic-F1 (red-black label) or Ad-Mock (black label). Note that mice expressing Magic-F1 showed increased running performance compared to Ad-Mock-injected mice. Muscle strength of treated and control animals was measured as time (left histogram) or distance to exhaustion (right histogram; n = 4, <italic>p</italic>&lt;0.05).</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003223.s001\"><label>Figure S1</label><caption><p>Production of Magic-F1 in eucaryotic systems. (A) After transient transfection with pIRES-neo-Magic-F1 plasmid, cells were washed and incubated with fresh serum-free medium; aliquots of medium after 3, 6 and 18 hours (lane 1, 2 and 3, respectively) were concentrated 100 times and subjected to Western blot analysis using anti-HGF antibodies. Cell lysate (lane 5) and mock conditioned medium (lane 4) were also analyzed. (B) Growth curves of CHO cells expressing Magic-F1 (#PM21) or transfected with an empty vector (CHO). (C) Western blot analysis of conditioned media from different CHO clones expressing Magic-F1 at different levels. (D) Quantification of Magic-F1 protein in the conditioned medium of clone # 21; a related recombinant protein, Metron Factor-1, was used as standard (Metr). (E) Immunoprecipitation of Magic-F1 protein from the conditioned medium of clone # 5, 7, 21 and 25. In some case (# 7f), following storage at −20°C, Magic-F1 could not be immunoprecipitated any more. In each lane we loaded 10 µl of medium concentrated 100 times.</p><p>(0.73 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003223.s002\"><label>Figure S2</label><caption><p>Quantitative RT-PCR analysis of Magic-F1-transfected C2C12 cells. The expression of 36 genes involved in myogenic differentiation was evaluated one day after transfection of C2C12 using quantitative real time PCR analysis. The results indicate that 30 out of the 36 analyzed genes were upregulated in C2C12 expressing Magic-F1 compared to controls, confirming an enhanced rate of differentiation induced by Magic-F1 expression.</p><p>(0.54 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003223.s003\"><label>Figure S3</label><caption><p>Magic-F1 enhances muscle regeneration. (A) Morphometric analysis on a tibialis anterior section shows a marked increase of the fiber area in Magic-F1 transgenic mice (red bar) relative to wild-type mice (blue bar). Note that the number of fibers with a larger cross sectional area is higher in Magic-F1 transgenic mice when compared to wild-type mice. This effect is evident in both regenerated and regenerating fibers (centrally nucleated) as showed in (B), where statistical analysis is reported.</p><p>(1.11 MB TIF)</p></caption></supplementary-material>" ]
[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>This work was supported in part by: FWO Odysseus Program n. G.0907.08; Wicka Funds n. zkb8720; the Italian Ministry of University and Scientific Research (grant n. 2005067555_003, COFIN 2006–08), the Muscular Dystrophy Association, Telethon, Association Francoise contre les Myopathies, Parent Project Onlus, CARIPLO.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"pone.0003223.g001\"/>", "<graphic xlink:href=\"pone.0003223.g002\"/>", "<graphic xlink:href=\"pone.0003223.g003\"/>", "<graphic xlink:href=\"pone.0003223.g004\"/>", "<graphic xlink:href=\"pone.0003223.g005\"/>", "<graphic xlink:href=\"pone.0003223.g006\"/>" ]
[ "<media xlink:href=\"pone.0003223.s001.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003223.s002.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003223.s003.tif\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["1"], "element-citation": ["\n"], "surname": ["Trusolino", "Comoglio"], "given-names": ["L", "PM"], "year": ["2002"], "article-title": ["Scatter-factor and semaphorin receptors: cell signalling for invasive growth. Nature Rev."], "source": ["Cancer"], "volume": ["4"], "fpage": ["289"], "lpage": ["300"]}]
{ "acronym": [], "definition": [] }
54
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 16; 3(9):e3223
oa_package/80/dd/PMC2528937.tar.gz
PMC2528938
18802456
[ "<title>Introduction</title>", "<p>Very late antigen-4 (VLA-4; α4 integrin; CD49d) is expressed on most leukocytes and plays an important role in leukocyte trafficking by interacting with vascular cell adhesion molecule-1 (VCAM-1; CD106) on endothelial cells to mediate tethering, rolling, firm adhesion and transendothelial migration ##REF##7534768##[1]##,##REF##7532110##[2]##. This interaction has also been implicated in the compartmentalisation of B cells into peripheral lymphoid tissue ##REF##12566419##[3]##, the association of neutrophils with bone marrow (BM) stromal cells ##REF##11520773##[4]##, the promotion of interactions between follicular dendritic cells (FDC) and B cells ##REF##1709674##[5]##, and more recently, in the formation of a docking structure that surrounds the B cell receptor (BCR) ##REF##16456548##[6]## and TCR ##REF##15263094##[7]## in the immunological synapse (IS) that forms between antigen presenting cells and antigen-specific B and T cells.</p>", "<p>Visceral leishmaniasis (VL) caused by <italic>Leishmania donovani</italic> infection in genetically susceptible C57BL/6 or BALB/c mice results in parasite replication in the liver, spleen and BM ##REF##10652464##[8]##. The liver is the site of an acute, resolving infection, associated with leukocyte recruitment to infected resident Kupffer cells (KC) and the subsequent generation of a localised inflammatory response (granuloma formation), that includes the production of IFNγ, TNF and reactive oxygen and nitrogen species, necessary for killing intracellular parasites ##REF##1541824##[9]##–##REF##11035737##[11]##. In contrast, a chronic infection is established in the BM and spleen, associated with major changes to tissue architecture in the latter organ, including the loss of marginal zone (MZ) macrophages and stromal cells in the periarteriolar lymphoid sheath (PALS) ##REF##12163368##[12]##,##REF##12436111##[13]##. Interestingly, despite long-term parasite persistence in the spleen, this tissue is an important site for early dendritic cell (DC) IL-12p40 production that plays a key role in the generation of anti-parasitic immunity required for the control of hepatic <italic>L. donovani</italic> replication ##REF##9180189##[14]##–##REF##9521077##[16]##.</p>", "<p>We recently showed that VCAM-1 was expressed on hepatic sinusoids during <italic>L. donovani</italic> infection in C57BL/6 mice. Furthermore, we demonstrated that VCAM-1 expression during infection required lymphotoxin alpha (LTα) and that in LTα-deficient C57BL/6 mice, the absence of VCAM-1 was associated with a failure of leukocytes to migrate from periportal areas to infected KC during granuloma formation, coincident with increased parasite growth ##REF##15579454##[17]##. In this study, we investigated whether the lack of VCAM-1 expression observed in <italic>L. donovani</italic>-infected mice deficient in LTα could explain their failure to recruit leukocytes into the liver and efficiently control parasite growth. Blockade of VCAM-1 or VLA-4 suppressed anti-parasitic immune responses and was associated with significantly higher hepatic parasite burdens. However, rather than directly mediating cellular recruitment to the liver during VL, our data indicate that VCAM-1 and VLA-4 play a role in rapid CD8<sup>+</sup> DC IL-12p40 production in the spleen following <italic>L. donovani</italic> infection, an event critical for the generation of effective anti-parasitic immunity.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Mice</title>", "<p>Inbred female C57BL/6 and BALB/c mice were purchased from the Australian Resource Centre (Canning Vale, Western Australia), and maintained under conventional conditions. All mice used were age-matched (6 to 10 weeks), and were housed under specific-pathogen free conditions. All animal procedures were approved and monitored by the Queensland Institute of Medical Research Animal Ethics Committee.</p>", "<title>Parasites and infection of mice</title>", "<p>\n<italic>L. donovani</italic> (LV9) was maintained by passage in BALB/c or B6.RAG-1<sup>−/−</sup> mice, and amastigotes were isolated from the spleens of chronically infected mice. Mice were infected by injecting 2×10<sup>7</sup> amastigotes intravenously via the lateral tail vein, killed at the times indicated in the text by CO<sub>2</sub> asphyxiation and bled via cardiac puncture. In experiments examining DC IL-12p40 protein production, mice were infected with 1×10<sup>8</sup> amastigotes intravenously, as previously reported ##REF##9521079##[15]##,##REF##16622017##[18]##,##UREF##0##[27]##. Spleens and perfused livers were removed and parasite burdens were determined from Diff-Quick-stained impression smears (Lab Aids, Narrabeen, Australia), and expressed in Leishman-Donovan units (the number of amastigotes per host nuclei multiplied by the organ weight) ##REF##9103448##[28]##. Liver and spleen tissue were also preserved in either RNA<italic>later</italic> (Sigma-Aldrich, Castle Hill, Australia) or Tissue-Tek O.C.T. Compound (Sakura, Torrance, USA). Hepatic mononuclear cells (MNC) were isolated immediately following death as previously described ##REF##15579454##[17]##.</p>", "<title>VCAM-1 and VLA-4 blockade</title>", "<p>Anti-VCAM-1 (MK2/7; CRL-1909, rat IgG1) ##REF##1713592##[29]## and anti-VLA-4 (P/S2; CRL-1911, rat IgG2b) ##REF##1997648##[30]## hybridomas were purchased from the American Type Culture Collection (Manassas, VA). Purified antibody was prepared from culture supernatants by protein G column purification (Amersham, Uppsala, Sweden) followed by endotoxin removal (Mustang membranes, Pall, East Hills, NY). For VCAM-1 and VLA-4 blockade, C57BL/6 mice were injected i.p. with 1 mg of appropriate mAb or purified control rat IgG (Sigma-Aldrich) on the day of infection and every three days thereafter, or as detailed in the text. This dosing regime was based on one previously used in a collagen-induced arthritis model ##REF##11982589##[31]##, except that 0.5 mg doses were used in this study. In our hands, 0.5 mg doses only achieved partial blockade of DC IL-12p40mRNA accumulation and hepatic anti-parasitic immunity, compared with 1 mg doses, hence our use of the increased amounts of mAb. We also found that the anti-VLA-4 mAb could be detected on the surface of splenic lymphocytes for at least 72 hours following injection of 1 mg into naïve mice (data not shown). Anti-VCAM-1 mAb and control rat IgG Fab fragments were generated using a commercial kit according to the manufacturer's instructions (Thermo Scientific, Rockford, IL).</p>", "<title>Histological response to hepatic infection</title>", "<p>Acetone-fixed liver sections (6 µm) were labelled with hamster antisera to <italic>L. donovani</italic> amastigotes at a dilution of 1 in 1000. Labelling was detected with a biotinylated goat anti-hamster antibody (Vector Laboratories, Burlingame, CA). Sections were developed with Vector-Elite ABC kit, followed by 3,3′-diaminobenzidine substrate kit (Vector Laboratories). Granuloma density was determined from 25 fields of view per mouse liver (×40 magnification), and the maturation of granulomas was scored around infected Kupffer cells, as described elsewhere ##REF##9780188##[32]##.</p>", "<title>Isolation of DC</title>", "<p>Spleens were digested in collagenase type IV (1 mg/ml; Worthington, Lakewood, NJ) and deoxyribonuclease I (0.5 mg/ml; Worthington) at room temperature for 45 minutes. Splenocytes were isolated by passing digested spleens through a 100 µm cell strainer, followed by red blood cell lysis (Sigma-Aldrich). CD11c<sup>+</sup> DC were positively selected from splenocyte preparations using magnetic-activated cell sorting (MACS) with metallo-conjugated anti-mouse CD11c antibodies (N418) and positive selection columns, according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). In some experiments, following MACS enrichment, DC were sorted into CD8α-positive and CD8α-negative populations by labelling with antibodies to CD11c, MHC-II and CD8α, and sorting on a MoFlo Cell Sorter (Dako, Botany, NSW, Australia), as shown in ##FIG##6##Figure 7##.</p>", "<title>Flow cytometry</title>", "<p>Liver MNC or splenocytes were harvested and pre-incubated with CD16/32 mAb (2.4G2; grown in-house) to avoid non-specific binding of antibodies to FcγR. For the staining of cell surface antigens, cells were incubated with fluorochrome-conjugated or biotinylated mAbs on ice for 30 minutes followed by streptavidin incubation for an additional 30 minutes when required. T cells, NKT cells and NK cells were enumerated with allophycocyanin (APC)-conjugated anti-TCRβ chain (H57-597), fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (GK1.5), phycoerythrin (PE)-conjugated anti-CD8α (H1.2F3), and biotinylated anti-NK1.1 (PK136). B cells were enumerated using FITC-conjugated anti-CD19 (6D5) and APC-conjugated anti-B220 (RA3-6B2). DC were enumerated with APC-conjugated anti-CD11c (N418) and FITC- or PE-conjugated anti-I-A/I-E (MHC-II; M5/114.15.2). All mAbs were purchased from Biolegend (San Diego, CA) or BD Biosciences (Franklin Lakes, NJ). Rat anti-mouse CR3 (5C6) and rat anti-mouse GR-1 (RB6 8C5) were grown and biotinylated in house, and used to enumerate monocytes and granulocytes, respectively. Biotinylated antibodies were detected using Alexa Fluor 488-conjugated streptavidin (Invitrogen Life Technologies, Mount Waverley, Australia). Flow cytometric analysis was performed on a FACScalibur flow cytometer and analysed using Cell Quest Pro Software (BD Biosciences). For intracellular IL-12p40 staining, splenocytes were incubated for 4 hours at 37°C in 10% (v/v) foetal calf serum, RPMI containing 10 µg/ml brefeldin A (Sigma-Aldrich) prior to cell surface labelling with antibodies to CD11c, CD8α and CD4 (all from BD Biosciences). Cells were then washed and fixed in 1% (w/v) paraformaldehyde, before being washed in FACS buffer containing 0.1% (w/v) saponin (BDH, Lutterworth, UK) and stained with PE-conjugated anti-IL-12p40 (C15.6) or an isotype control mAb (both from BD Biosciences).</p>", "<title>DC cytospins</title>", "<p>Sorted CD8<sup>+</sup> and CD8<sup>−</sup> DC (1×10<sup>5</sup>) in 100 µl FACS buffer were collected onto a glass slide using a Cytospin 3 centrifuge, according to the manufacturer's instructions (Shandon Scientific Ltd, Cheshire, UK), prior to staining with Diff-Quick (Lab Aids) to visualise host cell and parasite nuclei microscopically.</p>", "<title>Real Time Reverse Transcriptase-Polymerase Chain Reaction</title>", "<p>Total RNA was extracted from bone marrow, spleen or liver tissue using TRIzol reagent (Invitrogen Life Technologies), and an RNeasy Mini Kit with on-column DNase digestion (Qiagen, Valencia, CA). Total RNA was extracted from purified DC using an RNeasy Mini Kit with on-column DNase digestion (Qiagen), according to the manufacturer's instructions. RNA samples were reverse transcribed into cDNA using the cDNA Archive Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The number of IFNγ, TNF and NOS-2 cDNA molecules in each sample were calculated using Taqman Gene Expression Assays (Applied Biosystems), and the number of IL-12p40 (<named-content content-type=\"gene\">5′ CTTGCAGATGAAGCCTTTGAAGA</named-content> (forward) and <named-content content-type=\"gene\">5′ GGAACGCACCTTTCTGGTTACA</named-content> (reverse)), and HPRT (<named-content content-type=\"gene\">5′ GTTGGATACAGGCCAGACTTTGTTG</named-content> (forward) and <named-content content-type=\"gene\">5′GATTCAACCTTGCGCTCATCTTAGGC</named-content> (reverse)) (house-keeping gene) cDNA molecules in each sample were calculated by real-time reverse transcriptase-polymerase chain reaction (rtPCR) using Platinum Sybr Green Master Mix (Invitrogen Life Technologies). All real-time reverse transcriptase-polymerase chain reactions (rtPCR) were performed on a Corbett Research RG-3000 Rotor Gene (Corbett Life Sciences, Sydney, Australia). Standard curves were generated with known amounts of cDNA for each gene, and the number of cytokine molecules per 1000 HPRT molecules in each sample was calculated.</p>", "<title>Lymphocyte and DC trafficking experiments</title>", "<p>Mice were pre-injected with 100 µg FITC-dextran i.v. (200,000 MW, anionic, Invitrogen Life Technologies) to label marginal zone macrophages, followed by 1 mg of anti-VCAM-1 or rat IgG control antibody i.p. 24 hours later. Lymphocytes were isolated from naïve splenocytes using Histopaque 1083 (Sigma), according to the manufacturer's instructions. Splenic lymphocytes or CD11c<sup>+</sup> DC were labelled with Hoechst 33342, as described previously ##REF##12163368##[12]##. Mice were administered with 1×10<sup>7</sup> Hoechst 33342-labelled lymphocytes or 1×10<sup>6</sup> Hoechst 33342-labelled DC via the lateral tail vein 1 hour post mAb injection. Mice were sacrificed 3 hours following lymphocyte transfer and 24 hr following DC transfer, and spleens were removed and embedded in Tissue-Tek O.C.T. compound (Sakura). The distribution of Hoechst 33342-labelled cells was analysed in 20 µm sections mounted in Pro-long Gold anti-fade (Invitrogen Life Technologies) using a Carl Zeiss inverted fluorescent microscope under UV illumination.</p>", "<title>Confocal microscopy</title>", "<p>Tissue-Tek O.C.T. compound-preserved sections (6 µm) of spleen tissue were acetone fixed and labelled with anti-VCAM-1 (429; MVCAM.A, BD Bioscience) detected by direct conjugation to Alexa Fluor 647 using a monoclonal antibody labelling kit, or with a fluorochrome conjugated goat anti-rat antibody (both from Invitrogen Life Technologies). To identify cell populations the sections were then labelled with different combinations of rat antibodies to murine metallophilic macrophages (MOMA-1, Acris Antibodies, Hiddenhausen, Germany), marginal zone (MZ) macrophages (ERTR9, specific ICAM-3-grabbing nonintegrin-related 1 (SIGNR1), Bachem Ltd. Merseyside, UK), reticular fibroblasts (ERTR7, BMA Biomedicals, Augst, Switzerland), endothelial cells (Meca-32, BD Biosciences), FITC -conjugated CD11c (BD Biosciences) and Alexa Fluor 647-conjugated F4/80 (BD Biosciences). Fluorochrome conjugated goat anti-rat antibodies were used for detection of purified antibodies. Sections were mounted in Pro-long Gold anti-fade (Invitrogen Life Technologies) and visualized using a Carl Zeiss inverted LSM META 510 confocal microscope.</p>", "<title>Antigen re-stimulation of splenic CD4<sup>+</sup> T cells</title>", "<p>Splenic CD4<sup>+</sup> T cells were positively selected by MACS from splenocytes using metallo-conjugated anti-CD4 antibodies and positive selection columns, according to the manufacturer's instructions (Miltenyi Biotec). CD4<sup>+</sup> T cells (5×10<sup>4</sup> cells per well) were stimulated with 2×10<sup>6</sup> paraformaldehyde-fixed <italic>L. donovani</italic> amastigotes, and 1×10<sup>6</sup> irradiated, naïve C57BL/6 spleen cells at 37°C, 5% (v/v) CO<sub>2</sub>. After 72 hours of culture, cells were pulsed with 1 µCi [<sup>3</sup>H] thymidine for 18 hours, prior to measuring thymidine incorporation using a Betaplate reader, (Wallac, Turku, Finland).</p>", "<title>Statistical analysis</title>", "<p>The statistical significance of differences between groups was determined using a Mann Whitney test or an unpaired Student's <italic>t</italic> test using GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, CA) and p&lt;0.05 was considered statistically significant. The distribution of hepatic histological responses were compared using X<sup>2</sup> analysis with Microsoft Excel software. All data are presented as the mean values±standard errors unless otherwise stated.</p>" ]
[ "<title>Results</title>", "<title>VCAM-1/VLA-4 interactions are critical for efficient control of hepatic <italic>L. donovani</italic> infection</title>", "<p>To investigate the role of VCAM-1 in VL, C57BL/6 mice were administered anti-VCAM-1 mAb 5 hours prior to <italic>L. donovani</italic> infection and then every third day thereafter until 14 days p.i. Hepatic parasite burdens were significantly increased (p&lt;0.01) in these animals compared with rat IgG-treated controls at day 14 p.i. (##FIG##0##Figure 1A##). In addition, hepatic granuloma formation, an important process for efficient control of <italic>L. donovani</italic>\n##REF##1541824##[9]##, was significantly impaired in these mice with an increase in the frequency of infected KC and a decrease in the frequency of immature (IG) and mature granulomas (MG) (p&lt;0.01; ##FIG##0##Figure 1B##). As expected, this was associated with a dramatic decrease (p&lt;0.01) in leukocyte recruitment to the liver following VCAM-1 blockade (##FIG##0##Figure 1C##), with the recruitment of all leukocytes studied (CD4<sup>+</sup> T cells, CD8<sup>+</sup> T cells, NKT cells, NK cells, B cells, macrophages/monocytes, neutrophils and DC) being significantly and similarly reduced (data not shown). IFNγ, TNF and reactive nitrogen intermediates (RNI; measured using nitric oxide synthase (NOS-2) as a surrogate marker) are all critical for the control of <italic>L. donovani</italic> infection ##REF##1541824##[9]##–##REF##11035737##[11]##,##REF##15579454##[17]##, and mRNA encoding all of these molecules was significantly reduced (p&lt;0.05) in the livers of mice receiving anti-VCAM-1 mAb (##FIG##0##Figure 1D–F##) at day 14 p.i. To confirm that VLA-4 was the main integrin interacting with VCAM-1 during VL, we also blocked this molecule using antibody over the first 14 days of infection, and obtained very similar results (##FIG##1##Figure 2##). Together these data indicate that VCAM-1/VLA-4 interactions play an important role in the generation of hepatic immune responses following <italic>L. donovani</italic> infection.</p>", "<title>VCAM-1/VLA-4 interactions do not play a direct role in leukocyte recruitment to the liver following <italic>L. donovani</italic> infection</title>", "<p>To determine whether VCAM-1/VLA-4 interactions play a direct role in cellular recruitment to the liver following <italic>L. donovani</italic> infection, we delayed VCAM-1 or VLA-4 blockade until 3 days after infection. We chose this time point to begin blockade because no significant cellular recruitment to the liver had occurred before this time (3.85×10<sup>6</sup>±4.48×10<sup>5</sup> versus 4.22×10<sup>6</sup>±7.86×10<sup>5</sup> hepatic leukocytes in naïve versus day 3 p.i. mice, respectively). In contrast to blockade commenced prior to infection, this delayed blockade failed to have any effect on parasite burden (##FIG##2##Figure 3A##), the formation of granulomas (##FIG##2##Figure 3B and C##), or numbers of hepatic mononuclear cells (MNC; ##FIG##2##Figure 3D##), indicating that VCAM-1/VLA-4 interactions played no role in leukocyte recruitment to the liver. Therefore, the main role for VCAM-1/VLA-4 interactions in VL appeared to be in the generation of immune responses within the first 3 days of <italic>L. donovani</italic> infection.</p>", "<title>Early DC IL-12p40 production in the spleen following <italic>L. donovani</italic> infection</title>", "<p>We have previously demonstrated that DC IL-12p40 production in the spleen within 24 hours of <italic>L. donovani</italic> infection is critical for the efficient generation of immunity in the liver ##REF##9521079##[15]##,##REF##9521077##[16]##. However, the kinetics of DC IL-12p40 production, the relevance of other infected tissue sites for the generation of this cytokine and the specific DC sub-population responsible for IL-12p40 production remain unknown. Therefore, we first measured IL-12p40 mRNA levels in the spleen, liver and BM, the main sites for <italic>L. donovani</italic> infection, over the first 7 days of infection. We found that IL-12p40 production occurred predominantly in the spleen and peaked at 5 hours p.i. (##FIG##3##Figure 4A##). This pattern of production was also confirmed by analysis of IL-12p40 protein expression in spleen tissue sections (data not shown), as previously described ##REF##9521079##[15]##. The IL-12p40-producing DC were located in the PALS, as well in close proximity to the MZ (data not shown), as previously reported ##REF##9521079##[15]##,##REF##16622017##[18]##. To determine the specific DC subset producing IL-12p40, MACS-enriched DC from naive mice and <italic>L. donovani</italic>-infected mice 5 hours p.i., were labelled for CD11c, CD4 and CD8α, and intracellular IL-12p40 protein levels were measured by FACS. No IL-12p40 protein was detected in CD11c-negative cells from either naïve or infected animals (data not shown), thereby identifying DC as the main source of IL-12p40. Small numbers of IL-12p40-producing CD11c<sup>+</sup> DC were observed in naïve mice (##FIG##3##Figure 4B##). Five hours after <italic>L. donovani</italic> infection, there was a 2–3 fold increase in the number of IL-12p40-producing CD11c<sup>+</sup> DC, and virtually all of the increased IL-12p40 production could be attributed to the CD8<sup>+</sup> DC subset (##FIG##3##Figure 4B##). Together, these data demonstrate that parasite-induced IL-12p40 is produced in the spleen by CD8<sup>+</sup> DC within 5 hours of <italic>L. donovani</italic> infection.</p>", "<title>VCAM-1 co-localises with sinusoidal endothelial cells and MZ macrophages in close proximity with DC in the MZ of the spleen</title>", "<p>We next investigated where VCAM-1 was expressed in the spleen to identify cell populations that might influence the generation of immune responses to <italic>L. donovani</italic> infection, and in particular CD8<sup>+</sup> DC IL-12p40 production. Studies in the mouse spleen have reported VCAM-1 expression associated with individual cells in the red pulp ##REF##12130787##[19]##, within a broad zone in the MZ ##REF##12130787##[19]## and within the B and T cell zones, including on follicular dendritic cells ##REF##9738494##[20]##. We confirmed that the majority of VCAM-1 expression was in the red pulp region, and demonstrated that expression could also be detected on discreet cell populations in the MZ and white pulp (##FIG##4##Figure 5A##). In the red pulp, all F4/80<sup>+</sup> macrophages expressed VCAM-1 (##FIG##4##Figure 5B##). However, blood flowing into the spleen is released into the marginal sinus before flowing across the MZ into the red pulp and returning to the circulation via a venous route ##REF##1555921##[21]##, and the site where DC are first likely to encounter parasites and/or parasite antigens is the MZ ##REF##9521079##[15]##,##REF##16622017##[18]##. In the MZ, there was little overlap observed between staining for VCAM-1 and marginal metallophilic macrophages (MOMA-1<sup>+</sup>) (##FIG##4##Figure 5B##). In contrast, there was clear co-localisation of VCAM-1 expression with MZ macrophages (ERTR9<sup>+</sup>/SIGNR1<sup>+</sup>) (##FIG##4##Figure 5C##), and with sinusoidal endothelial cells (Meca-32<sup>+</sup>) located in the MZ (##FIG##4##Figure 5D##). Both VCAM-1<sup>+</sup> MZ macrophages and endothelial cells were located in close proximity to discrete areas of DC (CD11c<sup>+</sup>) (##FIG##4##Figures 5C and D##). However, no clear VCAM-1 expression was observed on these DC (##FIG##4##Figures 5C and D##), a finding also confirmed by FACS analysis of VCAM-1 expression by splenic DC (data not shown). There was some co-localisation of VCAM-1 with reticular fibroblasts (ER TR7<sup>+</sup>) throughout the spleen (data not shown). Isotype control mAbs matched in concentration to each mAb did not show any staining (not shown). These results indicate that the major VCAM-1<sup>+</sup> cells found in the MZ where DC expressing VLA-4 are first likely to contact parasites and/or parasite antigens are the sinusoidal endothelial cells and MZ macrophages.</p>", "<title>VCAM-1/VLA-4 interactions are not required for lymphocyte or DC migration into the spleen</title>", "<p>We next investigated whether VCAM-1 blockade modulated DC and/or lymphocyte trafficking in the spleen following <italic>L. donovani</italic> infection. Previous work showed that lymphocyte entry into the spleen was not prevented by integrin blocking antibodies ##REF##12566419##[3]##, and we also confirmed this result by measuring migration of labelled lymphocytes into the PALS following VCAM-1 blockade in naïve animals and after 5 hours of <italic>L. donovani</italic> infection (data not shown). However, the role of cell adhesion molecules in DC entry into the spleen has not been previously investigated. Therefore, we monitored migration of labelled DC into the spleen following VCAM-1 blockade in naïve mice and after 5 hours of <italic>L. donovani</italic> infection, but found no role for VCAM-1 in this process, regardless whether mice were infected or not (##FIG##5##Figure 6A–C##). Therefore, VCAM-1 blockade did not affect lymphocyte or DC migration into the spleen, although our data do not exclude a role for VCAM-1 in cell movement between distinct regions within the spleen.</p>", "<title>VCAM-1/VLA-4 interactions play a critical role in splenic DC IL-12p40 production</title>", "<p>To test whether VCAM-1/VLA-4 interactions play a direct role in DC IL-12p40 production, we blocked these molecules 12 hours prior to infection and measured IL-12p40 mRNA levels in DC isolated from the spleen at 5 hours p.i. (##FIG##6##Figure 7A##). In control-treated mice, DC IL-12p40 mRNA levels increased 2–3 fold 5 hours after <italic>L. donovani</italic> infection. VCAM-1 blockade inhibited 50–100% of DC IL-12p40 mRNA accumulation, while VLA-4 blockade reduced DC IL-12p40 mRNA levels by 50–90% (n = 4 experiments).</p>", "<p>However, given the co-localisation of DC with VCAM-1<sup>+</sup> cells in the spleen (##FIG##4##Figure 5##), it is possible that the ligation of Fcγ receptors (FcγR) on DC by anti-VCAM-1 mAbs could suppress IL-12p40 production, as previously reported for human and mouse DC ##REF##15240682##[22]##,##REF##12377934##[23]##. Therefore, we next generated anti-VCAM-1 Fab′ fragments that comprised the antigen binding region of the mAb, but have all FcγR binding domains removed, and hence, are unable to signal via FcγR. Splenic DC isolated from mice treated with control rat IgG Fab′ fragments 5 hours after <italic>L. donovani</italic> infection had a significant accumulation of IL-12p40 mRNA, relative to DC from naïve mice (##FIG##6##Figure 7B##). Importantly, IL-12p40 accumulation in splenic DC from mice treated with anti-VCAM-1 Fab′ fragments was significantly reduced (p&lt;0.01), compared with DC from control-treated mice (##FIG##6##Figure 7B##). The reduction of IL-12p40 mRNA accumulation with anti-VCAM-1 Fab′ fragments was not as effective as with anti-VCAM-1 mAb, and this most likely reflects the very short half-life of Fab′ fragments in plasma (around 1 hour) compared with mAbs (hours-days), and their rapid excretion by the kidney ##REF##14962817##[24]##,##REF##16528525##[25]##. Nevertheless, these data support the conclusion that specific blockade of VCAM-1 reduces DC IL-12p40 mRNA accumulation following <italic>L. donovani</italic> infection, and that this effect of anti-VCAM-1 mAb treatment was not caused by FcγR ligation on DC.</p>", "<p>To confirm that parasite-induced IL-12p40 mRNA accumulation in CD8<sup>+</sup> DC was the main target of VCAM-1 blockade, we next sorted these cells following MACS enrichment, based on expression of CD11c, MHC-II and CD8α (##FIG##6##Figure 7C##), from naïve animals or at 5 hours p.i. from mice infected with <italic>L. donovani</italic> that had received control rat IgG or anti-VCAM-1 mAb prior to infection. As expected, the majority of IL-12p40 mRNA accumulation occurred in CD8<sup>+</sup> DC, and levels increased approximately 2-fold at 5 hours p.i. Importantly, this increase did not occur in CD8<sup>+</sup> DC from mice in which VCAM-1 had been blocked (##FIG##6##Figure 7D##). There was some disparity between IL-12p40 mRNA accumulation (##FIG##6##Figure 7D##) and IL-12p40 protein levels (##FIG##3##Figure 4B##) in different DC subsets from naïve mice, whereby virtually all IL-12p40 mRNA accumulated in CD8<sup>+</sup> DC, but IL-12p40 protein expression was similar between DC subsets. This may reflect different rates of IL-12p40 mRNA turnover or protein retention in different splenic DC subsets. We also observed that a small proportion of CD8<sup>+</sup> DC were infected with <italic>L. donovani</italic> amastigotes at 5 hours p.i., and that infection of these cells was not reduced by anti-VCAM-1 mAb (0.50±0.10% versus 0.37±0.11% for mice receiving rat IgG or anti-VCAM-1 mAb, respectively, determined from cytospins).</p>", "<p>Our data indicate that VCAM-1/VLA-4 interactions are important for splenic CD8<sup>+</sup> DC IL-12p40 production 5 hours after <italic>L. donovani</italic> infection, and that this is important for the efficient generation of anti-parasitic T cell responses required for control of hepatic infection. To directly examine whether <italic>L. donovani</italic>-specific CD4<sup>+</sup> T cell activation was affected by blockade of VCAM-1/VLA-4 interactions, we next isolated splenic CD4<sup>+</sup> T cells at day 14 p.i., and re-stimulated them <italic>in vitro</italic> in the presence of naïve, irradiated APC and fixed <italic>L. donovani</italic> amastigotes. There was little proliferation of splenic CD4<sup>+</sup> T cells from naïve animals in response to parasite antigen (##FIG##6##Figure 7E##). However, significant parasite-specific proliferation was observed in CD4<sup>+</sup> T cells from control-treated mice, but this was significantly reduced in CD4<sup>+</sup> T cell from animals that had received either anti-VCAM-1 or anti-VLA-4 mAbs (##FIG##6##Figure 7E##). There was no difference in CD4<sup>+</sup> T cell proliferation in response to concanavalin A in any of the groups tested, indicating that blockade of VCAM-1/VLA-4 did not generally suppress or inactivate CD4<sup>+</sup> T cells. Together, these data indicate that CD8<sup>+</sup> DC require VCAM-1/VLA-4 interactions for IL-12p40 production, associated with the generation of effective anti-parasitic CD4<sup>+</sup> T cell responses required for the control of <italic>L. donovani</italic> growth in the liver.</p>" ]
[ "<title>Discussion</title>", "<p>The interaction between VCAM-1 and VLA-4 appears to be important for the outcome of hepatic <italic>L. donovani</italic> infection. However, these molecules play no role in the recruitment of leukocytes to the infected liver during VL, and there appears to be no clear role for LTα-dependent VCAM-1 expression on hepatic sinusoids. Instead, VCAM-1/VLA-4 interactions modulate IL-12p40 production by CD8<sup>+</sup> DC in the spleen within hours of parasite challenge. Blockade of VCAM-1, along with its physiological ligand VLA-4, resulted in reduced IL-12p40 production by CD8<sup>+</sup> DC. Studies with anti-VCAM-1 Fab′ fragments also indicate a role for VCAM-1/VLA-4 interactions in DC IL-12p40 production. Blockade of VCAM-1 with antibodies was also associated with reduced CD4<sup>+</sup> T cell proliferation in the spleen and impaired resistance to <italic>L. donovani</italic> in the liver. The spleen harbours a relatively low parasite burden at the early time points assessed in this study, and this was not affected by VCAM-1 or VLA-4 blockade (data not shown).</p>", "<p>DC IL-12p40 production shortly after <italic>L. donovani</italic> infection plays a key role in the generation of anti-parasitic immune mechanisms and is critical for the effective control of VL ##REF##9180189##[14]##,##REF##9521077##[16]##. Recently, both IL-12p70 and IL-23, each utilising the IL-12p40 subunit, were found to be functionally important cytokines for the control of <italic>L. donovani</italic> infection ##REF##16790814##[26]##. We have demonstrated that splenic CD8<sup>+</sup> DC are the major source of IL-12p40 following <italic>L. donovani</italic> infection, and that parasite-induced IL-12p40 production occurs transiently, peaking 5 hours after infection (##FIG##3##Figure 4##). This early IL-12p40 production is physiologically important because when it is blocked during the first 24 hours of infection there is a failure to effectively control parasite growth in the liver and spleen ##REF##9521077##[16]##.</p>", "<p>The location of DC in the spleen is important during this early phase of infection and DC are required to migrate from the MZ into the PALS within the first 5 hours of infection in order for efficient T cell priming and maximal IL-12p40 production to occur ##REF##16622017##[18]##. This pattern of cell movement supports a model whereby parasites are rapidly taken up by macrophages in the MZ ##REF##9521079##[15]##,##REF##16622017##[18]##, parasite antigen is then either transferred from these cells to DC or the DC directly acquire antigen in the MZ, and subsequently migrate into the PALS for T cell activation. Our data suggest that VLA-4/VCAM-1 interactions play a role in these events. Blockade of VCAM-1 did not affect splenic parasite burden in the first 24 hours of <italic>L. donovani</italic> infection (data not shown), suggesting that VLA-4/VCAM-1 interactions play no role in parasite uptake by macrophages. In addition, the acquisition of parasites by CD8<sup>+</sup> DC was not prevented by VCAM-1 blockade. Histological examination of spleen tissue indicated that red pulp macrophages were the main VCAM-1<sup>+</sup> cell population in the spleen (##FIG##4##Figures 5A and B##). However, these cells are spatially segregated from the IL-12p40-producing DC found in the MZ and T cell zones. In the MZ, sinus lining endothelial cells and MZ macrophages express VCAM-1, and importantly, are found in close proximity to DC (##FIG##4##Figures 5C and D##). VCAM-1 on endothelium can mediate cell adhesion and transendothelial migration ##REF##7534768##[1]##,##REF##7532110##[2]##, and early in <italic>L. donovani</italic> infection endothelial VCAM-1 may be involved in either the adhesion of DC in the MZ or the movement of these cells into the PALS after antigen acquisition. However, previous work has shown that lymphocyte entry into the spleen is not prevented by blocking any single integrin, including VLA-4 ##REF##12566419##[3]##, and we also observed no effect of VCAM-1 blockade on lymphocyte trafficking (data not shown) or on the retention of labelled naïve DC in the spleen in naïve animals and 5 hours after <italic>L. donovani</italic> infection (##FIG##5##Figure 6##). Therefore, a role for VCAM-1-mediated naïve DC retention in the MZ is unlikely.</p>", "<p>The other main VCAM-1<sup>+</sup> cell population in the MZ were the MZ macrophages (##FIG##4##Figure 5C##). These cells are highly phagocytic and rapidly take up <italic>L. donovani</italic> after infection ##REF##9521079##[15]##,##REF##16622017##[18]##. Furthermore, these cells are lost from the spleen after a chronic infection becomes established (day 21–28 p.i.) via a TNF-dependent mechanism, disrupting cellular movement in this organ ##REF##12163368##[12]##. Therefore, VCAM-1 on MZ macrophages could mediate DC movement from the MZ into the PALS. Alternatively, it could mediate interactions between DC and MZ macrophages, allowing uptake of parasite antigen by DC and/or activation of DC. Attempts to adoptively transfer splenic DC (5×10<sup>6</sup>) isolated from mice 5 hours post-<italic>L. donovani</italic> infection (time of peak IL-12p40 production) to mice receiving VCAM-1 blockade, thereby bypassing these early cellular interactions, failed to improve control of parasite growth, relative to control animals (data not shown). Although this result suggests that later cellular interactions might be VCAM-1 dependent, only a small proportion of transferred DC (less than 10%) were found in the spleen 24 hours after transfer. Therefore, we cannot exclude the possibility that the failure to overcome VCAM-1 blockade resulted from altered DC trafficking caused by infection or the fact that DC from infected mice are unable to traffic effectively back to the T cell zones within the spleen.</p>", "<p>VLA-4 has also been localised at the centre of the peripheral supramolecular activation complex (pSMAC) that surrounds the TCR-peptide-MHC complexes localised at the centre of the SMAC in the IS ##REF##15263094##[7]##. We failed to detect VCAM-1 expression by DC in the spleen either prior to or during infection, thus questioning the potential for VLA-4/VCAM-1 interactions in the SMAC of any IS that formed between DC presenting parasite antigen and <italic>L. donovani</italic>-specific T cells in the spleen after infection. However, we cannot rule out the possibility that physiologically relevant VCAM-1 expression on splenic DC is present, but beyond the detection limits of the histological and FACS methods we have employed. All conventional DC subsets in the spleen are capable of T cell activation, and the CD8<sup>+</sup> DC isolated from <italic>L. donovani</italic>-infected mice 5 hours p.i. promote IL-12/23p40-dependent skewing towards IFNγ production by responding CD4<sup>+</sup> T cells ##UREF##0##[27]##. In addition, the CD8<sup>+</sup> DC are capable of acquiring <italic>L. donovani</italic> amastigotes independent of VCAM-1 early after infection, and these infected cells are able to produce IL-12p40 (Maroof, unpublished). Our data also indicate that VCAM-1/VLA-4 interactions play an important role in the priming of parasite-specific CD4<sup>+</sup> T cells.</p>", "<p>Short-term (5 hr) experiments with anti-VCAM-1 Fab′ fragments suggested that FcγR ligation on DC was not responsible for the observed reduced DC IL-12p40 production. However, blockade of VCAM-1 and the reduction of DC IL-12p40 with Fab′ fragments was not as effective as whole mAb, perhaps due to their extremely short half-life and rapid excretion by the kidney due to their low molecular weight ##REF##14962817##[24]##,##REF##16528525##[25]##. Ideally we would have liked to confirm our results at day 14 with Fab′ fragments, as well as whole mAb. However, it would not be possible to interpret the results of such long-term experiments accurately due to the limitations discussed above.</p>", "<p>In conclusion, we have shown that VCAM-1/VLA-4 interactions modulate CD8<sup>+</sup> DC IL-12p40 production and may play a role in the activation of parasite-specific CD4<sup>+</sup> T cells during disease. Furthermore, our data indicate that VCAM-1 and VLA-4 are not directly involved in cellular recruitment to the liver during VL. These findings advance our understanding of the induction of cell-mediated immune responses following pathogen challenge, and identify a potential target for modulation to either enhance or suppress inflammation during disease.</p>" ]
[]
[ "<p>Conceived and designed the experiments: ACS GRH PMK CRE. Performed the experiments: ACS JED SHR KPM YZ FR WAS AM CRE. Analyzed the data: ACS GRH PMK CRE. Wrote the paper: ACS CRE.</p>", "<p>Vascular cell adhesion molecule-1 (VCAM-1) interacts with its major ligand very late antigen-4 (VLA-4) to mediate cell adhesion and transendothelial migration of leukocytes. We report an important role for VCAM-1/VLA-4 interactions in the generation of immune responses during experimental visceral leishmaniasis caused by <italic>Leishmania donovani</italic>. Our studies demonstrate that these molecules play no direct role in the recruitment of leukocytes to the infected liver, but instead contribute to IL-12p40-production by splenic CD8<sup>+</sup> dendritic cells (DC). Blockade of VCAM-1/VLA-4 interactions using whole antibody or anti-VCAM-1 Fab′ fragments reduced IL-12p40 mRNA accumulation by splenic DC 5 hours after <italic>L. donovani</italic> infection. This was associated with reduced anti-parasitic CD4<sup>+</sup> T cell activation in the spleen and lowered hepatic IFNγ, TNF and nitric oxide production by 14 days post infection. Importantly, these effects were associated with enhanced parasite growth in the liver in studies with either anti-VCAM-1 or anti-VLA-4 antibodies. These data indicate a role for VCAM-1 and VLA-4 in DC activation during infectious disease.</p>", "<title>Author Summary</title>", "<p>VCAM-1 and its major ligand VLA-4 are adhesion molecules required for the recruitment and movement of leukocytes within tissue. In this study, we have investigated the role of these molecules during an experimental infection with <italic>Leishmania donovani</italic>, a protozoan parasite that causes a chronic disease called visceral leishmaniasis. Surprisingly, we showed that VCAM-1 and VLA-4 were not required for leukocyte migration into the liver, a site of acute <italic>L. donovani</italic> infection. Instead, there was a requirement for these molecules to initiate cell-mediated immune responses in the spleen within the first 5 hours of infection. When VCAM-1 was blocked during infection, early dendritic cell production of IL-12p40, a potent pro-inflammatory cytokine required for control of <italic>L. donovani</italic>, was suppressed, associated with a reduced parasite-specific T cell response in the spleen, and impaired immunity and parasite clearance in the liver. These results are important because they identify a novel role for VCAM-1 and VLA-4 in the regulation of dendritic cell activation during infectious disease.</p>" ]
[]
[ "<p>We thank Grace Chojnowski and Paula Hall for expert assistance with flow cytometry, Kaye Wycherley from Walter and Eliza Hall Medical Research Institute for sub-cloning and selecting a high producing MK2/7 cell line, Jean-Pierre Levescue from the Mater Medical Research Institute for helpful discussion about VCAM-1 and VLA-4, and Andrew Boyd and Michael Gerometta from QIMR for helpful advice on the generation of Fab′ fragments. We also thank Ashraful Haque for critical review of the manuscript.</p>" ]
[ "<fig id=\"ppat-1000158-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000158.g001</object-id><label>Figure 1</label><caption><title>VCAM-1 is required for efficient control of <italic>L. donovani</italic> infection in the liver.</title><p>Female C57BL/6 mice were treated with anti-VCAM-1 mAb (closed bars) or control rat IgG (open bars) and infected with 2×10<sup>7</sup>\n<italic>L. donovani</italic> amastigotes i.v. Mice were injected i.p. with 1 mg of antibody prior to infection and every 3 days thereafter. Parasite burdens were determined in the liver at day 14 p.i. (A) and data represent the mean±SEM in Leishman Donovan units. The number and maturity of hepatic granulomas were estimated on day 14 liver sections stained with anti-<italic>L. donovani</italic> sera (B). Data represent the frequency of infected Kupffer cells (KC), immature granulomas (IG) and mature granulomas (MG) per liver. Hepatic mononuclear cells were isolated from naïve (hatched bars) and infected mice on day 14 p.i. and enumerated (C). mRNA was extracted from livers of naïve or day 14 p.i. VCAM-1 blocked or control mice, and accumulation of IFNγ (D), TNF (E) and NOS-2 (F) mRNA was detected by real-time RT-PCR and is expressed as mRNA molecule per 1000 HPRT molecules. One representative experiment of two performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). Statistical differences of p≤0.05 (*) or p&lt;0.01 (**) for control versus anti-VCAM-1 treatment are indicated.</p></caption></fig>", "<fig id=\"ppat-1000158-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000158.g002</object-id><label>Figure 2</label><caption><title>VLA-4 is required for efficient control of <italic>L. donovani</italic> infection in the liver.</title><p>Female C57BL/6 mice were treated with anti-VLA-4 mAb (closed bars) or control rat IgG (open bars) and infected with 2×10<sup>7</sup>\n<italic>L. donovani</italic> amastigotes i.v. Mice were injected i.p. with 1 mg of antibody prior to infection and every 3 days thereafter. Parasite burdens were determined in the liver at day 14 p.i. (A) and data represent the mean±SEM in Leishman Donovan units. The number and maturity of hepatic granulomas was estimated on day 14 liver sections stained with anti-<italic>L. donovani</italic> sera (B). Data represent the frequency of infected Kupffer cells (KC), immature granulomas (IG) and mature granulomas (MG) per liver. Hepatic mononuclear cells were isolated from naïve (hatched bars) and infected mice on day 14 p.i. and enumerated (C). mRNA was extracted from livers of naïve (hatched bars) or day 14 p.i. VLA-4 blocked (closed bars) or control mice (open bars), and accumulation of IFNγ (D), TNF (E) and NOS-2 (F) mRNA was detected by real-time RT-PCR and is expressed as mRNA molecule per 1000 HPRT molecules. One representative experiment of two performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). Statistical differences of p&lt;0.05 (*) or p&lt;0.01 (**) for control versus anti-VLA-4 treatment are indicated.</p></caption></fig>", "<fig id=\"ppat-1000158-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000158.g003</object-id><label>Figure 3</label><caption><title>VCAM-1/VLA-4 interactions are not required for leukocyte migration into the liver following <italic>L. donovani</italic> infection.</title><p>Female C57BL/6 mice were untreated (vertical hatched bar) or treated with anti-VCAM-1 mAb, anti-VLA-4 mAb (closed bars) or control rat IgG (open bars) and infected with 2×10<sup>7</sup>\n<italic>L. donovani</italic> amastigotes i.v. Mice were injected i.p. with 1 mg of antibody prior to infection and every 3 days thereafter (black bars), or starting from day 3 and every 3 days thereafter (grey bars). Parasite burdens were determined in the liver at day 14 p.i. (A) and data represent the mean±SEM in Leishman Donovan units. The number and maturity of hepatic granulomas was estimated on day 14 liver sections labelled with anti-<italic>L. donovani</italic> sera from VLA-4 blocked mice (B) and VCAM-1 blocked mice (C). Data represent the frequency of infected Kupffer cells (KC), immature granulomas (IG) and mature granulomas (MG) per mouse. Hepatic MNC were isolated from naïve (hatched bars) and infected mice on day 14 p.i. and enumerated (D). One representative experiment of two performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). Statistical differences of p&lt;0.01 (**) for control versus anti-VCAM-1 or anti-VLA-4 treatment are indicated.</p></caption></fig>", "<fig id=\"ppat-1000158-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000158.g004</object-id><label>Figure 4</label><caption><title>IL-12p40 is produced rapidly and transiently in the spleen by CD8<sup>+</sup> DC following <italic>L. donovani</italic> infection.</title><p>(A) Mice were infected with 2×10<sup>7</sup>\n<italic>L. donovani</italic> amastigotes i.v. and sacrificed at 2 hr, 5 hr, 12 hr, 24 hr, 72 hr, 5 days and 7 days p.i. mRNA was extracted from the spleen (squares), liver (inverted triangles) and bone marrow (triangles) at each time point and accumulation of IL-12p40 mRNA was detected by real-time RT-PCR and is expressed as mRNA molecule per 1000 HPRT molecules. Data are representative of 3 mice per time point. (B) Mice were infected with 1×10<sup>8</sup>\n<italic>L. donovani</italic> amastigotes i.v. and spleens were removed at 5 hr p.i. MACS-enriched DC were enumerated by labelling with anti-CD11c mAb and examined for expression of isotype control mAb or IL-12p40 (1,000,000 events collected, 10,000 events shown). Cells were then electronically gated, based on expression of IL-12p40, as indicated, and then examined for expression of CD4 and CD8 (5000 events shown). Gates were determined from an isotype control for IL-12p40, in which 0.8% of CD11c<sup>+</sup> cells were shown to be positive. Numbers below rectangular gates indicate the percentage of cells in that gate, while numbers in the upper right hand corners of FACS profiles indicate the percentage of cells in the indicated quadrants. One representative experiment of three performed with similar outcome is shown (n = 4 mice per treatment group in each experiment).</p></caption></fig>", "<fig id=\"ppat-1000158-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000158.g005</object-id><label>Figure 5</label><caption><title>VCAM-1 expression in the spleens of naive C57BL/6 mice.</title><p>(A) VCAM-1 localisation (green) in a tissue section from a naïve mouse. Marginal metallophilic macrophages were stained with MOMA-1 mAb (red) allowing visualisation of red pulp (RP), white pulp (WP) and marginal zone (MZ) regions of the spleen, as indicated (×100) The tissue section was mounted in media containing DAPI to stain cell nuclei (blue). (B-D) Staining for VCAM-1 expression, red pulp macrophages (F4/80<sup>+</sup>), MM macrophages (MOMA-1<sup>+</sup>), MZ macrophages (ERTR9/SIGNR1<sup>+</sup>), MZ sinus-lining endothelial cells (Meca-32<sup>+</sup>) and DC (CD11c<sup>+</sup>) (×630) is shown in colours indicated above panels. White arrows in (C) and (D) indicate areas where VCAM-1<sup>+</sup> cells (yellow) are in close proximity to DC (blue).</p></caption></fig>", "<fig id=\"ppat-1000158-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000158.g006</object-id><label>Figure 6</label><caption><title>VCAM-1/VLA-4 interactions are not critical for DC migration into the spleen.</title><p>Naïve C57BL/6 mice were injected with 100 µg FITC-dextran i.v. followed by 1 mg of control rat IgG (A) or anti-VCAM-1 mAb (B) i.p. 24 hr later. Hoechst 33342-labelled splenic CD11c<sup>+</sup> DC (1×10<sup>6</sup>) were administered i.v. 1 hr following mAb injection. The following day mice were either left as naïve or infected with 2×10<sup>7</sup>\n<italic>L. donovani</italic> amastigotes i.v. and spleens were removed 5 hr later (24 hr post-cell transfer). The distribution of Hoechst 33342-labelled cells was analysed in 20 µm sections and photographed under UV illumination (×100). Data are representative of one of two experiments performed (n = 3 mice per group). The number of Hoechst 33342-labelled cells was determined from 15 fields of view per mouse spleen (×25 magnification) (C).</p></caption></fig>", "<fig id=\"ppat-1000158-g007\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000158.g007</object-id><label>Figure 7</label><caption><title>VCAM-1/VLA-4 interactions are required for parasite-induced IL-12p40 mRNA accumulation by CD8<sup>+</sup> DC.</title><p>(A) C57BL/6 mice were treated with 1 mg anti-VCAM-1 mAb, anti-VLA-4 mAb or control rat IgG or (B) with 1 mg anti-VCAM-1 Fab fragments or control rat IgG Fab fragments the day prior to infection with 1×10<sup>8</sup>\n<italic>L. donovani</italic> amastigotes. CD11c<sup>+</sup> DC were enriched by positive selection by MACS from the spleens of naïve mice (hatched bars), or antibody treated mice (closed bars) or control treated mice (open bars) at 5 hr p.i. mRNA was extracted from MACS-enriched DC, and accumulation of IL-12p40 mRNA was detected by real-time RT-PCR. One representative experiment of four performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). All data is expressed as IL-12p40 mRNA molecules per 1000 HPRT molecules. (C–D) MACS-enriched CD11c<sup>+</sup> DC from naïve C57BL/6 mice (hatched bars) or <italic>L. donovani</italic>-infected mice at 5 hr p.i. treated with either control rat IgG (open bars) or anti-VCAM-1 (closed bars), were sorted based on CD11c and MHC-II expression, followed by separation into CD8α-positive and CD8α-negative populations. Percentages of each gated population are indicated. (D) mRNA was extracted from purified CD8<sup>+</sup> or CD8<sup>−</sup> DC populations, as indicated, and accumulation of IL-12p40 mRNA was detected by real-time RT-PCR. Data represent groups of pooled cells from 4 mice, repeated 3 times. (E) Proliferation of splenic CD4<sup>+</sup> T cells from naïve (hatched bars) or <italic>L. donovani</italic>-infected C57BL/6 mice (day 14 p.i.) that had received anti-VCAM-1 mAb, anti-VLA-4 mAb (closed bars) or control rat IgG (open bars), as indicated, in the presence of naïve irradiated splenic APC pulsed with fixed <italic>L. donovani</italic> amastigotes. One representative experiment of two performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). Data is presented as a stimulation index (SI) calculated by dividing the proliferation of each sample in response to parasite antigen by proliferation in culture media alone. Statistical differences of p&lt;0.01 (**) for antibody versus control treated mice are indicated.</p></caption></fig>" ]
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[ "<fn-group><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p>This work was supported by grants from the Australian NHMRC and the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR). CRE is an Australian NHMRC Career Development Fellow and GRH is an Australian NHMRC Practitioner Fellow.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"ppat.1000158.g001\"/>", "<graphic xlink:href=\"ppat.1000158.g002\"/>", "<graphic xlink:href=\"ppat.1000158.g003\"/>", "<graphic xlink:href=\"ppat.1000158.g004\"/>", "<graphic xlink:href=\"ppat.1000158.g005\"/>", "<graphic xlink:href=\"ppat.1000158.g006\"/>", "<graphic xlink:href=\"ppat.1000158.g007\"/>" ]
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[{"label": ["27"], "element-citation": ["\n"], "surname": ["Maroof", "Kaye"], "given-names": ["A", "PM"], "year": ["2007"], "article-title": ["Temporal regulation of IL-12p70 and IL-12-related cytokines in splenic DC subsets during Leishmania donovani infection."], "source": ["Infect Immun"]}]
{ "acronym": [], "definition": [] }
32
CC BY
no
2022-01-13 03:40:34
PLoS Pathog. 2008 Sep 19; 4(9):e1000158
oa_package/5c/23/PMC2528938.tar.gz
PMC2528939
18800165
[ "<title>Introduction</title>", "<p>Protein misfolding diseases are characterized by the formation of amyloid, which occurs through misfolding promoted by the conversion of a protein from its native to non-native state. Under the appropriate conditions, any proteins can form generic amyloid ##REF##11242064##[1]##. The ability of an oligomeric entity in amyloid to seed the polymerization of other proteins indicates that amyloid may disrupt cellular functions by interfering with the folding of other proteins ##REF##12702875##[2]##–##REF##16469881##[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6]##. Therefore, it is important to understand the molecular mechanism(s) underlying the seeding function of amyloid oligomers, whose pathogenic significance in protein misfolding diseases have been well supported. Because amyloid oligomers innately tend to aggregate, high-resolution elucidation of their structures through conventional physical techniques has been challenging. Novel insight on the structure of amyloid oligomers was made possible by the development of an anti-amyloid oligomer conformation-dependent antibody, A11 ##REF##12702875##[2]##. The fact that various amyloid oligomers are A11 immunopositive suggests that amyloid oligomers share a common structure and implies that various protein-misfolding diseases may have a common pathogenic mechanism ##REF##12702875##[2]##. In the present study, we identified proteins that contain the amyloid oligomer conformation by using the A11 antibody as a probe. One group of A11-reactive proteins commonly displayed anti-β-aggregation activity as opposed to the expected β-aggregation promoting activity of misfolded amyloid oligomers.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Immunoblot (Western blot and dot blot)</title>", "<p>Separated proteins by SDS-PAGE were transferred onto nitrocellulose membranes by using a wet system at 4°C. Alternatively, 3 µL of sample solution was dot-blotted onto membranes. After being blocked with milk, the blotted membrane was probed with anti-amyloid oligomer antibody A11 (1∶1000; Biosource, USA) ##REF##12702875##[2]## or 4G8 (1∶1000; Signet, USA). A11 and 4G8 antibody immunoreactivity were detected with HRP-conjugated anti-rabbit IgG (1∶2500) or anti-mouse IgG (1∶5000), respectively, followed by ECL. A replicated membrane probed with the secondary antibody alone did not display signals. Each panel displaying an immunoblot was from a single membrane and thus a single experiment. For presentation purposes, the squares containing each dot blot were arranged in a single column or row for figures.</p>", "<title>Aβ oligomer preparation</title>", "<p>Aβ oligomer was prepared as described previously ##REF##12702875##[2]##. Briefly, lyophilized Aβ40 peptide (Peptide Institute, Japan) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Wako, Japan) on ice and aliquoted to be frozen until use. Aβ was spin-vacuumed just prior to the experiment; dissolved in HFIP solution (final concentration: 10% (v/v) HFIP); and kept at room temperature under constant stirring. After 2 days, the tube was transferred to and maintained at 4°C.</p>", "<title>Thioflavin T (ThT) assay</title>", "<p>Lyophilized Aβ40 peptide was first dissolved in HFIP on ice. Most of the HFIP was vacuum evaporated before Aβ was dissolved in H<sub>2</sub>O; PBS; or artificial cerebrospinal fluid (aCSF: 124 mM NaCl, 2.5 mM KCl, 2 mM CaCl<sub>2</sub>, 2 mM MgSO<sub>4</sub>, 1.25 mM NaH<sub>2</sub>PO<sub>4</sub>, 26 mM NaHCO<sub>3</sub>, and 10 mM glucose). Solubilized Aβ40 (5 µM) was mixed with a protein dissolved in a respective buffer. Note that different buffer conditions resulted in different aggregation kinetics of Aβ. ThT (10 µM unless otherwise noted) was added to each solution to assess the formation of β-sheet structures. Samples were incubated at 37°C in 96-well plates; triplicate measurements were taken every 30 min with an ARVO multilabel spectrofluorometer (PerkinElmer, USA). Plates were shaken for 10 sec prior to every measurement. Excitation and emission wavelengths were 440 and 486 nm, respectively ##REF##8453378##[7]##. In each experiment, triplicate wells containing protein sample at each concentration without Aβ were made. Results from the measurement of these wells were not included in the figures because the values were significantly smaller than those of wells with Aβ.</p>", "<title>Luciferase refolding assay</title>", "<p>Twenty microliters of 250 nM luciferase (Sigma) in 100 mM Tris-HCl (pH 7.4) and 5 µL of 20 mM DTT in H<sub>2</sub>O were mixed in each well of a 96-well plate. For heat treatment, plates were sealed and placed in an incubator maintained at 45°C. After 5 min, the plates were taken out and kept at room temperature for 5 min. Next, 2.5 µL of 40 mM DTT (Nacalai Tesque), samples representing four different conditions, and 2.5 µL of 80 mM Mg-ATP (Sigma) in H<sub>2</sub>O were added to each well. The four samples were as follows: (1) 15 µL of H<sub>2</sub>O; (2) 5 µL of H<sub>2</sub>O, 5 µL of 12.5 µM GroEL, and 5 µL of 21 µM GroES; (3) 5 µL of 1 mg/mL A11, 5 µL of 12.5 µM GroEL, and 5 µL of 21 µM GroES; and (4) 5 µL of 1 mg/mL A11, and 10 µL of H<sub>2</sub>O. The plate was incubated at 30°C for 1 h. As soon as 5 µL of 2 mM luciferin (Nacalai Tesque) dissolved in 100 mM Tris-HCl (pH 7.4) was added to each well, the luminescence was measured with an ARVO multilabel spectrofluorometer (PerkinElmer). The average luminescence value from H<sub>2</sub>O-incubated wells was subtracted from the luminescence value measured from other wells. The percentage of luminescence recorded in each well was calculated, taking as 100% the average value measured from wells that were not heat treated. The percentages determined for each condition were averaged and defined as percent (%) luciferase activity. GroE represents the combination of both GroEL and GroES.</p>", "<title>Structural similarity search</title>", "<p>Ten PDB files of A11-immunoreactive proteins were retrieved. In the preliminary search, 214 potential sites on the surfaces of ten structures were detected among the areas where the main β-sheet chain was exposed to the surface (##TAB##0##Table 1##). Atoms in these sites were first classified into six physicochemical types using the PATTY algorithm ##UREF##3##[50]##. Structural alignment was carried out by aligning the spatial coordinates of atoms at every two sites and identifying pairs of atoms of the same physicochemical type. We then calculated similarity scores for these aligned structures. Twenty-three sites with relatively high similarity scores were determined using a clustering algorithm. For each structure, we selected sites having the highest similarity score. We then constructed a figure by performing multiple structure alignments on the ten sites having the highest similarity scores. Surface atoms were determined by calculating accessible surface areas (&gt;0 Å<sup>2</sup>) at 1.4 Å of probe atoms. The atoms were clustered using the single-linkage clustering algorithm with a cutoff value of 4 Å. Clusters formed by less than four atoms were removed. Surface atoms that existed within 4 Å from atoms of the remaining clusters were included. Each cluster was defined as a single site.</p>", "<title>Similarity score</title>", "<p>Each atom located on a structure's surface was given a feature vector. The feature vector describes the local physicochemical environment of a surface atom. Atoms were first classified into six physicochemical types using the PATTY algorithm ##UREF##3##[50]##: cation (<italic>AT</italic>1), anion (<italic>AT</italic>2), hydrogen-bond donor (<italic>AT</italic>3), hydrogen-bond acceptor (<italic>AT</italic>4), hydrophobic (<italic>AT</italic>5), and none of these (<italic>AT</italic>6). An atom that was both a hydrogen-bond donor and an acceptor (‘polar’ atom, according to the PATTY algorithm) was treated as 0.5 donor and 0.5 acceptor. The feature vectors were defined for each atom according to this atom-type classification. The feature vector <bold>C</bold>\n<italic><sub>i</sub></italic> of atom <italic>i</italic> was defined as:in which the summation was over all the solvent-accessible atoms (including atom <italic>i</italic>) of the protein, if <italic>d<sub>ij</sub></italic>/<italic>d<sub>c</sub></italic>≤1, and atom <italic>j</italic> is of type <italic>ATx</italic>; otherwise, . If atom <italic>j</italic> is both a donor and an acceptor and <italic>x</italic> = 3 or 4, <italic>a</italic> = 0.5; otherwise, a = 1. <italic>d<sub>ij</sub></italic> is the distance (in Å) between atom <italic>i</italic> and atom <italic>j</italic>. The parameter values <italic>d<sub>c</sub></italic> = 3.2 were used. The similarity between each pair of atoms from the two regions was assessed by <italic>s<sub>ij</sub></italic> = <italic>T<sub>C</sub></italic>(<italic>i</italic>, <italic>j</italic>), where atoms <italic>i</italic> and <italic>j</italic> were from regions <italic>a</italic> and <italic>b</italic>, respectively; and <italic>T<sub>C</sub></italic>(<italic>i</italic>,<italic>j</italic>) is the Tanimoto coefficient:\n</p>", "<p>Finally, a similarity score <italic>S</italic> was defined for the two regions <italic>a</italic> and <italic>b</italic>: <italic>S</italic> = Σ<italic>s<sub>ij</sub></italic>/min{<italic>N<sub>a</sub></italic>,<italic>N<sub>b</sub></italic>}, where <italic>s<sub>ij</sub></italic> is defined as above; <italic>N<sub>a</sub></italic> and <italic>N<sub>b</sub></italic> are the numbers of atoms of regions <italic>a</italic> and <italic>b</italic>, respectively; and the summation was over all atom pairs in the alignment except for those pairs of different atom types. Possible <italic>S</italic> values ranged from 0 to 1, where 1 represents the maximum similarity.</p>" ]
[ "<title>Results</title>", "<title>Amyloid oligomer conformation in chaperones</title>", "<p>A11 is an antibody that specifically detects the conformation of amyloid oligomers regardless of their amino acid sequence ##REF##12702875##[2]##. To determine whether proteins that contain the amyloid oligomer conformation exist in bacteria, we examined the A11 immunoreactivity of <italic>Escherichia coli</italic> cell lysates. We chose to examine bacterial proteins because extensive structural information about these proteins already exists in the protein data bank (PDB). We subjected <italic>E. coli</italic> DH5α cell lysates to PAGE analysis. To reduce false-positive signals, we used sample buffer that contained neither SDS nor reducing agents to solubilize the lysates although the disruption of conformation was predicted to some extent during SDS-PAGE. One gel was Western blotted with A11 to identify the molecular weights of A11-immunoreactive proteins (##FIG##0##Figure 1A##), and another gel was stained with coomassie brilliant blue (CBB). The bands in the CBB-stained gel corresponding to the A11-immunoreactive bands on the immunoblot were analyzed by tandem mass spectrometry (MS-MS). We identified one of these A11-immunoreactive bands to contain GroEL, a bacterial chaperonin ##REF##11884745##[6]##. Unlike other proteomics studies that have used sequence-dependent antibodies, in our study, the proteins detected in the cell lysates through this simple approach remain unconfirmed candidates of A11-reactive proteins. Indeed, the conformational specificity of A11 for each protein (purified and native forms) needs to be analyzed, since the cell lysis procedure might have affected protein conformation to unknown degrees.</p>", "<p>To confirm the A11 immunoreactivity of GroEL, we performed Western analysis on purified GroEL and observed that A11 strongly immunostained monomeric GroEL (##FIG##0##Figure 1B##). Although the sample buffer used in this PAGE analysis lacked SDS and reducing agents, the running buffer did contain SDS. In true native PAGE, which is performed without SDS, A11 mostly immunostained a high molecular weight band representing a GroEL oligomer complex, which is the expected functionally active form of GroEL in bacterial cytosol (##FIG##0##Figure 1C##). A11 antibody also strongly immunostained purified GroEL on dot blots (##FIG##0##Figure 1D##). This enabled us to exclude the possibility that the PAGE processing misfolded GroEL molecules that potentially could react with A11 and lead to false A11 positives. A11 antibody also immunostained purified recombinant human chaperones, including heat shock protein (Hsp) 27, 40, 70, 90; yeast Hsp104; and bovine heat shock cognate (Hsc) 70, albeit with varying intensity (##FIG##0##Figure 1D##). As expected, A11 antibody immunostained β-amyloid (Aβ) oligomer prepared from Aβ(1–40) but not monomeric Aβ. These results suggest that certain chaperone structures share a common amyloid oligomer conformation. Among the A11-reactive candidates, we were interested in identifying chaperones that normally display anti-aggregation activity as opposed to proteins, such as misfolded pathogenic amyloid oligomers, that induce β-aggregation of other proteins ##REF##12702875##[2]##–##REF##16469881##[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6]##.</p>", "<title>Proteins containing the amyloid oligomer conformation affect Aβ aggregation in opposite ways</title>", "<p>To confirm the seeding property of pathogenic Aβ oligomer, we assessed the β-aggregation kinetics of Aβ40 in the presence of preformed Aβ oligomer using a thioflavin T (ThT) fluorescence assay ##REF##8453378##[7]## (##FIG##1##Figure 2A##), and found that Aβ oligomer induced Aβ aggregation in a dose-dependent manner. To determine whether the chaperones we assessed with A11 antibody were functionally active and thus natively folded or misfolded like pathogenic amyloid oligomers, we examined the effects of Hsp27, Hsp70, and Hsp90 on Aβ-aggregation kinetics using the ThT assay ##REF##8453378##[7]## (##FIG##1##Figure 2B–D##). Aβ, a type of amyloid protein observed in Alzheimer's disease (AD), has been pathologically linked to these three chaperones ##REF##15611723##[8]##. As with other published studies ##REF##16973602##[9]##, ##REF##16635482##[10]##, we found that all three chaperones—Hsp27, Hsp70, and Hsp90—suppressed the β aggregation of Aβ in a dose-dependent manner to varying degrees. We did not observe fibrillar amyloid structures in Aβ samples incubated with each of these chaperones under an electron microscope (data not shown). To verify this chaperone activity ##REF##11884745##[6]##, we examined whether the anti-β-aggregation properties of Hsp was energy dependent (##FIG##1##Figure 2E, F##). Adding ATP to samples containing Hsp70 or Hsp70/Hsp40 at concentrations that alone do not show anti-β-aggregation activity caused Hsp70 and Hsp70/Hsp40 to significantly suppress Aβ aggregation. Most of the purified recombinant chaperones used here, including Hsp70 and Hsp40, have tested positive for their ATPase activity. The energy-dependent anti-β-aggregation activity of these chaperone samples indicates that A11-immunoreactive chaperones are indeed functional; thus, a significant number of chaperone molecules in these purified samples are natively folded ##REF##14685248##[4]##, ##REF##4124164##[11]##. In other words, the native structures of these chaperones possess the amyloid oligomer conformation. Therefore, there are at least two classes of A11-reactive proteins that contain the amyloid oligomer conformation: pathogenic amyloid oligomers that promote β aggregation and chaperones that suppress β aggregation.</p>", "<title>Heating affects the amyloid oligomer conformation and anti-β-aggregation activity of Hsp27</title>", "<p>Next, we examined how heat treatment alters the amyloid oligomer conformation of Hsp27 and the Hsp27-mediated suppression of Aβ aggregation (##FIG##2##Figure 3##). We assessed Hsp27 because of its strong A11 immunoreactivity on both dot blot and native-PAGE/Western blot analyses, and because of its pathological association with Aβ ##REF##15611723##[8]##. We heated Hsp27 dissolved in buffer to 80°C for 15 and 30 min and then subjected the sample to native-PAGE followed by Western blotting with A11 antibody. Heat treatment abolished A11 immunoreactivity of Hsp27 (##FIG##2##Figure 3A##), even though the peptide backbone of Hsp27 was intact (SDS-PAGE of heat-treated samples, ##FIG##2##Figure 3B##). ThT assay of samples containing Aβ and heat-treated Hsp27, however, showed that Hsp27-mediated suppression of Aβ aggregation disappeared concurrently with the reduction of amyloid oligomer conformation resulting from the heat treatment (##FIG##2##Figure 3C##). The heat-denaturing experiments simply suggested that the conformation-specific binding of A11, and thus the amyloid oligomer conformation—not non-specific interactions—was consistently observed as natively folded Hsp27 prevented β aggregation. These results certainly did not demonstrate that the A11-reactive domain within Hsp27 is responsible for suppressing Aβ aggregation. Both the structure and functional mechanisms of Hsp27 are unknown. Since the spherical and oligomeric forms of Hsp27 are active states ##REF##9029143##[12]##, Hsp27 could co-oligomerize with Aβ while suppressing amyloidogenesis. Heat-treated Hsp27 may have also formed amyloid that complexed with Aβ. Taken together, these data identify the amyloid conformation as the generic structural feature of natively folded chaperones directly correlating with the anti-β-aggregation activity of chaperones.</p>", "<title>A11 immunoreactivity and anti-β-aggregation activity in non-chaperone proteins</title>", "<p>Anti-β-aggregation activity is not unique to chaperones. Thus, if chaperones with anti-β-aggregation activity contain the amyloid oligomer conformation, we assumed that other proteins known to suppress Aβ aggregation might also contain the amyloid oligomer conformation. One such protein is transthyretin (TTR), which has been shown to sequester Aβ and prevent amyloid formation ##REF##8078889##[13]##, ##REF##12196559##[14]##. We confirmed the suppressive effect of TTR on Aβ aggregation in our model system (##FIG##3##Figure 4A##). Another protein with anti-Aβ-aggregation properties is α2-macroglobulin (α2MG). Although the genetic association between α2MG and AD remains controversial ##REF##9697696##[15]##, the anti-β-aggregation activity of α2MG against Aβ has been reported to occur <italic>in vitro</italic>\n##UREF##0##[16]##, ##REF##9489740##[17]##, as we also observed (##FIG##3##Figure 4B##). As expected, A11 antibody immunostained dot blots of TTR and α2MG (##FIG##3##Figure 4C##), indicating that both proteins possess the amyloid oligomer conformation. Although TTR is a protein that forms amyloid in pathogenic conditions, its A11 immunoreactivity has never been reported ##REF##14981241##[18]##. If a significant number of TTR molecules somehow misfold and bind to A11 in solution, they are more likely to induce or cross-seed Aβ aggregation, as other misfolded pathogenic amyloid oligomers ##REF##12702875##[2]##–##REF##16469881##[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6]##. In our experiment, this did not occur. Instead TTR suppressed Aβ aggregation (##FIG##3##Figure 4A##) ##REF##8078889##[13]##, ##REF##12196559##[14]##, indicating that the amyloid oligomer conformation within TTR is not misfolded but is natively folded.</p>", "<p>Analysis of the crystal structures of the TTR and α2MG receptor-binding domains reveal that both contain a β sandwich with a jelly-roll topology ##REF##201845##[19]##, ##REF##9634697##[20]##. The crystal structures of diphtheria toxin (DT) and exotoxin A (ETA) also have a homologous jelly-roll-like topology ##REF##1589020##[21]##, ##REF##3006045##[22]##. As expected, A11 antibody also immunostained both DT and ETA (##FIG##3##Figure 4C##). Surprisingly, these amyloid oligomer conformation-containing toxins also suppressed Aβ aggregation <italic>in vitro</italic> in a dose-dependent manner, even though they have no known physiological relationship to Aβ (##FIG##3##Figure 4D, E##). These results suggest that A11 immunoreactivity is not only found in chaperones but is also found among certain non-chaperone proteins possessing anti-β-aggregation activity.</p>", "<p>Comparative analyses require control experiments. Thus in the present study, we tested whether A11-immunonegative proteins fail to inhibit β aggregation. Different chain lengths of D-poly glutamic acids (pdE) and L-poly glutamic acids (plE) did not exhibit A11 immunoreactivity or inhibit β aggregation, instead they promoted β aggregation (##FIG##3##Figure 4F–H##). These results support the conformational specificity of A11 antibody binding. Indeed, plE mostly exists as a random coil at neutral pH ##REF##12296715##[23]##, the same pH we used in our control experiment. These results also suggest that A11 reactivity, and thus amyloid oligomer conformation, is not the only functional determinant of β-aggregation-promoting activity. Because we did not test the anti-β-aggregation activity of all purified and folded A11-non-reactive proteins, we could not exclude the possibility that an A11-non-reactive protein possessing anti-β-aggregation activity exists. For the same reason, our current results cannot exclude the possible existence of A11-reactive folded proteins that do not possess anti-β-aggregation activity. Therefore, we do not advance the idea that A11 immunoreactivity is a prerequisite for anti-β-aggregation activity.</p>", "<title>A11 affects GroE-assisted luciferase refolding and Hsp70-decelerated Aβ nucleation</title>", "<p>To determine whether the A11-binding site plays a functional role in protein folding, we first investigated the effects of A11 antibody on GroEL/ES (GroE)-assisted firefly luciferase refolding. Luciferase samples were heated at 45°C for 5 min. After cooling the samples at room temperature for 5 min and adding luciferin substrate, GroEL/ES and ATP facilitated luciferase refolding, which was evident by the increased luminescence in the samples (##FIG##4##Figure 5A##). Luminescence was not observed in heat-treated samples co-incubated with GroEL/ES and ATP in the presence of A11. This suggests that A11 prevented GroEL/ES-assisted refolding of heat-denatured luciferase. Surprisingly, A11 itself assisted luciferase refolding to a similar extent as GroEL/ES.</p>", "<p>Antibody-assisted refolding of proteins, including luciferase, has been reported previously in cases in which an antibody specific to a given protein facilitates antigen folding ##REF##15627377##[24]##, ##REF##15215528##[25]##. Therefore, we surmised that the A11-assisted refolding of luciferase we observed might involve A11 binding to luciferase, which also possesses a β motif ##REF##8805533##[26]##. Thus, we tested the A11 immunoreactivity of purified luciferase by dot blot (##FIG##4##Figure 5B##). The luciferase sample exhibited A11 immunoreactivity that was weaker than that of the positive GroEL control blotted on the same membrane. These results provide a possible explanation for the assisted refolding of luciferase by A11: Like other antibodies having chaperone-like activity, A11 may induce luciferase refolding through specific interactions. If this is the case, then the diminished refolding of luciferase in samples containing both chaperonin and A11 is more likely due to the interaction of A11 with chaperonin rather than the interaction of A11 with luciferase.</p>", "<p>Next, we examined how A11 affects Hsp70-mediated suppression of Aβ aggregation by assessing Aβ aggregation kinetics (##FIG##4##Figure 5C##). The presence of Hsp70 decelerated the nucleation of Aβ aggregation. This effect was suppressed by A11, even though elongation was not substantially affected. This result indicates that the A11-binding site on Hsp70 may play a role in inhibiting nucleation but not elongation during Aβ aggregation. Interestingly, A11 alone failed to substantially affect Aβ aggregation at the concentration used. It will be interesting to examine in further detail whether A11 assists or prevents the oligomerization of Aβ and other amyloidogenic proteins. The present results indicate that A11-binding sites on chaperones may be functionally important.</p>", "<title>Deduction of A11-epitope and amyloid oligomer conformation through structural similarity search</title>", "<p>Our analysis of A11 immunoreactivity and anti-β-aggregation activity of purified proteins led us to identify at least ten A11-reactive folded proteins with reported crystal structures. These included associated protein fragments and corresponding homologues from different species. Information about A11-immunoreactive proteins and their atomic structures may help us deduce the conformation shared by amyloid oligomers. To determine the A11 epitope and thus amyloid oligomer conformation, we searched each of the ten crystal structures for areas sharing the most surface physicochemical properties (see <xref ref-type=\"sec\" rid=\"s4\">Methods</xref>). For the similarity search, we placed two main restrictions according to the known properties of amyloid oligomer conformations. First, the search was limited to only structures having a β strand, which is a generally accepted basic component of amyloid. Second, the search was limited to only areas that are less influenced by amino acid side chains, because A11 immunoreactivity is conformation dependent not amino-acid-sequence dependent ##REF##12702875##[2]##. Areas showing the most similarity are shown in red for each of the ten crystal structures (##FIG##5##Figure 6## and ##SUPPL##0##Figure S1##). All areas were located at the side edge of the β-sheet conformation, which comprises most of larger β-structural motifs such as the β-sandwich motif. Some of the potential A11-binding areas were located at or near the suggested substrate-binding domain of chaperones (e.g., GroEL, Hsp70, Hsc70, Hsp40) ##REF##7935790##[27]##–##REF##14656432##[30]##, while other potential A11-binding areas were not (e.g., ClpB [Hsp104] and Hsp90) ##REF##14567920##[31]##–##REF##16625188##[33]##. The Aβ-binding site of TTR, previously identified through site-directed mutagenesis as well as through molecular modeling ##REF##8078889##[13]##, ##UREF##1##[34]##, partially matched the area proposed here as the A11 epitope. Interestingly, the displacement of this particular terminal β strand by a conformational change promotes fibril formation of TTR itself ##REF##12219081##[35]##. These observations support the hypothesis that the β-sheet edge, a common feature of the ten crystal structures we assessed, plays a key role in A11-reactive proteins. Indeed, the negative design of the β-sheet edge supposedly helps natural β-sheet proteins to avoid β aggregation ##REF##11880627##[36]##. Here, we propose that β-sheet edge in natively folded amyloid oligomers not only protects proteins from undergoing β aggregation but also prevents interacting molecules from undergoing β aggregation. Thus, in some cases this can be considered to be a positive design. How the amyloid oligomer conformation influences protein conformation still needs to be determined in the future. This can be achieved by investigating the detailed molecular interactions between chaperone/chaperone-like proteins and the A11 antibody and between these proteins and amyloidogenic proteins.</p>" ]
[ "<title>Discussion</title>", "<p>By using the A11 antibody, an antibody known to detect the conformation of amyloid oligomers ##REF##12702875##[2]##, we unexpectedly observed the amyloid oligomer conformation in a group of proteins whose folds were functionally characterized as native on the basis of their anti-β-aggregation activity. This contrasts with the misfolded pathogenic amyloid oligomers that promote β aggregation ##REF##12702875##[2]##–##REF##16469881##[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6]##. Because we propose a novel corollary to the original claim that A11 recognizes a common structure shared by oligomeric intermediates formed by a diverse range of amyloidogenic proteins ##REF##12702875##[2]##, our results were carefully examined.</p>", "<p>Could the recognition by A11 of the identified proteins be a non-specific effect caused, for example, by a small fraction of partially misfolded A11 antibody? We considered that this was unlikely because heat-denatured Hsp27 did not bind A11 (##FIG##2##Figure 3##). A11 reacted with some non-chaperone proteins (##FIG##3##Figure 4##). Moreover, none of the identified A11-immunoreactive proteins bound secondary antibody in samples lacking A11 (data not shown).</p>", "<p>Could the anti-β-aggregation activity of the identified proteins be induced by colloidal or crowding effects? We considered this to be unlikely because Hsp70/40 exhibited energy-dependent anti-β-aggregation activity (##FIG##1##Figure 2E##). Heat-treated Hsp27 (##FIG##2##Figure 3C##) and polyglutamate (##FIG##3##Figure 4G,H##) did not show the anti-β-aggregation activity. In addition, crowded GroE/A11 mixtures did not facilitate luciferase refolding more than less crowded GroE or A11 solutions (##FIG##4##Figure 5A##). Along similar lines, crowded Hsp70/A11 mixtures did not enhance anti-β-aggregation activity more than less crowded Hsp70 solutions (##FIG##4##Figure 5C##).</p>", "<p>Could A11 be recognizing traces of misfolded amyloidogenic intermediates of these proteins? We considered that this was unlikely because if the contaminating molecules of such misfolded amyloid intermediates were substantially present in the sample, then we would expect the A11-reactive proteins, especially non-chaperone proteins like toxins, not to suppress β aggregation (##FIG##3##Figure 4D, E##). Rather, we would expect them to promote β aggregation as Aβ oligomers did (##FIG##1##Figure 2A##), because misfolded amyloid oligomers characteristically promote oligomerization and fibrillization of other proteins, a suggested pathogenic mechanism underlying protein misfolding diseases ##REF##12702875##[2]##–##REF##16469881##[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6]##. A functional property that was commonly observed in the group of A11-reactive proteins was anti-β-aggregation activity, which differentiated these proteins from misfolded pathogenic amyloid oligomers, which promote β-aggregation. Together with the conformation-specific nature of A11 reactivity, this phenomenon led us to conclude that a significant number of molecules in the purified samples were neither misfolded nor unfolded, but were most likely natively folded.</p>", "<p>Does the notion of a “common structure” mean anything if it so diverse it encompasses the folded and misfolded proteins? Under the right conditions, any protein can form amyloid ##REF##11242064##[1]##. The formation of amyloid structures by poly amino acids supports the premise that amyloid formation occurs independent of specific side-chain contacts or amino acid sequences ##REF##12411486##[37]##. Although plE forms amyloid at pH 4.1, it exists mostly as a random coil at physiological pH ##REF##12296715##[23]##. Indeed, plE dissolved in a neutral pH buffer showed neither A11 immunoreactivity nor anti-β-aggregation activity, which is consistent with the conformational specificity of A11 binding (##FIG##3##Figure 4F–H##). The difference in the staining patterns in immunoblots probed with A11 and in CBB-stained gels of bacterial cell lysate samples (##FIG##0##Figure 1A##) indicates that A11 does not bind just any protein. Not all A11-reactive proteins in the bands were necessarily natively folded or retained their <italic>in vivo</italic> conformation, since lysing the cells can significantly affect the conformation of some proteins. We believe that A11 specificity does exist, as demonstrated in the original study ##REF##12702875##[2]##. It is most important to note, however, that the specificity of A11 is completely different from that of other antibodies that recognize specific amino acid sequences in that A11 reactivity depends on the three-dimensional conformation of a protein since A11 specifically binds oligomeric intermediates but not unfolded monomers or fibers of amyloidogenic proteins ##REF##12702875##[2]##. Thus, A11 reactivity is independent of an antigen's amino acid sequence, because A11 specifically binds oligomeric intermediates formed by a diverse range of amyloidogenic proteins. The conformational-dependent binding specificity of A11 also implies that the amyloid oligomer represents a generic conformation ##REF##12702875##[2]##. On the basis of this particular generic property of the A11 antibody, we believe that A11 antibody is also capable of binding a group of natively folded proteins, as we observed in the present study.</p>", "<p>What is the functional significance of having an amyloid oligomer conformation within natively folded proteins? Function-blocking experiments clarified the functional importance of A11-binding sites on chaperones (##FIG##4##Figure 5##). To further tackle this issue structurally, we took a computational approach. A functional property shared by pathogenic amyloid oligomers and chaperones is that both affect the conformation of unfolded proteins. This property might depend on the structural plasticity that is shared by amyloid and the substrate-binding domain of chaperonin, called the apical domain ##REF##9811807##[38]##–##REF##12065585##[41]##. The structural plasticity of amyloid is partially based on its polymorphism, the ability of one polypeptide to form aggregates of different structures ##REF##17198370##[39]##. Indeed, the polymorphic nature of amyloid sets it apart from other globular folded proteins with unique structures. Thus, it is unlikely that the amyloid oligomer conformation is represented by a unique structure.</p>", "<p>On the other hand, amyloid also exhibits isomorphism, the ability of different polypeptides to display similar morphologies ##REF##12702875##[2]##, ##REF##17198370##[39]##. Taking advantage of this property, we searched for areas containing similar physicochemical properties on the surfaces of the crystal structures of A11-reactive folded proteins (##FIG##5##Figure 6## and ##SUPPL##0##Figure S1##). Our analysis results were consistent with the proposed structures of amyloid protofilaments or precursors ##REF##17060612##[42]##–##REF##16302961##[46]##. Although the non-native amyloid precursor of β2-microglobulin is highly native-like, its edge strands contain perturbations that normally protect β-sandwich proteins from self-association ##REF##15811375##[43]##–##REF##16302961##[46]##. Structurally, the edges of a completely regular β-sandwich motif are inherently β-aggregation prone ##REF##11880627##[36]##. Coincidentally, our structural similarity search revealed that the β-sheet edge of GroEL was located in the apical domain. A pioneering study on GroEL suggested that chaperonins act by stabilizing non-native intermediates against off-pathway misfolding and aggregation ##REF##7935790##[27]##. Because β-sheet structures are primarily formed through main-chain hydrogen bonds ##REF##14685248##[4]##, ##REF##11884745##[6]##, the induction of β-strand formation in a substrate through the β-sheet edge in the apical domain of GroEL may normally function to prevent non-specific side-chain contacts and thus non-specific aggregation, but in certain cases promote β aggregation as reported previously ##REF##9391131##[47]##, ##REF##11697909##[48]##. We consider this idea to be speculative but consistent with the formation of β-strands by non-native substrates in a complex structure with Hsp40, and consistent with Hsp40-dependent amyloid assembly in cells ##REF##14656432##[30]##, ##REF##18480252##[49]##.</p>", "<p>Finally, we would like to mention evolutionary aspects of ‘native’ amyloid oligomers. The presence of the amyloid oligomer conformation within a natively folded protein indicates that the folding observed in this type of conformation is based on amino acid sequence, which is encoded in that particular protein's gene according to Anfinsen's hypothesis ##REF##4124164##[11]##. On the other hand, the unprecedented diversity of proteins containing amyloid oligomer conformations also indicates that the folding in these conformations may be caused by something else, especially in pathogenic conditions ##REF##11242064##[1]##, ##REF##12702875##[2]##, ##REF##14685248##[4]##. In evolution, aggregative properties of some proteins may have pressured the selection of anti-aggregation properties in some genes as a preventive measure. On the basis of the generic anti-aggregation characteristics of certain A11-reactive folded proteins, such as the chaperone family, we hypothesize that (1) the amyloid oligomer conformation has evolved over time as a means to prevent non-specific protein aggregations, and that (2) based on the structural commonality, amyloidogenic proteins may sporadically deviate from this system of preventing protein aggregation in some conditions such as aging in the extended life span of humanity today that has not been caught up by evolution.</p>" ]
[]
[ "<p>Conceived and designed the experiments: YY RM YM YRC TK. Performed the experiments: YY RM. Analyzed the data: YY RM YM YRC TK AT. Wrote the paper: YY RM AT.</p>", "<p>Recent in vitro and in vivo studies suggest that destabilized proteins with defective folding induce aggregation and toxicity in protein-misfolding diseases. One such unstable protein state is called amyloid oligomer, a precursor of fully aggregated forms of amyloid. Detection of various amyloid oligomers with A11, an anti-amyloid oligomer conformation-specific antibody, revealed that the amyloid oligomer represents a generic conformation and suggested that toxic β-aggregation processes possess a common mechanism. By using A11 antibody as a probe in combination with mass spectrometric analysis, we identified GroEL in bacterial lysates as a protein that may potentially have an amyloid oligomer conformation. Surprisingly, A11 reacted not only with purified GroEL but also with several purified heat shock proteins, including human Hsp27, 40, 70, 90; yeast Hsp104; and bovine Hsc70. The native folds of A11-reactive proteins in purified samples were characterized by their anti-β-aggregation activity in terms of both functionality and in contrast to the β-aggregation promoting activity of misfolded pathogenic amyloid oligomers. The conformation-dependent binding of A11 with natively folded Hsp27 was supported by the concurrent loss of A11 reactivity and anti-β-aggregation activity of heat-treated Hsp27 samples. Moreover, we observed consistent anti-β-aggregation activity not only by chaperones containing an amyloid oligomer conformation but also by several A11-immunoreactive non-chaperone proteins. From these results, we suggest that the amyloid oligomer conformation is present in a group of natively folded proteins. The inhibitory effects of A11 antibody on both GroEL/ES-assisted luciferase refolding and Hsp70-mediated decelerated nucleation of Aβ aggregation suggested that the A11-binding sites on these chaperones might be functionally important. Finally, we employed a computational approach to uncover possible A11-binding sites on these targets. Since the β-sheet edge was a common structural motif having the most similar physicochemical properties in the A11-reactive proteins we analyzed, we propose that the β-sheet edge in some natively folded amyloid oligomers is designed positively to prevent β aggregation.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>We thank Professor C. G. Glabe and his colleagues, Professor M. Taiji, and Takashima laboratory members for great advice and critical discussions. We are grateful for the support of BSI's Research Resources Center for MS-MS analysis.</p>" ]
[ "<fig id=\"pone-0003235-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003235.g001</object-id><label>Figure 1</label><caption><title>Amyloid oligomer conformation in chaperones.</title><p>(A) <italic>E. coli</italic> competent cells (strain DH5α; TOYOBO, Japan) were freeze-thawed three times by freezing the cells on ice at −180°C then thawing them at 37°C. The lysates were then mixed with sample buffer lacking SDS and reducing agents and then subjected to PAGE analysis. One gel was immunoblotted with A11 antibody. A11-immunoreactive bands (<italic>A–G</italic>) were excised from the CBB-stained gel, treated with trypsin, and analyzed by tandem-mass spectrometry. A candidate A11-reactive protein from band <italic>B</italic> was GroEL. The difference in staining patterns between the A11 immunoblot and CBB-stained gel indicates A11 antibody-binding selectivity. Note also that the proteins in cell lysates reacted with the A11 antibody are not necessarily all natively folded. Purified GroEL (23 µM) was mixed with sample buffer lacking both reducing agents and SDS, and then run on (B) a gel in a regular SDS-containing buffer or (C) a gel in a buffer lacking SDS (native PAGE). Gels were blotted onto a membrane that was subsequently probed with A11 under the same conditions. Note that the mobility of the molecular weight standard is different in (B) and (C). (D) The following solutions (3 µL each) were dot blotted onto a nitrocellulose membrane: 20 µM Aβ40 monomer, 20 µM Aβ40 oligomer, H<sub>2</sub>O, purified 23 µM GroEL, 37 µM Hsp27, 20 µM Hsp40, 14 µM Hsp70, 12 µM Hsp90, 10 µM Hsp104, and 10 µM Hsc70. GroEL, Hsp27, Hsp40, Hsp90, and Hsc70 were purchased from Stressgen (Canada). Hsp70 was from Sigma (USA). Hsp104 was from ATGen (South Korea). Each protein solution was blotted onto a single membrane that was probed with A11 under the same conditions. The individual dot blots were arranged into a single column.</p></caption></fig>", "<fig id=\"pone-0003235-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003235.g002</object-id><label>Figure 2</label><caption><title>Contrasting effects of Aβ oligomer and chaperones on Aβ aggregation.</title><p>(A) Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with 0, 0.5, 5, or 50 nM Aβ40 oligomer (i.e., “seed”) in H<sub>2</sub>O. Results from a sample containing only 50 nM Aβ40 oligomer is also shown. ThT fluorescence intensity indicates the level of Aβ aggregation. (B) Aβ40 (final concentration: 10 µM) dissolved in H<sub>2</sub>O was mixed with 0, 10, 100, or 1000 nM Hsp27 in buffer (1.08 mM Tris-HCl [pH 7.5], 541 µM NaCl, 54.1 µM EDTA, and 54.1 µM DTT). (C) Aβ40 (final concentration: 10 µM) dissolved in H<sub>2</sub>O was mixed with 0, 10, 100, or 1000 nM Hsp70 in buffer (2.7 mM KCl, 1.5 mM KH<sub>2</sub>PO<sub>4</sub>, 137 mM NaCl, and 8.1 mM Na<sub>2</sub>HPO<sub>4</sub>). (D) Aβ40 (final concentration: 5 µM) dissolved in H<sub>2</sub>O was mixed with 0, 5, 50, or 500 nM Hsp90 in buffer (3.9 mM Tris-HCl [pH 7.5], 7.8 mM NaCl, and 78.2 µM DTT). (E) Anti-β-aggregation activity of Hsp40 and Hsp70. Aβ40 (5 µM) dissolved in aCSF was mixed with either 10 nM Hsp70 in buffer A (15.1 µM KCl, 8.4 µM KH<sub>2</sub>PO<sub>4</sub>, 767 µM NaCl, and 45.3 µM Na<sub>2</sub>HPO<sub>4</sub>) or a mixture of 10 nM Hsp70 in Buffer A and 10 nM Hsp40 in Buffer B (10.8 µM KCl, 6.0 µM KH<sub>2</sub>PO<sub>4</sub>, 548 µM NaCl, and 32.4 µM Na<sub>2</sub>HPO<sub>4</sub>) in the presence or absence of 500 µM Mg-ATP. (F) Bar graph showing ThT fluorescence intensity of Aβ samples with or without chaperones after 17 h of incubation. Statistical significance was calculated by using a Student's t-test. *p = 0.035; **p = 0.00046. Data are presented as means+/−SEM.</p></caption></fig>", "<fig id=\"pone-0003235-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003235.g003</object-id><label>Figure 3</label><caption><title>Effects of heat treatment on amyloid oligomer conformation in Hsp27 and the anti-β-aggregation activity of Hsp27.</title><p>(A) Western blot of Hsp27 probed with A11 anti-amyloid oligomer conformation-dependent antibody. Hsp27 samples were heat treated at 80°C for 15 or 30 min. Heat-treated and untreated Hsp27 samples (10 µM) were mixed with sample buffer lacking SDS and reducing reagents, subjected to native PAGE using 4–20% gels, and separated proteins were immunoblotted with A11 antibody. Note that A11-immunoreactive Hsp27 bands migrated to a high molecular-weight level in native PAGE, because Hsp27 exists as large oligomers having chaperone-like activity ##REF##15611723##[8]##, ##REF##9029143##[12]##. (B) Heat-treated and untreated Hsp27 samples (10 µM) were mixed with sample buffer containing SDS and β-mercaptoethanol, and then subjected to SDS-PAGE using 4–20% gels. Gels were stained with coomassie brilliant blue (CBB). (C) Aβ-aggregation kinetics of samples containing heat-treated Hsp27. One buffer sample and three Hsp27 samples were diluted 100 times with H<sub>2</sub>O. Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with buffer (54 µM Tris-HCl [pH 7.5], 27 µM NaCl, 2.7 µM EDTA, and 2.7 µM DTT); untreated 50 nM Hsp27; 15-min heat-treated Hsp27; or 30-min heat-treated Hsp27. All samples were incubated in the presence of 10 µM ThT; triplicate measurements were taken every 30 min at excitation and emission wavelengths of 440 nm and 486 nm ##REF##8453378##[7]##, respectively. Data are presented as means+/−SEM.</p></caption></fig>", "<fig id=\"pone-0003235-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003235.g004</object-id><label>Figure 4</label><caption><title>Dose-dependent suppression of Aβ aggregation by A11-immunoreactive non-chaperone proteins.</title><p>(A) Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with 0, 50, 500, or 5000 nM TTR in 10 mM Tris-HCl (pH 7.4) with 0.9% NaCl. ThT fluorescence indicates the level of Aβ aggregation. (B) Aβ40 (final concentration: 5 µM) dissolved in H<sub>2</sub>O was mixed with 0, 5, 50, or 500 nM α2MG in buffer (1.45 mM Tris-HCl [pH 8.0], 9.4 mM glycine, and 5.8 mM trehalose). (C) Dot blot of 100 µM TTR, 13.8 µM α2MG, 17.2 µM DT, and 15.2 µM ETA (3 µl each) probed with A11 antibody ##REF##12702875##[2]##. (D) and (E) Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with buffer alone; 5, 50, or 500 nM DT; or ETA. For (D), the buffer contained 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. For (E), the buffer contained 10 mM sodium phosphate (pH 7.5) and 150 mM NaCl. (F) Dot blot of 100 µM pdE (approx. MW: 50 kDa); 10 mM plE (approx. MW: 1000 Da); and 50 µM Aβ40 oligomer (3 µl each) probed with A11, secondary antibody alone without primary antibody, and 4G8 antibody (which reacts to amino acid residues 17–24 of human Aβ). (G) and (H) Aβ40 (final concentration: 5 µM) dissolved in PBS was mixed with buffer alone; 0.05, 0.5, 5 µM pdE; or plE. Data are presented as means+/−SEM.</p></caption></fig>", "<fig id=\"pone-0003235-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003235.g005</object-id><label>Figure 5</label><caption><title>A11-mediated inhibition of GroE-assisted luciferase refolding and Hsp70-mediated decelerated Aβ nucleation.</title><p>(A) In a sample of GroE (final concentrations: 1.25 µM GroEL and 2.1 µM GroES) and luciferase (final concentration: 100 nM), about 30% of luciferase activity was recorded relative to the activity of luciferase samples without heat-treatment, indicating that GroE-assisted partial refolding of heat-denatured luciferase had occurred. Luciferase activity was not observed when heat-treated luciferase was co-incubated with both GroE and A11 (final concentration: 0.1 mg/mL). A11 antibody assisted the refolding of luciferase (n = 15 for GroE and n = 6 for GroE/A11 and A11 alone, respectively). Data are presented as means+/−SEM. (B) Three microliters of 250 nM GroEL, 2.5 µM luciferase, and H<sub>2</sub>O were blotted onto a nitrocellulose membrane. The membrane was incubated either with the A11 antibody in 5% skim milk or with just skim milk. (C) Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with or without Hsp70 (final concentration: 75 nM) and A11 (final concentration: 300 nM). All samples were incubated in the presence of 5 µM ThT. Triplicate measurements were taken every 30 min. Average fluorescence values are plotted. Error bars were omitted from the graph for clarity.</p></caption></fig>", "<fig id=\"pone-0003235-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003235.g006</object-id><label>Figure 6</label><caption><title>Sites with physicochemical properties of greatest similarity among ten A11-reactive proteins.</title><p>The crystal structures of ten A11-reactive proteins were aligned to identify the areas where the physicochemical properties of solvent-exposed atoms show the highest similarity (red). (A) GroEL [1aon]. (B) α2MG [1ayo]. (C) Hsp40 [1c3g]. (D) DnaK (Hsp70) [1dkz]. (E) TTR (prealbumin) [1dvq]. (F) ETA [1ikq]. (G) DT [1mdt]. (H) ClpB (Hsp104) [1qvr]. (I) Hsc70 [1yuw]. (J) Hsp90 [2cg9]. The PDB codes of the structures used in the structural similarity search are shown in square brackets.</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003235-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003235.t001</object-id><label>Table 1</label><caption><title>Potential surface sites on ten A11-reactive proteins in which the main β-sheet chain is exposed to the surface.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PDB code</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of sites</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1aon</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">114</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1ayo</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1c3g</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1dkz</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1dvd</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1ikq</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1mdt</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1qvr</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1yuw</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2cg9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">214</td></tr></tbody></table></alternatives></table-wrap>" ]
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[]
[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003235.s001\"><label>Figure S1</label><caption><p>Sites having the most similar physicochemical properties in the molecular structures of ten A11-reactive proteins. Because the aligned site in 1aon was hidden from view at the viewing angle we used for the other structures, only 1aon is shown from a different angle from other molecules. (A) GroEL [1aon]. (B) α2MG [1ayo]. (C) Hsp40 [1c3g]. (D) DnaK (Hsp70) [1dkz]. (E) TTR (prealbumin) [1dvq]. (F) ETA [1ikq]. (G) DT [1mdt]. (H) ClpB (Hsp104) [1qvr]. (I) Hsc70 [1yuw]. (J) Hsp90 [2cg9].</p><p>(8.92 MB TIF)</p></caption></supplementary-material>" ]
[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>This work was partly supported by Grant-in-Aid for Scientific Research on Priority Areas (Research on Pathomechanisms on Brain Disorders) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"pone.0003235.s001.tif\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["16"], "element-citation": ["\n"], "surname": ["Hughes", "Khorkova", "Goyal", "Knaeblein", "Heroux"], "given-names": ["SR", "O", "S", "J", "J"], "year": ["1995"], "article-title": ["\u03b12-macroglobulin associates with \u03b2-amyloid peptide and prevents fibril formation."], "source": ["Proc Natl Acad Sci USA"], "volume": ["95"], "fpage": ["3275"], "lpage": ["3280"]}, {"label": ["34"], "element-citation": ["\n"], "surname": ["Schwarzman", "Goldgaber"], "given-names": ["AL", "D"], "year": ["1994"], "article-title": ["Interaction of transthyretin with amyloid \u03b2-protein: binding and inhibition of amyloid formation."], "source": ["Ciba Found Symp"], "volume": ["199"], "fpage": ["146"], "lpage": ["160"]}, {"label": ["45"], "element-citation": ["\n"], "surname": ["Eakin", "Berman", "Miranker"], "given-names": ["CM", "AJ", "AD"], "year": ["2006"], "article-title": ["A native to amyloidogenic transition regulated by a backbone trigger."], "source": ["Nat Sruct Mol Biol"], "volume": ["13"], "fpage": ["202"], "lpage": ["208"]}, {"label": ["50"], "element-citation": ["\n"], "surname": ["Bush", "Sheridan"], "given-names": ["BL", "RP"], "year": ["1993"], "article-title": ["PATTY: A Programmable Atom Typer and Language for Automatic Classification of Atoms in Molecular Databases."], "source": ["J Chem Inf Comput Sci"], "volume": ["33"], "fpage": ["756"], "lpage": ["762"]}]
{ "acronym": [], "definition": [] }
50
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 18; 3(9):e3235
oa_package/01/93/PMC2528939.tar.gz
PMC2528940
18802457
[ "<title>Introduction</title>", "<p>\n<italic>Cryptococcus neoformans</italic> is an opportunistic fungal pathogen that causes life-threatening meningoencephalitis primarily in immunocompromised patients ##UREF##0##[1]##. <italic>C. neoformans</italic> is an obligate aerobe and its natural environment includes pigeon droppings, soil contaminated with avian guano ##UREF##0##[1]## and decaying tree barks ##REF##8732358##[2]##,##REF##8912173##[3]##. In laboratory conditions, atmospheric levels of oxygen (∼21%) are required for optimal growth of <italic>C. neoformans</italic> and lower oxygen concentrations lead to a significant reduction in cell growth ##REF##7790475##[4]##. Upon inhalation, <italic>C. neoformans</italic> disseminates to central nervous system and causes life-threatening meningoencephalitis mostly in patients with immune deficiency. It is well known that oxygen concentrations in the human brain and other anatomical sites are significantly lower compared to atmospheric levels ##REF##11718758##[5]##. Thus, in order to establish infection in the brain, <italic>C. neoformans</italic> needs to sense and adapt to low oxygen conditions. Even though the mechanisms involved in oxygen sensing and adaptation to low oxygen conditions have been studied in humans and other organisms, this important aspect towards understanding the pathobiology of <italic>C. neoformans</italic> remains elusive.</p>", "<p>In most eukaryotic organisms, molecular oxygen is essential for oxidative phosphorylation and biosynthetic processes. To survive in low oxygen conditions or hypoxia, organisms have evolved oxygen-sensing mechanisms that activate a complex set of responses. In mammals, a major effector of the hypoxic transcriptional response is the hypoxia inducible factor (HIF1α). Under high oxygen conditions, HIF1α is continuously degraded through hydroxylation while in low oxygen conditions, it is not hydroxylated thus avoiding degradation and activating the target genes ##REF##2849206##[6]##–##REF##11598268##[9]##.</p>", "<p>In mammalian systems, cobalt chloride has been widely used as the hypoxia-mimicking agent. Studies done so far have shown that the CoCl<sub>2</sub> mediated hypoxia-mimicking response is induced through the stabilization of HIF1α in the presence of oxygen ##REF##2825172##[10]##–##REF##8387214##[13]##. The absence of <italic>HIF1</italic> homolog in <italic>Saccharomyces cerevisiae</italic> indicates a different mode of oxygen sensing ##REF##17555402##[14]##. While CoCl<sub>2</sub> has been shown to have pleiotropic effects on cellular mechanisms in fungi, only a few of those have been linked to oxygen sensing ##REF##13229372##[15]##–##REF##13577873##[17]##.</p>", "<p>Recent work from our laboratory has established a link between sterol synthesis, oxygen sensing and CoCl<sub>2</sub> sensitivity in <italic>C. neoformans</italic>\n##REF##17645443##[18]##,##REF##17462012##[19]##. Under low sterol or low oxygen conditions, <italic>C. neoformans</italic> homologs of the mammalian SREBP (sterol regulatory element-binding protein) transcription factor and its binding partner SCAP (SREBP cleavage-activating protein), named Sre1 and Scp1 respectively, regulate the expression of several genes involved in ergosterol biosynthesis and iron homeostasis. Mutations in <italic>SRE1</italic> and <italic>SCP1</italic> genes resulted in reduced growth under low oxygen condition and these mutants were not able to establish infection in the mouse brain ##REF##17462012##[19]##. Interestingly, both <italic>sre1</italic> and <italic>scp1</italic> mutants show reduced growth on media containing CoCl<sub>2</sub>. Further characterization of these mutants demonstrated that the response to CoCl<sub>2</sub> in <italic>C. neoformans</italic> mimics certain aspects of the low oxygen condition by targeting enzymes in the sterol biosynthetic pathway ##REF##17645443##[18]##.</p>", "<p>In <italic>C. neoformans</italic>, apart from the genes involved in ergosterol biosynthesis, not many other genes have been identified that are required for adaptation to low oxygen as well as to high concentration of CoCl<sub>2</sub>. To identify genes involved in low oxygen response, our laboratory has taken two different approaches; 1) screening for the mutants directly under low oxygen conditions (manuscript in preparation) and 2) screening for the mutants that are sensitive to hypoxia-mimicking agent. In this study, we have explored the possibility of using CoCl<sub>2</sub> as an effective chemical to screen for other factors involved in oxygen sensing. Using a genetic screening approach, we have isolated cobalt chloride hypersensitive mutants. Through characterization of these mutants, we have shown a link between the ability of cells to respond to CoCl<sub>2</sub> and/or low oxygen conditions and mitochondrial function, sterol homeostasis, sensitivity to reactive oxygen species (ROS) and ubiquitination.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Strains, media, and growth conditions</title>", "<p>\n<italic>C. neoformans</italic> serotype D genomic sequencing strain; B-3501A (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www-sequence.stanford.edu/group/C.neoformans/index.html\">http://www-sequence.stanford.edu/group/C.neoformans/index.html</ext-link>) was used as the wild type strain. All the strains in this study were derived from this strain. The strains were maintained on YES and YES+geneticin (100 µg/ml) where necessary. YES medium consists of 0.5% (w/v) yeast extract, 2% glucose and supplements containing uracil, adenine, leucine, histidine and lysine (225 µg/ml). For CoCl<sub>2</sub> screening and sensitivity assays YES+0.7 mM CoCl<sub>2</sub> was used. For all FACS experiments and O<sub>2</sub> consumption assays yeast cells were grown to log phase (OD<sub>600</sub> = 0.5) at 30°C in YES medium then 1 mM CoCl<sub>2</sub> was added and grown further for 4 hrs at 30°C. Low-oxygen conditions (1% O<sub>2</sub>) were maintained using an Invivo<sub>2</sub> 400 workstation (Ruskinn) at 37°C.</p>", "<title>Construction and screening of library for CoCl<sub>2</sub>-sensitive mutants</title>", "<p>Since the frequency of random integration is very high by <italic>Agrobacterium tumefaciens</italic> mediated transformation (ATMT), a T-DNA insertion library of <italic>C. neoformans</italic> serotype D genomic strain (B-3501A) was made using ATMT ##REF##16260158##[20]##. The plasmid pYCC716 containing T-DNA fragment was used to create <italic>A. tumefaciens</italic> strain C603. This plasmid has a <italic>NEO</italic> gene conferring geneticin resistance. ATMT of <italic>C. neoformans</italic> was carried out as previously described ##REF##16260158##[20]##. To make a library, 30,000 individual transformants were picked and inoculated in 96 well plates containing YPD+50 µg/ml geneticin+200 µM cefotaxime. After 48 hr of growth at 30°C, glycerol stocks were made and stored at −80°C.</p>", "<p>To identify the genes involved in the sensitivity to CoCl<sub>2</sub>, we screened T-DNA insertion library of <italic>C. neoformans</italic>. The primary screening for mutants sensitive to CoCl<sub>2</sub> was carried out by replica spotting of the library consisting of 30,000 clones on YES+0.7 mM CoCl<sub>2</sub> and YES+geneticin plates. Mutants that failed to grow on CoCl<sub>2</sub> medium were selected for further analysis. A total 37 mutants (including 5 redundant isolates) were selected based on their sensitivity to CoCl<sub>2</sub>. Transformants selected for further analyses were streaked on selective YES+geneticin+cefotaxime medium.</p>", "<title>Mapping of T-DNA insertion site</title>", "<p>Isolation and analysis of genomic DNA was carried out as described previously ##REF##8007987##[21]##. To identify the number of insertion events in these transformants, Southern hybridization was done using the ORF of the <italic>NEO</italic> gene as a probe. Radioactive probes were prepared using StripEZ kit (Ambion, Austin, TX) according to manufacturer's manual. By using vectorette system (Sigma, Woodlands, TX), T-DNA insertion site was mapped in <italic>C. neoformans</italic> mutants and genomic sequence flanking the insertion site was obtained. BLAST analysis of these sequences was done to reveal which loci have been affected.</p>", "<title>Construction of complementation strains</title>", "<p>The wild-type gene for each affected gene was PCR amplified from B-3501A, cloned and sequenced. For complementation, using biolistic transformation method ##REF##8444802##[22]## T-DNA insertion mutants were transformed with the respective wild-type gene using <italic>NAT</italic> selection marker. Stable transformants were selected after repeated transfer on YES agar. PCR was used to identify integrative transformants containing an intact wild type gene and Southern blot analysis was used to confirm the transformation.</p>", "<title>Growth assays</title>", "<p>Plate assays were carried out to determine if the mutants are sensitive to an external source of ROS-generating chemicals. YES plates containing 25 mM of sodium dihydrogen citrate, pH 4.0 (buffered YES) were made with 0.5 mM of H<sub>2</sub>O<sub>2</sub> and 1mM of NaNO<sub>2</sub>. For other chemicals such as paraquat (0.25 mM) and diethyl maleate (2 mM), plates were made using regular YES. Cultures were either streaked or spotted and incubated at 30 and 37°C. For growth phenotype analysis serial dilutions of cultures were spotted on YES plates and incubated at 30°C, 37°C in ambient oxygen level and at 37°C under 1% O<sub>2</sub> for 3–4 d.</p>", "<title>Confocal microscopy</title>", "<p>Cells were grown overnight in YES medium, diluted in fresh YES to obtain initial OD<sub>600</sub> of 0.2–0.3 and incubated for an additional 4 h. After 4 h, cells were harvested and resuspended in YES medium containing the MitoTracker Red CMXRos stain (Molecular probes, Eugene, OR) in the final concentration of 100 nM. Cells were incubated at 30°C for 1 h. After staining, cells were washed 3 times with 1× PBS and resuspended in 1× PBS. For mitochondrial DNA staining, cells were suspended in 10 mM HEPES buffer, pH 7.4, containing 5% glucose. SYTO 18 mitochondrial stain (Molecular probes, Eugene, OR) was added to a final concentration of 10 µM and cells were incubated at room temperature for 15–20 min. Before proceeding for microscopy, cells were washed and suspended in HEPES buffer. Images were collected on a Leica SP5 confocal microscope (Leica Microsystems, Exton, PA, USA) using a 100× oil immersion objective NA 1.4 zoom 4. Fluorochromes were excited using an Argon laser for SYTO18 and a 561 nm diode laser for MitoTracker Red CMXRos. Differential interference contrast (DIC) images were collected simultaneously with the fluorescence images using the transmitted light detector. Images were processed using Leica LAS-AF software (version 1.8.2 build 1465).</p>", "<title>ROS measurement</title>", "<p>Cells were grown overnight in YES medium at 30°C, diluted in fresh YES and allowed to grow till OD<sub>600</sub> reaches 0.4–0.5. Dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes, Eugene, OR) at a final concentration of 10 µM was added for an additional 2 h to load the dye into cells. After 2 h, one set was supplemented with 1 mM CoCl<sub>2</sub> and both sets were further incubated at 30°C for 4 h. Cells were harvested, washed 3 times with 1× PBS and an equal number of cells (2×10<sup>6</sup>) were resuspended in 1 ml of PBS. Fluorescence was then analyzed using a Becton- Dickinson FACScan Flow Cytometer. The parameters for Fluorescence-activated cell sorting (FACS) were set at excitation of 488 nm and for emission FITC channel using standard FL-1 filter (530/30 nm).</p>", "<title>Oxygen consumption assays</title>", "<p>Cells were grown overnight in YES medium at 30°C, diluted in fresh YES to get starting OD<sub>600</sub> of 0.2–0.3 and incubated for an additional 4 h. After 4 h, one set was supplemented with 1 mM CoCl<sub>2</sub> and both sets were further incubated at 30°C for 4 h. Cells were harvested and washed with PBS and an equal number of cells (5×10<sup>7</sup>) were resuspended in 2 ml of YES and YES+CoCl<sub>2</sub>. Cells were then loaded into a sealed 2.0 ml glass chamber. Changes in oxygen tension were measured at room temperature with a Clark-type oxygen electrode, and the respiratory rate was calculated as a change in oxygen tension over time. In order to quantify mitochondrial respiration oxygen consumption, respiratory inhibitor Antimycin A (1 µg/ml) was used to block mitochondrial respiration.</p>", "<title>Gene expression profiling</title>", "<p>Microarray slides were purchased from an academic consortium co-ordinated by T. Doering, C. Hull and J. Lodge at the University of Washington-St Louis. Arrays contain 7,738 70 bp DNA oligomers designed to uniquely recognize each gene in the <italic>C. neoformans</italic> serotype D genome plus control oligomers. Each oligomer is printed in duplicate. Overnight cultures of wild-type (B-3501A) strain was refreshed and grown in YES for 5 h and incubated for an additional 2 h in the presence or absence of 0.6 mM CoCl<sub>2</sub>. Wild-type strain does not show any growth inhibition at this concentration when treated only for 2 h. RNA was extracted from yeast cells using Trizol (Invitrogen, Carlsbad, CA), treated with RNAse-free DNase (Ambion, Austin, TX) for the removal of genomic DNA, and purified with RNeasy MinElute cleanup kit (Qiagen, Valencia, CA). RNA was labeled and hybridized as described before ##REF##17645443##[18]##. Data were collected using a GenePix 4000B scanner. For data analysis, features were examined and flagged using Genepix Pro 6.0 software (Axon Instruments, Foster City, CA) and confirmed manually. Data were further analyzed in mAdb database at <ext-link ext-link-type=\"uri\" xlink:href=\"http://mAdb.niaid.nih.gov\">http://mAdb.niaid.nih.gov</ext-link> (NIAID). Three biological repeats were performed using three independent RNA sets isolated from cells cultured on different days and the dye-reverse hybridizations were performed for all 3 sets. One set of RNA was also subjected to technical repeats. All statistically significant genes were identified by significance analysis of microarrays (SAM) using a mean false discovery rate (FDR) of 5% ##REF##11309499##[23]##. Only statistically significant genes were used for data analysis. To simplify presentation of the results, ##FIG##7##Figure 8## shows categorization of statistically significant genes identified by SAM, that have average changes greater than 2-fold. ##SUPPL##0##Table S1## lists the statistically significant genes identified by SAM that have average changes of greater than 2-fold in the wild-type strain upon CoCl<sub>2</sub> treatment. All statistically significant genes identified by SAM are given in ##SUPPL##1##Table S2## in the supplementary material. The array data has been submitted at Gene Expression Omnibus (GEO) site (accession no. GSE11390).</p>" ]
[ "<title>Results</title>", "<title>Construction of T-DNA insertional library and screening for cobalt chloride sensitive mutants</title>", "<p>\n<italic>Agrobacterium tumefaciens</italic>–mediated transformation is a very useful technique to study <italic>Cryptococcus</italic>. The high frequency of random integrations by ATMT ##REF##16260158##[20]##,##REF##15075272##[24]## enabled the construction of a T-DNA insertional library using the <italic>C. neoformans</italic> serotype D genomic strain (B-3501A). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable (unpublished data). The library consists of 30,000 individual transformants.</p>", "<p>To identify the genetic loci involved in the sensitivity to CoCl<sub>2</sub>, the T-DNA insertion library was screened for mutants sensitive to CoCl<sub>2</sub>. A total of 37 mutants that failed to grow on CoCl<sub>2</sub> medium were selected for further analysis. Sequence analysis of the genomic sequence flanking the insertion site revealed that the T-DNA was inserted in a wide array of genes (##TAB##0##Table 1##). Based on the sensitivity towards CoCl<sub>2</sub>, mutants could generally be categorized into 2 types: mutants that either showed complete growth inhibition or those that exhibited partial growth on media containing 0.7 mM CoCl<sub>2</sub> (##TAB##0##Table 1##). From the mutants listed in ##TAB##0##Table 1##, mutants 146G2, 92D9, 135G10, 132H6, 17B1, 238D8, and 72B10 showed partial growth on medium containing cobalt chloride. All other mutants were hypersensitive to cobalt chloride and did not show any growth on CoCl<sub>2</sub> containing plates (##TAB##0##Table 1##). ##FIG##0##Figure 1## shows the sensitivity pattern of some of these mutants from each group. To establish a link between a mutation and the CoCl<sub>2</sub> sensitive growth phenotype, some of the mutants were complemented with wild-type gene. These complemented strains, when grown on CoCl<sub>2</sub> containing plates, were no longer hypersensitive to CoCl<sub>2</sub> and the growth was comparable to the wild-type strain (##FIG##1##Figure 2##). This indicates the defects in these genes result in the sensitivity towards cobalt chloride. In this screening, both <italic>sre1</italic> and <italic>scp1</italic> mutants were isolated several times and it has previously been demonstrated that the deletion of each of these two genes leads to CoCl<sub>2</sub> sensitivity and these genes are involved in oxygen sensing in <italic>C. neoformans</italic>\n##REF##17645443##[18]##. The repeated isolation of <italic>sre1</italic> and <italic>scp1</italic> mutants in the screen strongly attests to the suitability of this screening approach in the identification of genes responsible for growth under low oxygen conditions.</p>", "<title>CoCl<sub>2</sub>-hypersensitive mutants are unable to grow in low oxygen conditions</title>", "<p>To determine if the CoCl<sub>2</sub> sensitive mutants also showed reduced growth in low oxygen conditions, cells were spotted on YES plates and incubated under different conditions: 30°C+ambient air (21% O<sub>2</sub>), 37°C+21% O<sub>2</sub>, and 37°C+1% O<sub>2</sub>. 37°C was chosen in this set of experiments in order to mimic the human body temperature. In the selected mutant pool, 10 of 32 mutants either failed to grow or grew poorly at 37°C, exhibiting a temperature-sensitive (Ts) phenotype (##FIG##2##Figure 3A## and ##TAB##0##Table 1##). Most interestingly, the remaining non-Ts mutants showed reduced or no growth in low oxygen conditions (##FIG##2##Figure 3B## and ##TAB##0##Table 1##). These results showed that screening for CoCl<sub>2</sub> sensitivity is a useful approach to find mutants sensitive to low oxygen and suggested a strong correlation between CoCl<sub>2</sub> sensitivity and sensitivity to low-oxygen conditions in <italic>C. neoformans</italic>. To identify the cellular processes involved in CoCl<sub>2</sub> and/or low oxygen sensitivity, we categorized these mutants into different functional groups as shown in ##TAB##0##Table 1##. Clearly, the processes involved in CoCl<sub>2</sub> and/or low oxygen sensitivity are complex.</p>", "<title>Mitochondrial membrane permeability defect</title>", "<p>We noted that seven mutants listed in ##TAB##0##Table 1## are directly related to mitochondrial function. In the mammalian system, cobalt chloride is known to affect mitochondrial function ##REF##10773083##[11]##, ##REF##14027961##[25]##–##REF##16643849##[27]##. Cobalt has also been shown to target mitochondria and induce respiratory deficiency in yeast ##REF##13229372##[15]##,##REF##13577873##[17]##,##REF##4190017##[28]##,##REF##12191769##[29]##. Various aspects of mitochondrial dysfunction can be detected by analyzing the mutants for perturbed mitochondrial membrane potential and efficiency of respiration. First, we examined all the cobalt chloride sensitive mutants for any perturbation in mitochondrial membrane potential by using mito-tracker dyes such as CMXRos. Confocal microscopy results showed that six different mutants were unable to retain the mito-tracker dye indicating the presence of dysfunctional mitochondria (##FIG##3##Figure 4A, red stain##). We observed heterogeneity in the cell size of these strains that maybe resulted from mutation caused by insertion of T-DNA. Four mutants belonged to the group involved in mitochondrial function in ##TAB##0##Table 1## (10D12, 161B3, 69G9, and 66G6). Both genes affected in 10D12 and 161B3 mutants have been studied in detail in <italic>S. cerevisiae</italic>\n##REF##16164973##[30]##–##REF##9587671##[32]##. Locus CNC05260 (mutant 10D12) encodes subunit f of the F(0) sector of mitochondrial F<sub>1</sub>F<sub>0</sub> ATP synthase. The other locus, CNM01080 (mutant 161B3), encodes ATP∶ADP antiporter that catalyzes the exchange of ADP and ATP across the mitochondrial inner membrane. In the mutant 69G9, a tRNA lysine gene involved in mitochondrial protein synthesis ##REF##16738406##[33]##,##REF##8797799##[34]## is disrupted. The locus CNC04010 (mutant 66G6) encodes a hypothetical protein that has hypoxia induced protein conserved region (HIG_1_N domain). Two mutants (146G2 and 67A3) that also failed to accumulate the CMXRos dye did not belong to the group involved in mitochondrial function (##TAB##0##Table 1##). Mutant 146G2, containing a T-DNA insertion in <italic>ERG9</italic> gene encoding squalene synthase (CNM00870), also showed a defect in mitochondrial membrane potential. As the name suggests, this enzyme is involved in ergosterol biosynthesis and joins the two farnesyl pyrophosphate moieties to form squalene ##REF##11111085##[35]##. The hypothetical protein encoded by CNA00940 (mutant 67A3) has two <italic>trans</italic>-membrane domains and a weak homology to the t-RNA synthetase subunit. To determine whether these mutants lacked mitochondria, yeast mitochondrial DNA specific dye Syto18 was used for staining. Accumulation of Syto18 in the cells indicated that all 6 mutants contained mitochondrial DNA (##FIG##3##Figure 4B, green stain##). All the other mutants listed in ##TAB##0##Table 1## were also analyzed for reduced membrane potential by staining with CMXRos. The staining profile of these mutants was similar to that of wild type cells indicating that mitochondrial membrane potential was not disturbed in the rest of mutants (unpublished data).</p>", "<title>CoCl<sub>2</sub>-sensitive mutants have respiratory defects</title>", "<p>As mentioned above, another way to analyze mitochondrial dysfunction is to assess the efficiency of respiration, which can be accomplished by measuring oxygen consumption. From the mutants listed in ##TAB##0##Table 1##, all of the mutants in “mitochondrial function/energy metabolism” category and selected mutants from other categories were chosen to assay the rate of oxygen consumption. As presented in ##FIG##4##Figure 5##, 9 mutants, 155B3, 146G2, 10D12, 135G10, 161B3, 69G9, 94F1, 46E10, and 92D9 showed a greater than 40% reduction in the rate of oxygen consumption compared to that of wild type in the absence of any chemical treatment. Four of these mutants, 146G2, 10D12, 161B3, and 69G9 also had a mitochondrial membrane potential defect as demonstrated in ##FIG##3##Figure 4##. Even though mitochondrial membrane potential seemed unperturbed in the other 5 mutants, the reduction in oxygen consumption indicated those mutations seemed to have caused severe respiratory defects. Interestingly, mutant 66G6 had a mitochondrial membrane potential defect but showed only close to a 20% reduction in oxygen consumption rate compared to wild type in the absence of any chemical treatment. Among the rest of the mutants, for instance, 95C11 and 215F4 showed only a ∼15%–20% reduction while mutants 297F11, 48B7, and 72B10 had similar rates of oxygen consumption compared to that of the wild type.</p>", "<p>To understand if the reduced oxygen consumption is due to a defect in the core respiration process, antimycin A was used. Antimycin A blocks mitochondrial respiration by inhibiting complex III of the electron transport chain and the use of antimycin A revealed the rate of oxygen consumption in the core respiration process. In wild-type cells, addition of antimycin A resulted in a 30% reduction in oxygen consumption. In mutants 155B3, 146G2, 10D12, 135G10, 161B3, and 69G9, the addition of antimycin A did not induce further reduction compared to the control samples (##FIG##4##Figure 5##). Similarly, mutants 94F1, 66G6, and 95C11 showed no significant difference in respiration rates between antimycin A treated and non-treated samples (<italic>p</italic>&lt;0.02). These results suggest the reduction in the oxygen consumption in these mutants is due to the block in the antimycin A sensitive pathway and the genes targeted in these mutants are responsible for proper functioning of core respiration. It is not clear why in mutants 215F4 and 297F11 the oxygen consumption rate was close to the wild type and yet not sensitive to antimycin A treatment. In a second group of mutants, respiration was sensitive to antimycin A treatment. In the mutant 46E10 antimycin A caused 80% reduction in oxygen consumption while in mutants 72B10, 92D9, and 48B7 it was 30%, 40%, and 65%, respectively. This significant reduction in oxygen consumption in the presence of antimycin A in these 4 mutants indicates unlike above-mentioned mutants, cells are able to respire through the core electron transport chain.</p>", "<p>Next, the cells were treated with cobalt chloride and assayed for oxygen consumption in the presence and absence of antimycin A. Compared to antimycin A, treatment with cobalt chloride showed an increased reduction in oxygen consumption in wild type. Addition of antimycin A to the cells grown in the presence of CoCl<sub>2</sub> did not show any further reduction in the rate of oxygen consumption in the wild type as well as in all the mutants. This suggested that cobalt chloride strongly inhibits both antimycin A sensitive and antimycin A insensitive respiration pathways in all strains. Unlike in mutants where CoCl<sub>2</sub> showed a significant effect on respiration compared to antimycin A alone, the reduction in the rate of respiration due to antimycin A or CoCl<sub>2</sub> or in combination of both was similar in mutants 46E10 and 48B7. This indicates that in these mutants, antimycin A insensitive respiration pathways are more severely affected than the respiration through antimycin A sensitive core electron transport chain (ETC). These results indicate that 12 of the 15 genes affected in cobalt chloride–sensitive mutants influence oxygen consumption either through antimycin A sensitive mitochondrial respiration or antimycin A insensitive respiration.</p>", "<title>Mutants show elevated ROS levels and are sensitive to oxidants</title>", "<p>Cobalt is one of the transition metals known to generate a spectrum of reactive oxygen species (ROS) in the Fenton reactions and subsequent lipid peroxidation ##REF##7744317##[36]##,##REF##9720310##[37]##. Generation and accumulation of ROS in the cellular environment due to cobalt chloride creates an imbalance in the oxidative state of the cells. This ROS-induced oxidative stress could be another factor that is leading to cobalt chloride sensitivity in the mutants. To investigate the possibility that the mutants have abnormal ROS production, the fluorescent dye H2DCFDA was employed. This probe increases its fluorescence when oxidized by ROS. Cells preloaded with H2DCFDA were treated with cobalt chloride and analyzed by FACS along with non-CoCl<sub>2</sub>–treated cells. Upon CoCl<sub>2</sub> treatment, we observed only a slight increase in ROS levels in the wild type cells. One interesting observation to note here is that in four mutants, their intrinsic ROS levels were higher (1.5- to 2.3-fold increase in the fluorescence intensity) than that of wild type even without CoCl<sub>2</sub> exposure (10D12, 66G6, 155B3, and 161B3; ##FIG##5##Figure 6##). Compared to cobalt chloride treated wild-type cells, some mutants such as 46E10, 92D9, 95C11, and 135G10 showed an increase in ROS levels after incubation with CoCl<sub>2</sub> (1.2- to 2.2-fold increase in fluorescence intensity; ##FIG##5##Figure 6##). Therefore, most of the mutants have increased ROS levels either with or without CoCl<sub>2</sub> treatment.</p>", "<p>Cellular antioxidant system influences the ability of cells to tolerate various oxidants. Unlike in wild-type cells, if the antioxidant system is not functioning properly in the mutants, they can exhibit hypersensitivity towards ROS and RNS (reactive nitrogen species) generating chemicals. To determine how CoCl<sub>2</sub>-sensitive mutants respond to the external source of ROS/RNS, growth was monitored on media containing H<sub>2</sub>O<sub>2</sub>, sodium nitrite, paraquat, and diethyl maleate. These four reagents act on different cellular targets and generate reactive oxygen/nitrogen species. H<sub>2</sub>O<sub>2</sub> has the ability to directly damage nucleic acids, proteins, and lipids ##REF##9885153##[38]##,##REF##9252331##[39]##. The effect of H<sub>2</sub>O<sub>2</sub> was analyzed at pH 4.0 and both 30°C and 37°C. 28 of the 29 mutants showed increased sensitivity towards H<sub>2</sub>O<sub>2</sub> at 37°C compared to 30°C (##FIG##6##Figure 7A## and ##TAB##0##Table 1##). Few mutants such as 95C11, 69G9, and 94F1 showed increased sensitivity even at 30°C. Sodium nitrite is known to generate nitric oxide (NO) at pH 4.0 ##REF##10859695##[40]##,##UREF##1##[41]##. Nitric oxide reacts with different chemicals inside the cells forming reactive nitrogen species (RNS). The targets for RNS include protein-bound metal centers, thiols, DNA, lipids etc. ##REF##8845445##[42]##. NO has also been shown to inhibit complex III and complex IV of the respiratory chain ##REF##9073575##[43]##,##REF##11245784##[44]##. Based on plate assays, NO affected the growth in 25 out of 29 CoCl<sub>2</sub>-sensitive mutants (##FIG##6##Figure 7B## and ##TAB##0##Table 1##). Paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride) is widely used to induce superoxide generation in cells. After its entry in mitochondria, paraquat is reduced by complex I leading to superoxide generation and subsequently extensive mitochondrial damage ##REF##18039652##[45]##. ##FIG##6##Figure 7C## and ##TAB##0##Table 1## show that 28 of the 32 tested mutants are sensitive to paraquat. Diethyl maleate (DEM) is a glutathione-depleting agent and glutathione is one of the important non-enzymatic antioxidants that binds and inactivates ROS directly to protect cells against the toxic effects of ROS ##REF##8095770##[46]##. Exposure of cells to DEM leads to protein denaturation and increased intracellular ROS levels ##REF##9314607##[47]##. The majority of mutants tested (28 of the 32) showed sensitivity towards DEM (##FIG##6##Figure 7D## and ##TAB##0##Table 1##). Furthermore, not all the mutants showed sensitivity to all four chemicals (##TAB##0##Table 1##). This was expected because based on the gene affected, different antioxidant systems would produce different outcomes in growth inhibition. Importantly, all the mutants showed sensitivity to at least one of the four ROS/RNS generating chemicals suggesting that all the CoCl<sub>2</sub> sensitive mutants had a defect in handling the stress generated by ROS/RNS-producing reagents.</p>", "<title>Transcriptional profile of cobalt chloride treated wild-type cells</title>", "<p>To gain an insight into the molecular basis for the effect of cobalt chloride treatment on <italic>C. neoformans</italic> cells, a whole genome microarray experiment was performed. Cells were harvested after 2 h of CoCl<sub>2</sub> treatment. All genes with statistically significant differences in expression between wild type cells with CoCl<sub>2</sub> and without CoCl<sub>2</sub> were identified by significance analysis of microarray (SAM) using a mean false discovery rate (FDR) of less than 5% as described in Experimental Procedures. Of the 6,660 detectable genes analyzed, 979 genes were identified as significant genes by a mean FDR of 5% with SAM analysis. Of the 979 genes, 314 genes were up- or down-regulated by at least 2-fold. 42% of these genes have been annotated as hypothetical or expressed genes. The remaining 182 genes were categorized into different groups based on their annotated functions and presented in the form of a pie chart (##FIG##7##Figure 8##). The complete list of 314 genes is provided in the supplementary material (##SUPPL##0##Table S1##).</p>", "<p>As the figure suggests, genes involved in mitochondrial functions form a major category (30%). Upon examination of the transcriptional profile of genes involved in mitochondrial function, it is apparent that CoCl<sub>2</sub> repressed the expression of genes involved in the electron transport chain. ##TAB##1##Table 2## lists the genes encoding ETC components (complex I through IV) that were down-regulated at least 2-fold upon cobalt chloride treatment. Along with ETC component genes, CoCl<sub>2</sub> treatment also led to a significant reduction in the expression of the gene encoding alternative oxidase (<italic>AOX1</italic>). Alternative oxidase bypasses complex III and IV by carrying out ubiquinol oxidation and reduction of dioxygen to water ##REF##1883834##[48]##,##REF##12689632##[49]##. As shown above, the respiration rate was reduced by ∼65% when the wild-type cells were treated with CoCl<sub>2</sub>. The possible reason for this significant reduction may be in part due to the down-regulation of ETC genes as well as alternative oxidase in response to CoCl<sub>2</sub>.</p>", "<p>Along with the genes involved in mitochondrial function are other groups of genes required for ergosterol biosynthesis, iron/copper homeostasis, oxidative stress, proteasome/ubiquitination function, carbohydrate metabolism, various transporters, specific transcription/translation, and some cellular metabolic processes. These data suggest that CoCl<sub>2</sub> can induce a wide range of response in <italic>C. neoformans</italic>.</p>" ]
[ "<title>Discussion</title>", "<p>This study investigated the role of CoCl<sub>2</sub> as an effective chemical to screen for probable pathways involved in oxygen sensing in <italic>C. neoformans</italic>. We show that most of the CoCl<sub>2</sub> sensitive mutants are also sensitive to low oxygen conditions. These mutants fall into various functional categories including; mitochondrial function, sterol biosynthesis, vesicular transport, carbohydrate metabolism, and other cellular functions. Based on the characterization, we report cobalt chloride sensitivity and/or sensitivity to low oxygen conditions are strongly influenced by mitochondrial function, ability of cells to deal with ROS production, ubiquitination, and sterol homeostasis. These data highlight the complex nature of CoCl<sub>2</sub> hypersensitivity and/or oxygen sensing and adaptation process in <italic>C. neoformans</italic>.</p>", "<p>Many mutants obtained in our CoCl<sub>2</sub> sensitivity screen were unable to retain Mitotracker dye indicating defective membrane potential. Genes affected in 3 of these mutants (10D12, 161B3, and 146G2) are related to structural components of the mitochondrial membrane. These results suggested proper maintenance of mitochondrial membrane potential is essential for <italic>C. neoformans</italic> to tolerate the high concentration of CoCl<sub>2</sub> and to adapt to low oxygen conditions.</p>", "<p>Another major defect in many of our CoCl<sub>2</sub> sensitive mutants is the impaired oxygen consumption. It is clear that CoCl<sub>2</sub> not only affected antimycin A sensitive core respiration but also inhibited antimycin A insensitive alternative respiration. Importantly, many of the cobalt chloride sensitive mutants have a defect in oxygen consumption through either mode of respiration, core or alternative. Cobalt chloride has the ability to induce a respiratory deficiency in cells ##REF##13229372##[15]##,##REF##13577873##[17]##. In response to specific inhibitors and oxidative stress, fungi are known to utilize alternative components of the respiratory chain consisting of alternative NADH dehydrogenases to bypass complex I and an alternative oxidase to bypass complex III and IV ##REF##11245784##[44]##. Although we do not have mutants that belong to the alternative respiration pathway, we isolated a complex I NADH∶ubiquinone oxidoreductase mutant (135G10). In addition, four more mutants defective in mitochondrial membrane potential (146G2, 10D12, 69G9, and 161B3) also showed only antimycin insensitive respiration. These data indicate that the presence of an intact respiration system is required for <italic>C. neoformans</italic> to handle the high concentrations of CoCl<sub>2</sub>. If respiration is affected so severely in the presence of CoCl<sub>2</sub>, it is possible that CoCl<sub>2</sub> treatment affects the expression of genes encoding respiration related proteins. As described in ##TAB##1##Table 2##, expression of a wide array of genes for subunits in complex I through III along with other genes involved in aerobic metabolism (<italic>FIXC</italic>, <italic>HEMD</italic>, <italic>HEM1</italic>) were down-regulated upon CoCl<sub>2</sub> treatment. In addition, our array data showed that the <italic>AOX1</italic> gene was significantly repressed, while 10 different alternative dehydrogenases that belong to class II NADH∶ubiquinone oxidoreductases were up-regulated in CoCl<sub>2</sub> treated cells. Two of the oxidoreductases listed in ##TAB##1##Table 2##, (CNA05000 and CNI02360) have been shown to be up-regulated in a <italic>SRE1</italic>-dependent manner under 1% oxygen ##REF##17462012##[19]##. Interestingly, in <italic>S. cerevisiae</italic> under normoxic conditions aerobic genes are needed to encode proteins involved in aerobic metabolism such as oxidative phosphorylation while in low oxygen conditions hypoxic response genes are induced so as to allow the cells to utilize oxygen efficiently. These gene products involved in oxygen utilizing pathways include alternate cytochrome subunits and oxidases, and enzymes such as reductases and desaturases for heme, sterol, and fatty acid biosynthesis ##REF##1579104##[50]##. Our data here indicate in <italic>C. neoformans</italic> CoCl<sub>2</sub> affects the expression of genes that are partially overlapping with genes expressed in oxygen limiting conditions. Perhaps, by regulating the expression of these genes in response to CoCl<sub>2</sub>, the cells can utilize oxygen more efficiently.</p>", "<p>Many of the mutants in this study showed sensitivity to various ROS producing agents and a change in the intracellular ROS levels either with or without CoCl<sub>2</sub> treatment. This increase in ROS levels in the mutants as well as hypersensitivity towards oxidants could be the cumulative effect of mutations they are harboring and the presence of the ROS inducing chemicals in the environment. To eliminate ROS from the cellular environment, a number of antioxidant systems such as superoxide dismutase, catalase, thioredoxin, glutathione, mannitol are in play ##UREF##2##[51]##,##REF##10647118##[52]##. The sensitivity towards ROS generating chemicals in the mutants indicates that either the antioxidant systems are not functioning properly in these strains or irrespective of functional antioxidant system, cells are unable to handle the elevated levels of ROS. There may be some correlation between the ability of various chemicals to increase ROS levels and their increased toxicity to a CoCl<sub>2</sub> sensitive mutant. In <italic>Saccharomyces cerevisiae</italic>, mitochondrial function has been shown to be required for resistance to oxidative stress ##REF##9237633##[53]##. Hence, there is a possibility that some of the mutants are sensitive to oxidants due to defects in mitochondrial function and not due to the antioxidant status of the cell. The mitochondrial respiratory chain is the main site of cellular ROS production. It has been reported that disruption of the electron transport chain leads to an increase in ROS levels in mammals as well as in <italic>S. cerevisiae</italic>\n##REF##12237311##[54]##–##REF##17627464##[56]##. Among our category of mitochondrial function mutants, although 135G10, the complex I NADH∶ubiquinone oxidoreductase mutant did not show elevated levels of ROS in log phase cells, this strain displayed increased ROS levels upon CoCl<sub>2</sub> treatment compared to the wild type. Furthermore, two strains with a mitochondrial membrane potential defect (10D12 and 161B3) show increased levels of ROS in log phase cells even in the absence of CoCl<sub>2</sub>. These data support the notion that a perturbance in mitochondrial function can lead to intracellular accumulation of reactive oxygen species. Mitochondrial ROS generated during hypoxia and in the presence of CoCl<sub>2</sub> have been implicated in cellular oxygen sensing in metazoans as well as in <italic>S. cerevisiae</italic> suggesting a role of mitochondria in oxygen sensing ##REF##17627464##[56]##–##REF##9751731##[58]##. Therefore, like in hypoxic conditions in other systems, it is probable that CoCl<sub>2</sub> also induces mitochondrial ROS levels in <italic>C. neoformans</italic> leading to the cross talk between mitochondria and the nucleus, which ultimately results in changes of gene expression profile. Since the mutants showing abnormal ROS levels are also sensitive to low oxygen conditions, it is possible that in oxygen depriving conditions, due to a mutation in a respective gene in the mutant, signals transduced through ROS are not able to activate a cascade of genes that sustain growth under low oxygen conditions.</p>", "<p>Ubiquitination has been shown to be involved in targeting of nuclear-encoded pre-proteins into mitochondria, intracellular localization of macromolecules and degradation of superfluous/denatured proteins ##REF##17707817##[59]##,##REF##16135527##[60]##. In <italic>S. cerevisiae</italic>, in addition to these general functions, functional proteasomes and ubiquitin-dependent pathways are necessary for degradation of aerobic proteins (iso-1-cytochrome c) and modification of hypoxic proteins (Spt23, Mga2) ##REF##9395529##[61]##,##REF##12440977##[62]##. When <italic>C. neoformans</italic> cells were treated with CoCl<sub>2</sub>, we observed up-regulation of genes involved in proteasome function and ubiquitination suggesting similar mechanisms may be taking place (##SUPPL##0##Table S1##). In addition, complementation of clone 94F1 with CNH00220 locus encoding ubiquitin protein ligase rescued the CoCl<sub>2</sub> sensitive phenotype (##FIG##1##Figure 2##), supporting the involvement of ubiquitination in handling the stress induced by cobalt chloride and/or low oxygen conditions in <italic>C. neoformans</italic>.</p>", "<p>One of the early studies that suggested antagonism between iron and cobalt in fungal systems was carried out in the 1950s using <italic>Neurospora crassa</italic> as a model system ##REF##13229372##[15]##. Further studies in the same system showed growth inhibition due to cobalt could be reversed to some extent by iron ##REF##13986674##[63]## and cobalt induced iron deficiency resulted in the formation of siderochrome ##REF##5938658##[16]##,##REF##14240595##[64]##. In <italic>S. cerevisiae</italic>, even at sub-lethal cobalt concentrations, iron regulon is induced resulting in immediate expression of iron transporter genes to increase intracellular iron content. This response is similar to iron starvation conditions ##REF##12176980##[65]##. In mammalian systems, it is known that Co<sup>2+</sup> can be substituted for the iron in porphyrin ring of oxygen-requiring enzymes leading to the lower affinity for oxygen and mimicking hypoxia environment ##REF##2849206##[6]##,##REF##454362##[66]##,##REF##7082380##[67]##. In light of these studies in yeast, molds and mammals, gene expression profile in relation to iron/copper homeostasis made sense. Our data showed when wild-type cells are exposed to CoCl<sub>2</sub>, genes encoding high affinity iron permeases (<italic>FRE3</italic>, <italic>CFT1</italic>, <italic>CFO1</italic>, <italic>CFT2</italic>) as well as siderophore transporters (<italic>SIT1</italic>) were significantly up-regulated (##SUPPL##0##Table S1##), mimicking the iron deprivation response, although none of our CoCl<sub>2</sub> mutants showed significant growth reduction in iron limiting conditions (unpublished data). As suggested in other systems, cobalt may also exert its antagonistic effects on iron metabolism in <italic>C. neoformans</italic> by competing with Fe<sup>2+</sup> thereby affecting heme dependent and heme independent proteins. Transcription profile of cells treated with CoCl<sub>2</sub> also revealed that genes involved in sterol regulatory pathways such as <italic>ERG1</italic>, <italic>ERG3</italic>, <italic>ERG25</italic>, <italic>OLE1</italic>, <italic>NCP1</italic>, <italic>SCS7</italic> are significantly induced (##SUPPL##0##Table S1##). These are oxygen-responsive genes and have been shown to be induced in low oxygen conditions in <italic>C. neoformans</italic> as well as in <italic>S. cerevisiae</italic>\n##REF##17462012##[19]##,##REF##9510529##[68]##. Recent studies in <italic>C. neoformans</italic> showed the deletion of high affinity iron permeases (Cft1 and Cft2) leads to sterol synthesis defects ##REF##18282105##[69]##. Previous work from our laboratory identified an oxygen-sensing mechanism in <italic>C. neoformans</italic>, in which Sre1p and Scp1p are required for normal growth under low oxygen ##REF##17462012##[19]##. In our CoCl<sub>2</sub> sensitive mutant screen, we identified two additional mutants related to sterol biosynthesis; squalene synthase (146G2) and C22-sterol desaturase (234E1), which are also sensitive to low oxygen conditions. Thus, in <italic>C. neoformans</italic> also iron-cobalt antagonism may disrupt iron and sterol homeostasis affecting the ability of cells to survive under high CoCl<sub>2</sub> concentrations and/or low oxygen conditions.</p>", "<p>Here, we have explored the possibility of using CoCl<sub>2</sub> to understand the mechanism of adaptation to low oxygen conditions in <italic>C. neoformans</italic>. We propose CoCl<sub>2</sub> may be mimicking hypoxia like conditions by inhibiting many of the mitochondria-related functions. These data have highlighted the complexity of oxygen sensing in <italic>C. neoformans</italic>. The unpublished data from our lab suggest involvement of a respiratory chain is only one aspect of oxygen sensing and there are multiple pathways involved in oxygen sensing which are not related to CoCl<sub>2</sub> sensitivity (unpublished data). Further studies on the complex interactions between sterol and iron homeostasis, mitochondrial function and oxygen sensing, using this diverse set of CoCl<sub>2</sub> sensitive mutants will shed light on the pathobiology of <italic>C. neoformans</italic>, especially in the brain environment.</p>" ]
[]
[ "<p>Conceived and designed the experiments: SSI YCC KJKC. Performed the experiments: SSI YCC HL CMM MLL. Analyzed the data: SSI YCC HL. Wrote the paper: SSI YCC KJKC.</p>", "<p>\n<italic>Cryptococcus neoformans</italic> is an environmental fungal pathogen that requires atmospheric levels of oxygen for optimal growth. For the fungus to be able to establish an infection, it must adapt to the low oxygen concentrations in the host environment compared to its natural habitat. In order to investigate the oxygen sensing mechanism in <italic>C. neoformans</italic>, we screened T-DNA insertional mutants for hypoxia-mimetic cobalt chloride (CoCl<sub>2</sub>)-sensitive mutants. All the CoCl<sub>2</sub>-sensitive mutants had a growth defect under low oxygen conditions at 37°C. The majority of mutants are compromised in their mitochondrial function, which is reflected by their reduced rate of respiration. Some of the mutants are also defective in mitochondrial membrane permeability, suggesting the importance of an intact respiratory system for survival under both high concentrations of CoCl<sub>2</sub> as well as low oxygen conditions. In addition, the mutants tend to accumulate intracellular reactive oxygen species (ROS), and all mutants show sensitivity to various ROS generating chemicals. Gene expression analysis revealed the involvement of several pathways in response to cobalt chloride. Our findings indicate cobalt chloride sensitivity and/or sensitivity to low oxygen conditions are linked to mitochondrial function, sterol and iron homeostasis, ubiquitination, and the ability of cells to respond to ROS. These findings imply that multiple pathways are involved in oxygen sensing in <italic>C. neoformans</italic>.</p>", "<title>Author Summary</title>", "<p>\n<italic>Cryptococcus neoformans</italic> is an obligate aerobic fungus that requires atmospheric levels of oxygen (21%) for optimal growth. However, the fungus is able to cause life-threatening brain infections in humans, where the oxygen tension is significantly lower than 21%. To understand the pathobiology of <italic>Cryptococcus neoformans</italic>, it is important to explore the molecular mechanisms adopted by the fungus to survive under low oxygen conditions. By using cobalt chloride, a hypoxia-mimicking agent, we isolated a number of mutants that are unable to grow in the presence of 0.7 mM CoCl<sub>2</sub> as well as at low oxygen conditions. In this study, we show that mitochondria play an important role for <italic>C. neoformans</italic> to survive in low oxygen conditions. We demonstrate that mutants harboring mutations in the genes related to mitochondrial functions have mitochondrial membrane permeability defect and lowered respiration rate and are more sensitive to stress generating chemicals, in addition to their inability to survive at low oxygen conditions. Finally, we also show that when wild-type cells are exposed to hypoxia-mimicking cobalt chloride, expression of genes involved in respiration and iron and sterol homeostasis, as well as ubiquitination, changes significantly.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>We thank biological imaging section and flow cytometry section in NIAID for helping us with confocal microscopy and FACS analysis. We thank Dr. Robert Balaban and Dr. Stephanie French at NHLBI for helping us with oxygen consumption assays. We are grateful to Dr. Ashok Varma for critical reading of this manuscript.</p>" ]
[ "<fig id=\"ppat-1000155-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.g001</object-id><label>Figure 1</label><caption><title>Cobalt chloride sensitive phenotype of T-DNA insertional mutants.</title><p>Ten fold serial dilutions of wild type and T-DNA insertional mutant strains were spotted on YES and YES+CoCl<sub>2</sub> plates and incubated at 30°C for 3 d.</p></caption></fig>", "<fig id=\"ppat-1000155-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.g002</object-id><label>Figure 2</label><caption><title>Complementation of CoCl<sub>2</sub> sensitivity phenotype.</title><p>Individual mutant was transformed with the corresponding wild type gene. Original and complemented strains were patched on YES and YES+CoCl<sub>2</sub> plates and incubated at 30°C for 3 d.</p></caption></fig>", "<fig id=\"ppat-1000155-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.g003</object-id><label>Figure 3</label><caption><title>CoCl<sub>2</sub> sensitive mutants exhibit hypoxia sensitive phenotype.</title><p>(A). Mutants showing temperature sensitive phenotype. Yeast cells were serially diluted and spotted on YES and incubated at 30°C and 37°C for 3 d. (B). Mutants showing hypoxia sensitive phenotype. To check growth in low oxygen conditions, serial dilutions of wild type and mutant cells were spotted on YES and plates were incubated at 30°C, 37°C, and 37°C+1%O<sub>2</sub> for 3 d.</p></caption></fig>", "<fig id=\"ppat-1000155-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.g004</object-id><label>Figure 4</label><caption><title>Confocal microscopy for mitochondrial staining.</title><p>(A) Cells were grown to log phase and stained with mito-tracker dye CMXRos. Red color in the wild type shows the normal membrane permeability and diminished red color in the mutants suggested defect in mitochondrial membrane permeability. (B) Cells were grown to log phase and stained with SYTO18 dye for mitochondrial DNA. Green color indicates the presence of mitochondrial DNA. Experiments were done at least two times using mito-tracker red and SYTO18 dyes for these strains. Scale bar represents 3 µm.</p></caption></fig>", "<fig id=\"ppat-1000155-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.g005</object-id><label>Figure 5</label><caption><title>Oxygen consumption is reduced in yeast grown in CoCl<sub>2</sub>-containing medium.</title><p>Yeast cells were grown as described in <xref ref-type=\"sec\" rid=\"s2\">Materials and Methods</xref>. A total of 5×10<sup>7</sup> washed cells were suspended in 2 ml media, and changes in oxygen tension were measured at room temperature using a Clark type electrode. Samples were measured with no addition of chemical (control), with antimycin A (A+), with CoCl<sub>2</sub> (Co+), or with CoCl<sub>2</sub> and antimycin A (Co+, A+).</p></caption></fig>", "<fig id=\"ppat-1000155-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.g006</object-id><label>Figure 6</label><caption><title>CoCl<sub>2</sub>-sensitive mutants accumulate reactive oxygen species (ROS).</title><p>Wild-type and mutant cells were grown to log phase at 30°C, loaded with H2DCFDA and exposed to CoCl<sub>2</sub> for 4 h. ROS accumulation was assessed by flow cytometry. Each panel represents the FACS analysis result of each strain; filled peaks (black) represent fluorescence intensity of untreated cells while line peaks (grey line) represent the fluorescence intensity of CoCl<sub>2</sub>-treated cells on the FL1-H scale. All the FACS experiments were done multiple times, and data presented here are a representative of the experiments.</p></caption></fig>", "<fig id=\"ppat-1000155-g007\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.g007</object-id><label>Figure 7</label><caption><title>CoCl<sub>2</sub>-sensitive mutants are sensitive to ROS-generating reagents.</title><p>Serial dilutions of yeast cells were spotted onto buffered, pH 4.0 YES with and without 0.5 mM H<sub>2</sub>O<sub>2</sub> and incubated at 30°C and 37°C for 3 d (A), and with and without 1.0 mM NaNO<sub>2</sub> and incubated at 30°C for 3 d (B). Wild-type and mutant strains were streaked on YES containing 0.25 mM paraquat (C) and YES+2 mM diethyl maleate plates (D). Plates were incubated at 30°C for 3 d.</p></caption></fig>", "<fig id=\"ppat-1000155-g008\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.g008</object-id><label>Figure 8</label><caption><title>Pie chart.</title><p>Pie chart shows the functional categories and distribution of the 182 genes whose expression has changed more than 2-fold in response to CoCl<sub>2</sub>. Annotations were obtained from the NCBI database (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ncbi.nlm.nih.gov\">http://www.ncbi.nlm.nih.gov</ext-link>), with additional hand editing based on BLAST searches. Numbers in parentheses refer to the percentage of genes in each class and do not include the genes that are annotated hypothetical or expressed.</p></caption></fig>" ]
[ "<table-wrap id=\"ppat-1000155-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.t001</object-id><label>Table 1</label><caption><title>A list of CoCl<sub>2</sub> sensitive mutants isolated from T-DNA insertion library and their growth phenotypes.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clone no.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Locus ID</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Description<xref ref-type=\"table-fn\" rid=\"nt101\">a</xref>\n</td><td colspan=\"6\" align=\"left\" rowspan=\"1\">Growth<xref ref-type=\"table-fn\" rid=\"nt102\">b</xref> on</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">CoCl<sub>2</sub>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37°C/1%O<sub>2</sub>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">H<sub>2</sub>O<sub>2</sub>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NaNO<sub>2</sub>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Paraquat</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">DEM</td></tr></thead><tbody><tr><td colspan=\"9\" align=\"left\" rowspan=\"1\">\n<bold>Sterol Biosynthesis Pathway</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10E11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNC00950</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SCAP<xref ref-type=\"table-fn\" rid=\"nt103\">1</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">87A1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNJ02310</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SREBP<xref ref-type=\"table-fn\" rid=\"nt103\">1</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">146G2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNM00870</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Squalene synthase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td colspan=\"9\" align=\"left\" rowspan=\"1\">\n<bold>Genes Involved in Mitochondrial Function/Energy Metabolism</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10D12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNC05260</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">H+ transporting ATP synthase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">nd*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">nd*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">46E10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA00760</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">tRNA (guanine-N2-)-methyltransferase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">66G6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNC04010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HIG_1_N domain family</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">69G9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNE00180</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">t-RNA Lysine</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">92D9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNJ01940</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Dihydrofolate reductase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">135G10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND04070</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NADH∶ubiquinone oxidoreductase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">161B3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNM01080</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ATP∶ADP antiporter</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td></tr><tr><td colspan=\"9\" align=\"left\" rowspan=\"1\">\n<bold>Vesicular Transport</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">132H6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF00890</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Importin beta-4 subunit</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">155B3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA06920</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">V ATPase subunit H</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">252C2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNC07180</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vacuolar protein sorting 54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">239E3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNJ03270</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clathrin heavychain 1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td colspan=\"9\" align=\"left\" rowspan=\"1\">\n<bold>Regulatory Function Related</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">27C2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNK03380</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fungal Zn(2)-Cys(6) domain family</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">215F4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF01510</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense-mediated mRNA decay factor</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">238D8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNN00160</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Two-component protein-histidine kinase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">293A4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNM01040</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">C6 transcription factor</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td></tr><tr><td colspan=\"9\" align=\"left\" rowspan=\"1\">\n<bold>Enzymes/Transporters</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">72B10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNG01540</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hexose transport related protein</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">103H12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNN01350</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ATP dependent clp protease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">148C4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF00750</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Seroheme synthase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">262F2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNM00800</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Amino acid transporter</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">297F11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF03950</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Myo-inositol oxygenase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td></tr><tr><td colspan=\"9\" align=\"left\" rowspan=\"1\">\n<bold>Mutants with Multiple Insertion Sites</bold>\n<xref ref-type=\"table-fn\" rid=\"nt104\">2</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">29A6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA07630</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hypothetical</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">nd*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">nd*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">94F1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNH00220</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ubiquitin protein ligase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">209A11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNH03300</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Microtubule binding protein</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">234E1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF03720</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">C-22 sterol desaturase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNG02960-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hypothetical-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">-CNG02950</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-Dihydrodipicolinate synthetase</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td colspan=\"9\" align=\"left\" rowspan=\"1\">\n<bold>Hypothetical and Expressed Proteins</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">17B1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNB03300</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hypothetical protein</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">nd*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">nd*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">48B7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNL04460</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hypothetical</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">67A3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA00940</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Expressed protein</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Ts</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">95C11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF01170</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Expressed protein</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">262A5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNG02200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hypothetical</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>−/+</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>+/−</bold>\n</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"ppat-1000155-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000155.t002</object-id><label>Table 2</label><caption><title>Effect of CoCl<sub>2</sub> on expression of respiration-related genes.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Locus ID</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Fold Change<xref ref-type=\"table-fn\" rid=\"nt106\">*</xref>\n</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Description<xref ref-type=\"table-fn\" rid=\"nt105\">a</xref>\n</td></tr></thead><tbody><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">\n<bold>Complex I</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNH02730</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.54</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADH-ubiquinone oxidoreductase (subunit D)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNB01310</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−2.10</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADH-ubiquinone oxidoreductase (subunit G)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND04070</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.93</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADH-ubiquinone oxidoreductase 51 kDa subunit (NuoF)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF03360</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.45</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADH-ubiquinone oxidoreductase 30.4 kDa subunit, putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND01070</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.61</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADH dehydrogenase (ubiquinone)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNC07090</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.91</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADH dehydrogenase (subunit E) putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNE03960</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.62</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADH ubiquinone oxidoreductase (subunit NDUFA12) putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNM01810</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.69</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">ETC complex I subunit conserved region, putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNM02270</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.77</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">ETC complex I subunit conserved region, putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNE02800</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.70</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADH dehydrogenase (subunit B), putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNH01030</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.18</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADH dehydrogenase 10.5 K chain</td></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">\n<bold>Complex II</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA03530</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.80</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Succinate dehydrogenase (ubiquinone) (Sdh3 subunit)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNG03480</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−3.40</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Succinate dehydrogenase iron-sulfur (Sdh2 subunit)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNI03270</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.54</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Succinate dehydrogenase flavoprotein subunit precursor (Sdh1)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNB00800</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−2.01</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Mitochondrial inner membrane protein (Sdh4 subunit)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNJ00140</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−3.12</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Succinate dehydrogenase/fumarate reductase, flavoprotein subunit</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND02060</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.57</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Succinate-semialdehyde dehydrogenase I, GabD</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF03900</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.32</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Succinate-semialdehyde dehydrogenase I, Alddh</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA05580</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.27</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Complex I protein (LYR family)</td></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">\n<bold>Complex III</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF00630</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.40</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Electron transporter, cytochrome <italic>c</italic>\n<sub>1</sub>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNH02740</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.17</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">ubiquinol-cytochrome C reductase complex (Qcr9 subunit)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNG00860</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.19</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Mitochondrial processing peptidase beta (Cor1 subunit)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNB01620</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.88</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">L-lactate dehydrogenase, cytochrome <italic>b</italic>\n<sub>2</sub> (Cyb2)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND04430</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.12</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">ubiquinol-cytochrome C reductase complex (Qcr7 subunit)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNF03560</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.27</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">ubiquinol-cytochrome C reductase complex (Qcr6 subunit)</td></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">\n<bold>Cytochrome</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA06950</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−3.46</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Electron carrier, Iso-1-cytochrome <italic>c</italic>, (Cyc1)</td></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">\n<bold>Other Enzymes</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA01500</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−2.35</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Alternative oxidase 1 (Aox1)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND02080</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−2.27</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">FMN-dependent dehydrogenase, putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA04420</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−2.69</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">oxidoreductase (Dehydrogenases (flavoproteins), FixC)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND02030</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.09</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Methylmalonate-semialdehyde dehydrogenase [acylating], putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNM01770</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.56</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Aconitate hydratase (AcnA_Mitochondrial)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNK02510</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.34</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">uroporphyrinogen-III synthase, (HemD)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNI03590</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−1.69</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">phosphoenolpyruvate carboxykinase</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNB04260</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">−2.27</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">5-aminolevulinate synthase, (Hem1)</td></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">\n<bold>Alternative Respiration Related</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNI00360</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.06</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">NADH dehydrogenase, (FAD-containing subunit, classII)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND02280</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.43</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">oxidoreductase (aldehyde/oxo group of donors, NAD or NADP as acceptor)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNC06220</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.39</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">glycerate-and formate-dehydrogenase (NAD or NADP as acceptor)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNI02360</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.46</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">NADPH dehydrogenase 2 (Oye2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNB03640</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.58</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">oxidoreductase (NADH dehydrogenase, FAD-containing subunit)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNM00370</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.13</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">Aryl-alcohol dehydrogenase</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA05000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.63</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">NADH dehydrogenase (FAD-containing subunit, class II), putaive</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND00720</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.76</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">NADH-flavin reductase, putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CND02380</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.63</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">NADPH dehydrogenase, putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNC00200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.12</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">Methylase in ubiquinone/menaquinone biosynthesis, putative (UbiE)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNA04340</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.81</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">Mitochondrial hypoxia responsive domain protein, putative</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNE02820</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.62</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">Mitochondrial ATPase, putative (AFG1_ATPase)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CNE03260</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.02</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">Transcriptional activator (Hap3)</td></tr></tbody></table></alternatives></table-wrap>" ]
[]
[]
[]
[]
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[ "<supplementary-material content-type=\"local-data\" id=\"ppat.1000155.s001\"><label>Table S1</label><caption><p>Gene expression upon 0.6 mM CoCl<sub>2</sub> treatment.</p><p>(444 KB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"ppat.1000155.s002\"><label>Table S2</label><caption><p>All statistically significant genes obtained by SAM analysis that were affected by 0.6 mM CoCl<sub>2</sub> in wild type.</p><p>(78 KB XLS)</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"nt101\"><label>a</label><p>Annotations were obtained from NCBI database (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ncbi.nlm.nih.gov\">http://www.ncbi.nlm.nih.gov</ext-link>) with additional hand editing based on BLAST searches.</p></fn><fn id=\"nt102\"><label>b</label><p>\n<bold>+</bold>, growth similar to WT; <bold>+/−</bold>, slight reduction in growth compared to WT; <bold>−/+</bold>, significant reduction in growth compared to WT; <bold>−</bold>, no growth. <bold>Ts</bold>, temperature sensitive, mutants did not grow at 37°C. <bold><sup>*</sup></bold>, phenotype could not be determined because these mutants did not grow at pH 4.0.</p></fn><fn id=\"nt103\"><label>1</label><p>Multiple mutants were obtained with same insertion sites.</p></fn><fn id=\"nt104\"><label>2</label><p>Based on Southern analysis, these mutants have 2 or more T-DNA insertions. Only the loci that were able to produce PCR product and sequenced were shown.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt105\"><label>a</label><p>Annotations were obtained from NCBI database (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ncbi.nlm.nih.gov\">http://www.ncbi.nlm.nih.gov</ext-link>) with additional hand editing based on BLAST searches.</p></fn><fn id=\"nt106\"><p><bold>*:</bold> Fold change values have been presented as log<sub>2</sub> values.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p>This study was supported by funds from the intramural program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"ppat.1000155.g001\"/>", "<graphic xlink:href=\"ppat.1000155.g002\"/>", "<graphic id=\"ppat-1000155-t001-1\" xlink:href=\"ppat.1000155.t001\"/>", "<graphic xlink:href=\"ppat.1000155.g003\"/>", "<graphic xlink:href=\"ppat.1000155.g004\"/>", "<graphic xlink:href=\"ppat.1000155.g005\"/>", "<graphic xlink:href=\"ppat.1000155.g006\"/>", "<graphic xlink:href=\"ppat.1000155.g007\"/>", "<graphic xlink:href=\"ppat.1000155.g008\"/>", "<graphic id=\"ppat-1000155-t002-2\" xlink:href=\"ppat.1000155.t002\"/>" ]
[ "<media xlink:href=\"ppat.1000155.s001.xls\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"ppat.1000155.s002.xls\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["1"], "element-citation": ["\n"], "surname": ["Kwon-Chung", "Bennett"], "given-names": ["KJ", "JE"], "year": ["1992"], "source": ["Medical Mycology"], "publisher-loc": ["Philadelphia, PA"], "publisher-name": ["Lea & Febiger"]}, {"label": ["41"], "element-citation": ["\n"], "surname": ["Jolly"], "given-names": ["WL"], "year": ["1964"], "source": ["The inorganic chemistry of nitrogen"], "publisher-loc": ["New York"], "publisher-name": ["W.A. Benjamin, Inc"], "fpage": ["68"], "lpage": ["79"]}, {"label": ["51"], "element-citation": ["\n"], "surname": ["Toledano", "Delaunay", "Biteau", "Spector", "Azevedo", "Hohmann", "Mager"], "given-names": ["MB", "A", "B", "D", "D", "S", "WH"], "year": ["2003"], "article-title": ["Oxidative stress responses in yeast."], "source": ["Topics in current genetics"], "publisher-name": ["Springer"], "fpage": ["242"], "lpage": ["303"]}]
{ "acronym": [], "definition": [] }
69
CC0
no
2022-01-13 03:40:34
PLoS Pathog. 2008 Sep 19; 4(9):e1000155
oa_package/98/8c/PMC2528940.tar.gz
PMC2528941
18797503
[ "<title>Introduction</title>", "<p>Successful conservation management of large mammals has the ironic consequence of problems associated with overpopulation ##UREF##0##[1]##. This is particularly so with fragmented, small populations or with keystone species that, at high population densities, can impose negative impacts on the system ##REF##14561329##[2]##. A key uncertainty that emerges is what limits such populations naturally, and whether such limitation will occur at the same levels in human modified systems (e.g. with fences or artificial water) compared to natural systems ##UREF##0##[1]##. Some species may be resource limited, displaying density dependent responses ##REF##11375487##[3]##. Others may be top-down limited by predators ##UREF##1##[4]##. Long-lived species may also be limited by environmental catastrophes, such as drought, flood, fire or disease, which can cause sudden and, at times, significant shifts in population size and dynamics over a very short time, if the effects of such catastrophic impacts on demographics are of sufficient frequency and intensity ##UREF##2##[5]##. Although there is some theoretical and empirical evidence that drought may limit elephant populations ##UREF##3##[6]##, there has been no evaluation of the role that fire may play. Due to their rare occurrence, evaluation of the impacts of such events on population dynamics and individual responses is also rare.</p>", "<p>Fire is commonly applied for ecosystem management in savannas and arson fires occur regularly ##UREF##4##[7]##. Whilst the impact of fire on plant mortality has been extensively researched, there is little research that has assessed the influence of fire on mortality in animals or the welfare issues associated with fire in savanna systems. Given elephants are highly intelligent and social mammals, fire, or other severe disturbances, may also precipitate behavioural or physiological responses. For example, high elephant poaching caused heavily stressed elephants to form larger groups than unstressed elephants ##UREF##5##[8]##.</p>", "<p>The extremely hot, dry, windy (“hot-fire”) conditions experienced towards the end of the dry season in Pilanesberg National Park (PNP), a small (570 km<sup>2</sup>), fenced reserve in South Africa facilitated the spread of an uncontrolled wild fire. The area had a 1–2 year fuel load, with the last pre-fire rains falling in May 2005. Below average (∼630 mm p.a.) annual rainfall of 554 mm was recorded during the 2004/5 wet season, while 824 mm fell in 2003/4 and 411 mm in 2002/3. On 21 September 2005, ambient midday air temperature was 34°C, while wind speed was generally strong but variable in direction. The fire entered the western boundary of the Park near Tlatlaganyane village on 20 September 2005 and within two days had moved across an area of approximately 61 km<sup>2</sup>. This catastrophic fire resulted in the mortality of 29 and injury to 18 elephants, unprecedented in PNP, where few natural elephant mortalities had occurred prior to this event ##UREF##6##[9]##, ##UREF##7##[10]##. This event provided us with the opportunity to assess the potential influence of severe fires in which animals become trapped on the behavioural, physiological and demographic responses of the elephant population. We provide an assessment of (1) the behavioural and physiological responses of the elephants to this large disturbance event, and (2) the potential for rare, stochastic mass-mortality events to limit population size. We examined movement and herd association patterns of six GPS-collared breeding herds, and evaluated physiological response through faecal glucocorticoid metabolite (stress) levels ##UREF##8##[11]##, ##UREF##9##[12]##. We investigated population size, structure and projected growth rates.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Study site</title>", "<p>Pilanesberg National Park (PNP; 25°24′S, 27°08′E; 570 km<sup>2</sup>), North West Province, South Africa, is located within the transition zone of Kalahari Thornveld in the west and Bushveld in the east ##UREF##28##[34]##. The habitat consists mainly of savanna ranging from broadleaf/<italic>Acacia</italic> thickets to open grassland. There are several dams within the Park, one major perennial river system and many ephemeral tributaries and streams. The region has summer rainfall of approximately 630 mm p.a. Geologically, PNP is an extinct volcanic crater formed over 1 200 million years ago and is an example of an alkaline ring complex ##UREF##29##[35]##. The weathering of this complex has created a rugged, hilly landscape, with steep slopes and deep valleys (##FIG##4##Figure 5##). PNP is open to tourists, but has large “wilderness” areas where there is no tourist access, limited management tracks, and is rarely traversed by people (##FIG##4##Figure 5##). This wilderness zone comprises approximately half of the total area of PNP. Elephant were introduced to PNP between 1981 and 1998 ##UREF##30##[36]##. As of early September 2005, the PNP population totalled 165 individually identified elephants, of which 37 were independent adult bulls and 128 were part of 18 relatively stable matriarchal family groups. All individuals in the population were known from unique ear notches and tusk configuration. Pre-fire, the population was made up of 86 juveniles under 10 years of age (56 males, 30 females), 23 10–20 year old adults (10 males, 13 females), as well as 29 adult females and 27 adult males between the ages of 20 and 42 (oldest elephants in population). There had been no mortality from old age, with the first expected to occur in around 15 years time ##UREF##31##[37]##.</p>", "<title>Behavioural response</title>", "<p>We investigated both short-term and long-term responses of the elephants to a fire event that caused elephant injury on the afternoon of 21 September 2005. Short-term responses were investigated over a four and ten-day period pre- and post-fire. Longer-term responses were assessed over three months pre- and post-fire. Prior to the fire, GPS-collars had been fitted to six elephant cows, belonging to different breeding herds within the PNP population. The movement of these elephants (CE03, CE13, CE32, CE61, CE81 and CE88) was assumed to depict the movement behaviour of the breeding herd to which they belonged ##UREF##11##[14]##. Location points were taken at similar times of the afternoon for each cow every day. Members of two of these breeding herds were injured in the fire (CE03 and CE88). CE03 was severely injured, with more than 45% total body surface area (TBSA) burned and CE88 was less severely injured, sustaining burn injuries to approximately 20% TBSA ##UREF##10##[13]##. Analyses pre-fire included data before fire injury occurred, while post-fire analyses included data post-injury.</p>", "<p>All statistical analyses in this paper were performed in SPSS 15.0 (SPSS Inc., Chicago, Illinois, USA) with α = 0.05. In the case of parametric tests, assumptions were tested and satisfied. The work was approved by the Animal Ethics Committee of the University of KwaZulu-Natal.</p>", "<title>Daily displacement</title>", "<p>The distance moved by the collared elephants each day (24 hour fixes) was calculated using polylines in the Animal Movement Extension ##UREF##32##[38]## to ArcView 3.2 (ESRI Inc., Redlands, California, USA). We considered this shortest line between the two readings as an index of daily displacement, and refer to this value as daily displacement hereafter.</p>", "<p>To test whether there was an initial flight directly after the fire, mean daily displacement of all cows over four days before and after the fire was compared using a paired samples t-test. Four day mean daily displacement of injured cows (CE03, CE88) as well as uninjured cow in close proximity to fire during injury (CE32) was tested with a paired-samples t-test and the same was done for four day mean daily displacement of uninjured cows (CE13, CE61, CE81).</p>", "<p>The mean daily displacement during a ten day period before the fire was compared among cows using one-way ANOVA, and the same was done for mean daily displacement for a ten day period, post-flight, after the fire (i.e. day 5–14). A paired-samples t-test was performed on each cow's daily displacement ten days before and post-flight, after the fire. Variability in displacement was assessed using coefficient of variation (CV) for each cow's daily displacement over ten days, pre- and after flight, post-fire; these were contrasted using a paired-samples t-test. We performed the same contrasts of daily displacement from a three month period directly before the fire (21 June 2005–21 September 2005) and after the fire (22 September 2005–22 December 2005).</p>", "<title>Home range</title>", "<p>Each cow's 24-hourly locations for a period of three months pre- and post-fire were mapped in ArcView 3.2. We calculated Kernel home ranges (core home range enclosed by the 50% probability contour and 95% home range enclosed by the 95% probability contour) in animal movement extension SA 2.1 ##UREF##32##[38]##, using least-squares cross-validation (LSCV) smoothing. Both 50% core and 95% home ranges were compared before versus after the fire using paired-samples t-tests. Percentage of overlap between the 95% home range before the fire and after the fire was calculated for each cow according to the following equation ##UREF##33##[39]##:where A<sub>ab</sub> is the area of overlap between home range before the fire (A<sub>b</sub>) and home range after the fire (A<sub>a</sub>). Percentage overlap data, together with the percentage of locations of each cow in either the wilderness or tourist zones of PNP for three months pre- and post-fire, were used to establish whether a shift in home range had occurred subsequent to the fire and to ascertain if the elephants avoided the tourist zone after the disturbance. A paired-samples t-test was used to compare the percentage location of collared cows in the wilderness zone pre- and post-fire.</p>", "<p>An increase in home range size has been reported for elephants in semi-arid environments during the wet season, due to increased access to areas with ephemeral water sources in the wet season ##UREF##34##[40]##, ##UREF##35##[41]##. The first spring rains fell in PNP on 4 November, 2005. In order to examine whether there was a change in home range size and location after the rain (and therefore establish whether any change could be attributed to a seasonal shift in home range alone), kernel home range was calculated for 44 day periods before the fire (9 August–21 September 2005), after the fire but before the rain (22 September–4 November 2005), as well as after the rain (5 November–18 December 2005). Areas of 50% and 95% home ranges for each collared cow over these time periods were compared (paired-samples t-test).</p>", "<p>Approximately 70% of PNP was burnt during the 2005 dry season. Thus during the late dry season (post-fire and before the rains), the availability of forage was similar throughout the Park in terms of fire-impacted vegetation. We therefore did not consider the post-burn condition of the vegetation as a bias to elephant movement decisions over the study period.</p>", "<title>Herd fission/fusion</title>", "<p>To determine whether the breeding herds showed a ‘fission’ or ‘fusion’ response (i.e. whether breeding herds came together or dispersed, respectively) following the fire, we compared the number of matriarchs (where one matriarch indicates the presence of one herd) seen together in the three-month period pre- and post-fire. A herd's grouping tendency was represented by the percentage fusion, calculated as the number of sightings of a particular herd with other breeding herds, as a percentage of the total number of sightings of that herd. For each herd, percentage fusion was calculated before the fire and after the fire. Because injured and uninjured herds showed opposite fusion trends, a t-test on the difference between pre- and post-fire percentage fusion was used to compare injured and uninjured collared herds. Injured cow fusion response over three months post-fire was further broken down into the first two months post-fire, and the month following that, to examine any change in association pattern on partial recovery from injury.</p>", "<title>Physiological stress response</title>", "<p>Severe, persistent stress can cause glucocorticoid levels to increase and remain elevated ##UREF##36##[42]##. The measurement of glucocorticoid metabolite levels in elephant faeces has proven a useful non-invasive way of investigating stress levels in African elephants ##UREF##9##[12]##, ##REF##11205170##[43]##, ##REF##14511986##[44]##. A total of 171 fresh (i.e. &lt;6 hours since deposition) elephant faecal samples were collected randomly from the PNP population in the three-month periods before and after the fire. Some samples were collected from known individuals at the time of deposition. Those that were not, were classified according to the approximate age of the elephant from which they came ##UREF##37##[45]##, where age was estimated from dung bolus size. Average dung bolus diameter greater than 16 cm was considered to belong to adult bulls; all adult cows in the PNP population, with the exception of one, were below 30 years of age at the time of sample collection, corresponding to dung bolus diameters of approximately 14 cm. For anonymous samples with a bolus &gt;16 cm in diameter, the sample was considered to originate from an adult bull if it was from a site where a single track indicated the presence of a large, solitary elephant. Samples were assigned to cows if they were collected from a site where tracks indicated breeding herd activity and if bolus diameters were between 10–14 cm. Samples with a dung bolus diameter &lt;10 cm were considered to belong to juveniles.</p>", "<p>Faecal glucocorticoid metabolite levels were measured using methods involving the use of a corticosterone I<sup>125</sup> radioimmunoassay (RIA) kit (MP Biomedicals, Costa Mesa, California, USA) ##UREF##38##[46]##, ##REF##11121291##[47]##. This assay has been validated and used for elephants ##UREF##9##[12]##, ##REF##11121291##[47]##.</p>", "<p>Differences in glucocorticoid levels among adult males, adult females and juveniles, pre- and post-fire, were compared using two-way ANOVA. Repeated measures ANOVA was not used because samples before and after the fire were not necessarily, and likely improbably, from the same individuals.</p>", "<p>The effect of season on stress levels was examined to establish whether any change in stress hormone level after the fire was consistent with the onset of the first spring rains and thus a seasonal change in stress hormone level. An analysis (two-way ANOVA) of stress hormone levels was carried out over 44 day periods post-fire (i.e. before the rain 22 September–4 November 2005 and after the rain 5 November–18 December 2005). Wet and dry season stress levels for different elephant states (juveniles, adult cows and adult bulls) were compared.</p>", "<title>Demography</title>", "<p>The effect of the fire on PNP elephant population size and age structure was assessed by accounting for all mortalities (categorized according to age and sex) and comparing population size and age structure before the fire with that after the fire. A G-test was used to assess if age structure was different, by separating total count data into 10-year and 4-year age classes and comparing the number of elephants in each age class pre- and post-fire.</p>", "<p>The potential for fire to limit the population was considered using a probabilistic age and state model ##UREF##39##[48]##. The model was used to calculate population size over 30 years, a time-period relevant for conservation management decision-making, using population data (1) before the fire, and (2) after the fire. The model was also used to determine how often a fire of this nature would need to occur for long-term population growth rate to be reduced to zero over a period of 300 years, to allow for ample reproductive generations and growth. Population growth rate was calculated using projected population size from demographic data before the fire and after the fire, according to the following standard equation for exponential population growth:where r = (ln <italic>Nt</italic>\n<sub>2</sub>–ln <italic>Nt</italic>\n<sub>1</sub>)/<italic>t</italic> and <italic>Nt</italic>\n<sub>1</sub> and <italic>Nt</italic>\n<sub>2</sub> are population size at the beginning and end of the time interval in question, respectively; and <italic>t</italic> is the length of the time span in years.</p>", "<p>The model incorporated aspects of the life history of individuals and the following important demographic parameters according to acceptable values from the literature: maximum expected lifespan of 60 years ##UREF##14##[18]##, ##UREF##31##[37]##, female age at sexual maturity of ten years ##UREF##7##[10]##, ##UREF##25##[31]##, average calving interval for the population of four years ##UREF##25##[31]##, ##UREF##40##[49]##, age at menopause of 50 years ##UREF##31##[37]##, ##UREF##41##[50]## and a 1:1 sex ratio of newborns ##UREF##25##[31]##, ##UREF##40##[49]##. These parameters were a slightly conservative estimate of those estimated from past elephant demographic patterns in PNP ##UREF##7##[10]##.</p>", "<p>The model used was a probabilistic matrix model, where numbers of individuals of different ages were transitioned through specific biological states, i.e. males; sexually immature females; sexually mature, non-pregnant females; pregnant females (in first or second year of pregnancy); females in the first, second, third or fourth year post-parturition. Males were aged but not transitioned through specific states. All parameters other than average calving interval were input as probabilities for each age and state, determined by comparison of input probability value with one obtained from a random number generator that produced a normal distribution of values between 0 and 1. The statistical variation introduced by the probabilistic approach was determined by repeating each simulation 500 times and the means and standard deviations were calculated from these replicate simulations. The population was recorded at the end of each year of a simulation.</p>" ]
[ "<title>Results</title>", "<title>Behavioural response</title>", "<title>Daily displacement</title>", "<p>There was no significant difference in mean daily displacement over four days before versus after the fire for all cows (t<sub>5</sub> = −1.238, <italic>P</italic> = 0.271). However, injured herds (CE03, CE88) and a herd in close proximity to the fire at time of injury (CE32) moved significantly further per day after the fire than before (t<sub>2</sub> = −6.915, <italic>P</italic> = 0.020), while there was no significant difference in mean daily displacement of uninjured herds (CE13, CE61, CE81) over four days before versus after the fire (##FIG##0##Figure 1##).</p>", "<p>There was no significant difference in daily displacement among collared cows over a ten-day period before the fire (F<sub>5, 54</sub> = 0.305, <italic>P</italic> = 0.908). However, there was a significant difference in daily displacement among collared cows over the ten-day period, post-flight, after the fire (F<sub>5, 54</sub> = 9.346, <italic>P</italic>&lt;0.0005). Injured cows (CE03 and CE88) moved at a significantly slower rate in the ten days after the fire (t<sub>9</sub> = 4.486, <italic>P</italic> = 0.002; t<sub>9</sub> = 2.756, <italic>P</italic> = 0.022 respectively), compared with ten-day daily displacement before the fire (##FIG##0##Figure 1##). Uninjured cows CE61, CE81, CE32 and CE13, did not show a significant change in daily displacement in the ten day period before versus after the fire (t<sub>9</sub> = −0.450, <italic>P</italic> = 0.663; t<sub>9</sub> = −0.930, <italic>P</italic> = 0.928; t<sub>9</sub> = 1.084, <italic>P</italic> = 0.307; t<sub>9</sub> = 0.745, <italic>P</italic> = 0.476 respectively) (##FIG##0##Figure 1##). There was no significant difference in the coefficients of variation (CV) of daily displacement for the ten-day periods pre- and post-fire (t<sub>9</sub> = −2.064, <italic>P</italic> = 0.094), but a general trend of increased variability is evident for those herds involved in the fire (CE03, CE88) or those close to the fire when injuries occurred (CE32) (##FIG##0##Figure 1##).</p>", "<p>There was no significant difference in daily displacement among collared cows over a three-month period before the fire (F<sub>5, 540</sub> = 1.709, <italic>P</italic> = 0.131). However, there was a significant difference in daily displacement among collared cows over a three-month period post-fire (F<sub>5, 540</sub> = 5.720, <italic>P</italic>&lt;0.001). The daily displacement over three months for injured cows CE03 and CE88, as well as CE88's new matriarch CE13, were not statistically different, while daily displacement for CE88 and CE13 was not statistically different from uninjured cows (CE81, CE61 and CE32) (##FIG##0##Figure 1##). Uninjured cows CE81 and CE61 showed significant increase in their daily displacement during three months post-fire (t<sub>90</sub> = −3.664, <italic>P</italic>&lt;0.001; t<sub>90</sub> = −3.830, <italic>P</italic>&lt;0.001) (##FIG##0##Figure 1##). Severely injured cow CE03 showed a significant decrease in three-month daily displacement post-fire (t<sub>90</sub> = −3.240, <italic>P</italic>&lt;0.0005) (##FIG##0##Figure 1##). Less severely injured cow CE88, matriarch CE13 and uninjured cow CE32 showed no significant difference in three month daily displacement before versus after the fire (t<sub>90</sub> = −1.337, <italic>P</italic> = 0.185; t<sub>90</sub> = −0.747, <italic>P</italic> = 0.457; t<sub>90</sub> = −1.641, <italic>P</italic> = 0.104 respectively) (##FIG##0##Figure 1##). There was a significant difference between pre- and post-fire CV in daily displacement over three months (t<sub>9</sub> = −2.984, <italic>P</italic> = 0.031), with a general trend of increase in variability post-fire (##FIG##0##Figure 1##).</p>", "<title>Home range</title>", "<p>There was no significant difference in home range size before and after the fire among all cows (50% kernel size, t<sub>5</sub> = 0.505, <italic>P</italic> = 0.635; 95% kernel home range, t<sub>5</sub> = −0.024, <italic>P</italic> = 0.982). Only severely injured cow CE03 reduced the size of her core home range (36.1 km<sup>2</sup> to 6.3 km<sup>2</sup>) and 95% home range (305.9 km<sup>2</sup> to 71.6 km<sup>2</sup>) dramatically after the fire and her home range shifted from the central areas of the Park to the south-eastern wilderness area (##FIG##1##Figure 2##).</p>", "<p>Cows spent significantly more time in the wilderness areas of the Park in the three months after than in the three months before the fire (t<sub>5</sub> = −4.510, <italic>P</italic> = 0.006). Percentage overlap of home ranges indicated a shift in home range location post-fire (##FIG##1##Figure 2##).</p>", "<p>There was no significant difference in core (50% kernel) range size over the 44 day period before versus after the fire (t<sub>5</sub> = −1.290, <italic>P</italic> = 0.267). However, the size of 95% kernel home range differed significantly over this time period (t<sub>5</sub> = −3.753, <italic>P = </italic>0.020), with most cows having a larger 95% home range after the fire. Home range size for the 44 day period after the first spring rains was not significantly different to home range size before the rain (50%: t<sub>5</sub> = 0.096, <italic>P</italic> = 0.928; 95%: t<sub>5</sub> = 0.654, <italic>P</italic> = 0.542). Percentage overlap of before rain 95% home range and after rain 95% home range was 47.2%, 80.5%, 73.2%, 90.7%, 64.8% and 78.1% for CE03, CE13, CE32, CE61, CE81 and CE88 respectively. Therefore, all except severely injured cow CE03 had similar 95% home range location before versus after the rain. This suggests that the change in season post-fire was not the reason for the change in 95% home range we observed.</p>", "<title>Herd fission/fusion</title>", "<p>The time spent associating with other herds pre- and post-fire was significantly different for uninjured versus injured cows (t<sub>4</sub> = −3.675, <italic>P</italic> = 0.021). For the first two months post-fire, fission behaviour was exhibited by injured cows, with CE03 and CE88 spending only 10.3 % and 34.7 % of their time associating with other herds respectively, compared with 91.2 % and 62.2 % respectively before the fire. In the third month post-fire, CE03 exhibited increased fusion behaviour, with association time increasing from 10.3 % to 43.8 % and CE88 joined uninjured collared cow CE13 (permanent association to August 2008). Uninjured cows generally exhibited greater fusion behaviour after the fire.</p>", "<title>Physiological stress response</title>", "<p>While pre- versus post-fire measurement, in general, had no significant effect on stress hormone levels (F<sub>1, 133</sub> = 0.261, <italic>P</italic> = 0.610), there was a significant difference among elephant age-sex classes (i.e. juvenile, adult bull, adult cow) (F<sub>2, 133</sub> = 16.155, <italic>P</italic>&lt;0.001). There was also a significant interaction between pre- versus post-fire and elephant age-sex class (F<sub>2, 133</sub> = 4.240, <italic>P</italic> = 0.016). Before the fire, adult cow and juvenile stress levels were not significantly different, but were both significantly lower than adult bull stress levels (##FIG##2##Figure 3##). Cow stress levels increased significantly post-fire but juvenile and bull stress levels were unchanged by the fire (##FIG##2##Figure 3##).</p>", "<p>Evaluation of stress hormone levels before versus after the first Spring rain fell showed that there was no significant change in stress hormone levels between wet and dry 44 day periods (F<sub>1, 98</sub> = 0.015, <italic>P</italic> = 0.902).</p>", "<title>Demography</title>", "<p>Five family units and six independent adult bulls suffered burn injuries (47 individuals), of which 29 mortalities occurred (17.6% of pre-fire population total) (##TAB##0##Table 1##). Five juveniles between six and ten years of age, 11 adult females and two adult males recovered from their burn injuries (##TAB##0##Table 1##). Fifteen infants (≤3 years old), seven weaned calves (4 to 10 years old), four adult females and three adult males died, either as a direct result of their burn injuries, or from euthanasia implemented by Park authorities due to the severity of their injuries (##TAB##0##Table 1##). Initial post-fire assessment resulted in the euthanasia of two of the four adult females and all three adult males, with veterinarians deciding on strict euthanasia criteria which included &gt;50% burns to total body surface area, marked oedema, eschars, severe supparative oozing and severe impairment of mobility due to burn lesions. Seventeen of the injured juveniles were taken to a holding facility off-site and their wounds treated. Only two of these elephants survived and were released back into the Park. Of the fifteen juveniles that died, ten were euthanazed, with euthanasia criteria including &gt;50% burns to total body surface area, large skin surface area with open tissue, comparative behavioural records indicating severe pain and distress, collapse without recovery after revival, as well as low blood protein and calcium. Euthanasia was only considered in cases where recovery was impossible (criteria for recovery see ##UREF##10##[13]##) and thus mortality can be considered representative of natural fire mortality.</p>", "<p>Age structure, classified according to 10-year and 4-year age classes, was significantly different after the fire than before (10-year age classes: G<sub>3</sub> = 70.637, <italic>P</italic>&lt;0.001; 4-year age classes: G<sub>3</sub> = 71.598, <italic>P</italic>&lt;0.001). Model projections over 30 years showed no change in projected population growth rates achieved using demographic data before the fire, as well as demographic data after fire mortalities were accounted for (6.5% p.a.). It took four years for the projected population to recover to the pre-fire population size of 165 individuals (##FIG##3##Figure 4##). In the absence of fire, the population was projected to grow to 303, 577, and 1079 individuals in 10, 20, and 30 years respectively (##FIG##3##Figure 4##). Taking into account the effects of fire on the population structure, the population was projected to reach 255, 485, and 903 individuals in the same timeframes (##FIG##3##Figure 4##). For this type of mortality event to reduce long-term population growth rate to 0%, it would be required at a frequency of every three to four years (##FIG##3##Figure 4##).</p>" ]
[ "<title>Discussion</title>", "<p>A large disturbance event causing catastrophic injury and mortality has consequences that can significantly affect the functioning and behaviour of an elephant population. In response to a catastrophic fire in PNP, injured elephant cows showed an initial short-term (lasting about four days) flight response post-fire, a longer-term (over about ten days to three months) decrease in daily displacement, a shift in home range, social withdrawal, seclusion to non-tourist areas, and significantly higher stress levels. However, behavioural responses were not limited to injured individuals alone. Uninjured cows also showed altered physiological and behavioural responses post-fire. These cows had significantly raised stress levels, a general increase in daily displacement, more variability in daily distance moved, withdrawal to non-tourist areas and a herd fusion response. Injured herds therefore may have signalled their distress to uninjured herds. Elephant family groups that show a high frequency of association have been known to act in a co-coordinated manner, due to the complex social behaviour and long-range communication used by elephants ##UREF##11##[14]##, ##REF##10877888##[15]##. The stress of injury, together with social disruption due to the loss of and injury to family members is likely to have affected the behaviour of injured breeding herds. This is additional to the increased vulnerability of injured juveniles to predation, or the compromised ability of injured adult cows to protect their young, which would have increased stress levels. The incidence of elephant calf predation has been found to increase during times of drought when nutritional stress and dehydration facilitates the circumstances where calves can lag behind the herd and become vulnerable to predators ##UREF##12##[16]##. Injured calves were seen alone in PNP after the fire (pers. obs.), increasing their vulnerability to predation.</p>", "<p>The physiological and behavioural responses apparent in the PNP population post-fire are consistent with elephant reactions to stressful conditions. Breeding herds showed raised stress levels and a fusion response to cow immobilizations and high-volume tourist activity in PNP ##UREF##6##[9]##. Working elephants in a safari operation had high stress hormone levels associated with transportation and episodic loud noises, such as lightning and thunderstorms and human-induced activities, with baseline levels of faecal glucocorticoid metabolites for adult elephants in PNP of approximately 25 ng.g<sup>−1</sup>\n##UREF##9##[12]##. Heavily stressed elephants, responding to high levels of poaching, formed larger groups than unstressed elephants ##UREF##5##[8]##. Stress responses to culling in Kruger National Park were initial flight, taking elephants outside of home ranges ##UREF##13##[17]##, as well as the movement of elephants into and out of culling regions in response to culling events ##UREF##14##[18]##, ##UREF##15##[19]##. As these studies indicate, elephants are stressed by human-induced and natural disturbances. Stressed animals alter their behaviour in an attempt to eliminate the stressor. Thus, shifts in home range and seclusion to non-tourist areas are predictable, adaptable responses to disturbance. Therefore, a fire event resulting in elephant mortality has the potential to induce severe behavioural and physiological stress responses (see ##UREF##16##[20]## review of trauma effects on neuroendochrinological development of elephants, and subsequent non-normative behaviour). Whereas drought may cause elephant mortalities over an extended period of time ##UREF##17##[21]##, fire mortality occurs within a short time period after the event. Long-term elephant behavioural response to fire mortality may therefore persist, due the dramatic and traumatic nature of the event ##UREF##16##[20]##.</p>", "<p>The demographic impact of fire on the PNP elephant population predominantly involved the mortality of juveniles (76% of total mortality). Among large herbivore populations where predators are absent, high temporal variation in juvenile survival is often seen, with fairly constant adult survival ##REF##21238011##[22]##–##UREF##18##[24]##. In systems where large predators are present, both adult and juvenile survival responds to environmental variability, due to interactions between resource availability, population size and predation pressure ##UREF##1##[4]##. Without constant predation pressure, the natural mortality patterns often seen in African elephant populations during stochastic events, such as droughts, follows that of the classic mortality pattern seen in predator-free large ungulate populations, which mainly involves juveniles ##UREF##17##[21]##, ##UREF##19##[25]##, ##UREF##20##[26]##. Fire therefore functions in a similar manner to other environmental catastrophes.</p>", "<p>Population structure prior to the fire was significantly different post-mortality, due to predominant mortality in the juvenile age-class. In effect, the loss of a high proportion of juveniles serves not only to lower population size, but also to increase calving interval where the lost calf creates a gap between siblings. However, among large herbivore populations, population growth is most sensitive to adult mortality, especially that of prime-aged females ##REF##21238011##[22]##–##UREF##18##[24]##, ##UREF##23##[29]##. The mortality of only four adult females from the PNP population as a result of the fire meant that projected population growth rate remained unchanged. Therefore, the ability of this type of stochastic, catastrophic mortality exhibited in the PNP fire to limit population size or growth would require higher or more frequent mortality, would need to include a higher proportion of adult females ##UREF##15##[19]##, ##UREF##23##[29]##, or cause demographic delays such as a decline in conception rates, increased inter-calving interval, or increased age at sexual maturity ##UREF##7##[10]##, ##UREF##24##[30]##.</p>", "<p>In order to reduce the PNP elephant population growth rate to zero, this type and level of mortality event would be required at a frequency of approximately three to four years. This gives an indication of the resilience of elephant populations to environmental perturbation. The demographic response of populations to episodic mortality is influenced by the life-history characteristics of the species. Elephant life-history is typical of large-bodied ungulates in that these mammals have long generation times, low fecundity and high adult survival ##UREF##24##[30]##, ##UREF##25##[31]##. In elephant populations, a unique combination of life-history traits prolong demographic response to environmental disturbance ##UREF##24##[30]## and maximum population growth rate tends to be maintained until the very limit where forage resources can no longer support the population before density dependent feedbacks occur ##UREF##0##[1]##, ##UREF##26##[32]##, ##UREF##27##[33]##. Therefore, stochastic mortality alone has the potential to limit short-term population size, but is unlikely to affect population growth over the long-term. However, in combination with density-dependent effects, elephant populations may be limited by environmental catastrophes and the stochasticity brought about by temporal variation in resources. Thus when populations are close to carrying capacity, and background mortalities are higher and fecundities lower than observed here, less intense mortality would be required to achieve a stabilizing effect, and longer intervals between periodic catastrophes could still result in fire-induced mortalities influencing demographics substantially. Catastrophic fires are likely to be rare events with expected return in the order of decades ##UREF##21##[27]##. Therefore in isolation these events will not provide population regulation, but in combination with other stochastic environmental events and density-dependent feedbacks, they may play a role in population limitation. Thus removal of fire from the system in some actively managed nature reserves may not only be detrimental to the vegetation ##UREF##4##[7]##, ##UREF##22##[28]##, but also the dynamics of herbivore populations where fire mortality may be avoided. These fire events should not be considered as negative catastrophes but instead as integral to the savanna system, with the potential to make infrequent but positive contributions to the regulation of abundant herbivore populations.</p>", "<p>Burning during the late dry season, under “hot-fire” conditions when fires can be very intense, can result in catastrophic mortality of large mammal species. Arson fires during these times have the potential to impact not only the vegetation of the area, but also raise welfare concerns over any animals affected by the fire due to the significant stress responses and behavioural changes which may occur. The conservation status and abundance of species will influence fire management requirements. The contribution of fire mortality to abundant game species (e.g. blue wildebeest (<italic>Connochaetes taurinus</italic>), impala (<italic>Aepyceros melampus</italic>)) population dynamics may be less problematic than to that of threatened species such as the black rhino (<italic>Diceros bicornis</italic>), which would conversely require extreme awareness of the need to prevent potentially intense catastrophic fires to ensure minimum mortality impacts. If herbivore populations are fairly stable, even a rare catastrophic fire could cause a shift in population dynamics that, in combination with the current factors causing regulation, may cause a population decline. Therefore, the integration of the conservation implications of intense, hot-fire suppression (i.e. removal of a potential contributing factor to natural population regulation), the welfare concerns of arson fires and burning during “hot-fire” conditions into fire management strategies for conservation areas is important.</p>" ]
[]
[ "<p>Conceived and designed the experiments: LAW RS. Performed the experiments: LAW. Analyzed the data: LAW RLM. Contributed reagents/materials/analysis tools: LAW JM RJW SJvR BP. Wrote the paper: LAW RS.</p>", "<p>In predator-free large herbivore populations, where density-dependent feedbacks occur at the limit where forage resources can no longer support the population, environmental catastrophes may play a significant role in population regulation. The potential role of fire as a stochastic mass-mortality event limiting these populations is poorly understood, so too the behavioural and physiological responses of the affected animals to this type of large disturbance event. During September 2005, a wildfire resulted in mortality of 29 (18% population mortality) and injury to 18, African elephants in Pilanesberg National Park, South Africa. We examined movement and herd association patterns of six GPS-collared breeding herds, and evaluated population physiological response through faecal glucocorticoid metabolite (stress) levels. We investigated population size, structure and projected growth rates using a simulation model. After an initial flight response post-fire, severely injured breeding herds reduced daily displacement with increased daily variability, reduced home range size, spent more time in non-tourist areas and associated less with other herds. Uninjured, or less severely injured, breeding herds also shifted into non-tourist areas post-fire, but in contrast, increased displacement rate (both mean and variability), did not adjust home range size and formed larger herds post-fire. Adult cow stress hormone levels increased significantly post-fire, whereas juvenile and adult bull stress levels did not change significantly. Most mortality occurred to the juvenile age class causing a change in post-fire population age structure. Projected population growth rate remained unchanged at 6.5% p.a., and at current fecundity levels, the population would reach its previous level three to four years post-fire. The natural mortality patterns seen in elephant populations during stochastic events, such as droughts, follows that of the classic mortality pattern seen in predator-free large ungulate populations, i.e. mainly involving juveniles. Fire therefore functions in a similar manner to other environmental catastrophes and may be a natural mechanism contributing to population limitation. Welfare concerns of arson fires, burning during “hot-fire” conditions and the conservation implications of fire suppression (i.e. removal of a potential contributing factor to natural population regulation) should be integrated into fire management strategies for conservation areas.</p>" ]
[]
[ "<p>We thank Pilanesberg National Park management (North-West Parks and Tourism Board) for logistical support and permission to undertake this study. Special thanks to Hans Meier for the use of ParkControl© software, facilitating elephant location in the field, and to Karen Trendler for valuable information on euthanasia criteria and juvenile elephant response to severe burn wounds. Brian van Wilgen contributed valuable comments to the manuscript.</p>" ]
[ "<fig id=\"pone-0003233-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003233.g001</object-id><label>Figure 1</label><caption><title>Behavioural response to fire giving mean daily displacement (±95% CL) of each collared cow over: (A) 4 days, (B) 10 days and (C) 3 months, before and after the fire.</title><p>Coefficients of variation (CV (%)) of daily displacement before or after the fire are given above or below the upper or lower limit of CL bars. Pre-fire CV's are located above if the value of pre-fire mean±95% CL is located above (i.e. is greater than) post-fire mean±95% CL. Where mean±95% CL pre- and post-fire are equal, pre-fire CV's are located below CL bar.</p></caption></fig>", "<fig id=\"pone-0003233-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003233.g002</object-id><label>Figure 2</label><caption><title>Behavioural response to fire showing core home range and 95% home range kernels for:</title><p>uninjured adult cows (A) CE13, (B) CE81, (C) CE61, (D) CE32 and injured adult cows (E) CE88 and (F) CE03 three months (a) before and (b) after injury in a fire on 21 September 2005 in Pilanesberg National Park. The percentage overlap between 95% home ranges before and after the fire was: (A) 80.5, (B) 72.7, (C) 49.7, (D) 86.0, (E) 74.1 and (F) 52.5 respectively.</p></caption></fig>", "<fig id=\"pone-0003233-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003233.g003</object-id><label>Figure 3</label><caption><title>Physiological response to fire indicated by glucocorticoid metabolite (stress) levels (mean±95% CL) for adult bulls, adult cows and juveniles before and after the fire.</title><p>Sample size (n) is shown above each category.</p></caption></fig>", "<fig id=\"pone-0003233-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003233.g004</object-id><label>Figure 4</label><caption><title>The effect of fire on the future Pilanesberg National Park elephant population:</title><p>(A) comparative modelled projection over a 30 year period using population data before and then after the fire in September 2005; (B) effect of three year and four year fire frequency on population size over a 300 year period using population data before the fire, and the mortality parameters associated with this fire event.</p></caption></fig>", "<fig id=\"pone-0003233-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003233.g005</object-id><label>Figure 5</label><caption><title>Map of Pilanesberg National Park incorporating 20 m contours, tourist roads, management tracks, the site where elephants were injured in a fire on 21 September 2005 and the approximate extent of the fire.</title></caption></fig>" ]
[ "<table-wrap id=\"pone-0003233-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003233.t001</object-id><label>Table 1</label><caption><title>Demographic response to fire: elephant mortality and survival recorded after a fire in Pilanesberg National Park on 21 September 2005, giving the herd of origin and an estimate of elephant age are given (as of December 2005).</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Herd</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Collared cow</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Age of individuals in different categories <xref ref-type=\"table-fn\" rid=\"nt101\">*</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Injured and died</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Injured and survived</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gold</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CE 57</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1, <bold>2</bold>, <bold>3</bold>, 4, 8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>4</bold>, <bold>8</bold>, 8, <bold>10</bold>, <bold>25</bold>, <bold>30</bold>, <bold>35</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Monica</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CE 98</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1, <bold>2</bold>, 4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>10</bold>, <bold>12</bold>, <bold>30</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Red</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CE 07</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>2</bold>, 2, 3, <bold>35</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8, <bold>12</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Sheena</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CE 88</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2, 2, <bold>3</bold>, 4, 4, 6, <bold>30</bold>, <bold>30</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8, <bold>20</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yellow</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CE 03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1, <bold>1</bold>, <bold>2</bold>, 4, 5, <bold>30</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>15</bold>, <bold>42</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adult bulls</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">12, 15, 20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12, 15</td></tr></tbody></table></alternatives></table-wrap>" ]
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[ "<table-wrap-foot><fn id=\"nt101\"><label>*</label><p>Females are indicated in bold.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>Funding received from Amarula (Distell (Pty) Ltd), PPC Cement, National Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></fn-group>" ]
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[]
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{ "acronym": [], "definition": [] }
50
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 17; 3(9):e3233
oa_package/6c/4d/PMC2528941.tar.gz
PMC2528942
18795098
[ "<title>Introduction</title>", "<p>Molecular analyses have been used to study higher-level relationships among the Scleractinia only comparatively recently, in part because of technical difficulties. Rates of molecular evolution in mitochondrial genes are extremely slow in anthozoans ##REF##12453233##[1]##, and finding informative nuclear markers is generally challenging. Even so, results to date have revolutionized our understanding of relationships, suggesting that first, traditional subordinal classifications are largely unsupported by molecular data ##UREF##0##[2]##–##UREF##1##[4]##; second, many families are not monophyletic ##UREF##0##[2]##–##REF##16221328##[7]##; and third, the order Corallimorpharia, an anthozoan group lacking skeletons, may be imbedded in the skeleton-possessing scleractinians ##REF##16754865##[8]##, but see ##UREF##3##[9]## and ##UREF##4##[10]##. Despite these findings, it remains the case that no study to date has examined the phylogeny of Scleractinia using a consistent combination of genes for most genera (including both Pacific and Atlantic representatives) across most families. Here we provide such an analysis, focusing on those scleractinian taxa with large numbers of zooxanthellate species, as well as members of the Corallimorpharia and other anthozoan outgroups.</p>", "<p>We analyzed 127 species in the order Scleractinia (Online Supporting Information, ##SUPPL##0##Table S1##), representing 75 genera and 17 families, 16 of which have important reef-building species. This represents a substantial expansion of a previous study ##REF##14985760##[6]## that considered only seven families of robust corals. We included a member of the azooxanthellate family Fungiacyathidae because of its possibly close relationship with corallimorpharians ##UREF##5##[11]##, but did not analyze members of the largely azooxanthellate Rhizangiidae and Caryophylliidae or other exclusively azooxanthellate families studied by others ##UREF##6##[12]##, ##UREF##7##[13]##, as our focus was on reef-building groups. To test the recently suggested hypothesis that the skeletonless Corallimorpharia are imbedded within the Scleractinia, we analyzed seven genera in this order, including members of both the Corallimorphidae and Discosomatidae ##UREF##8##[14]##. Outgroups consisted of members of the hexacorallian orders Antipatharia, Actiniaria, and Zoanthidea, which are the closest relatives of the Scleractinia and Corallimorpharia ##UREF##5##[11]##. The initial analyses were based on two mitochondrial genes: cytochrome oxidase I (<italic>cox1</italic>) and cytochrome b (<italic>cob</italic>). A subset of taxa was analyzed using two nuclear gene regions: ß-tubulin and/or parts of the nuclear ribosomal genes (it was not possible to amplify both nuclear genes for all taxa). The nuclear analyses were targeted to test mitochondrial results that strongly contradicted traditional morphological classification. Unless noted otherwise, all conclusions are supported by both the mitochondrial dataset (##FIG##0##Fig. 1##) and at least one of the two nuclear datasets (##FIG##1##Figs. 2##, ##FIG##2##3##).</p>" ]
[ "<title>Materials and Methods</title>", "<title>Collections and DNA extraction</title>", "<p>We collected 15 cm×15 cm samples from the following locations: Bocas del Toro, Republic of Panama (Caribbean coast); Carrie Bow Cay, Belize; Togian Island, Indonesia; Aka Island, Okinawa and Shirahama, Wakayama, Japan; Yeiliu, Suao, Kenting National Park and Penghu Island, Taiwan (see Supporting Information ##SUPPL##0##Table S1##). Total DNA extraction method is as described in Fukami et al. ##REF##14985760##[6]##.</p>", "<title>DNA sequencing</title>", "<p>The mitochondrial <italic>cox1</italic> gene was amplified by polymerase chain reaction (PCR) with previously reported primers MCOIF and MCOIR ##REF##14985760##[6]## for most “robust” corals. For a few “robust” coral species (especially pocilloporids), “complex” corals, corallimorpharians and other hexacorallians, combinations (e.g. primer pair of AcMCOIF and SeaMCOIR for PCR) of the following newly designed primers were used: AcMCOIF (<named-content content-type=\"gene\">5′-GAC ATG GCT ATT TTT AGC CT-3′</named-content>), SeaMCOIF (<named-content content-type=\"gene\">5′-CTA CTA ATC ATA AAG ATA TCG G-3′</named-content>), AcMCOIR (<named-content content-type=\"gene\">5′-AAG CAT AGG AGT GTC GTC TAA TC-3′</named-content>), SeaMCOIR (<named-content content-type=\"gene\">5′-CAA AGT CAG AGT ATC GTC TTG G-3′</named-content>).</p>", "<p>Mitochondrial <italic>cob</italic> was amplified by PCR with previously reported primers MCytbF and MCytbR for “robust” corals ##REF##14985760##[6]##, and with newly designed primers AcCytbF (<named-content content-type=\"gene\">5′-GCC GTC TCC TTC AAA TAT AAG-3′</named-content>) or MCytbF, and AcCytbR (<named-content content-type=\"gene\">5′-AAA AGG CTC TTC TAC AAC-3′</named-content>) for “complex” corals and other hexacorallians except for the three sea anemone species. For the sea anemones, SeaCytbF (<named-content content-type=\"gene\">5′-GTG GAA CTT CGG TTC TTT ATT-3′</named-content>) and SeaCytbR (<named-content content-type=\"gene\">5′-ATA CAG AGG CTA ATT GTC C-3′</named-content>) were used for PCR.</p>", "<p>The basic protocol for mitochondrial gene amplifications was 94°C for 120 s, followed by 30 cycles at 94°C for 45 s, 55°C for 45 s and 72°C for 90 s, ending with a final phase of 72°C for 5 min. In the case of amplification failure, the following protocol was used: 94°C for 120 s, followed by first 10 cycles at 94°C for 45 s, 45°C for 45 s and 72°C for 90 s, and next 20 cycles at 94°C for 45 s, 45°C for 45 s and 72°C for 90 s, ending with a final phase of 72°C for 5 min. PCR products were directly sequenced as described previously ##REF##14985760##[6]##.</p>", "<p>The ß-tubulin gene (intron and exon regions) was amplified, cloned, and sequenced following the methods described previously ##REF##14985760##[6]##.</p>", "<p>The ribosomal DNA (rDNA) segment containing the 3′-end of 18S, internal transcribed spacers, 5.8S, and the 5′-end of 28S was amplified, cloned, and sequenced following the methods described elsewhere ##UREF##9##[15]##, ##UREF##17##[26]##.</p>", "<p>DNA sequence accession numbers of all genes analyzed here (AY722757, AY722761, AY722775, AY722781, AY722785, AY722793-6, AB441193-AB441421) are listed in the ##SUPPL##0##Table S1## (Online Supporting Information).</p>", "<title>DNA phylogenetic analyses</title>", "<p>For mitochondrial DNA, we conducted phylogenetic analyses on the dataset [total 1383 bp, combined <italic>cox1</italic> (607 bp) and <italic>cob</italic> (776 bp) without gaps; 666 parsimony-informative sites] by searching for optimal topologies, as measured by the criteria maximum likelihood (ML) and parsimony (MP) methods, as well as by taking a Bayesian approach. For the ML and Bayesian analyses, we assumed a model of nucleotide evolution obtained by using the Akaike Information Criterion (AIC) as implemented in ModelTest ##REF##9918953##[27]##. The most appropriate model of nucleotide evolution for the mitochondrial datasets was the GTR with gamma (G) and invariant (I) parameters (GTR+I+G). The software GARLI ##UREF##18##[28]## was used to search for optimal ML topologies (10 replicate searches started with random trees) and to conduct bootstrap analyses (200 replicates). The program TNT ##UREF##19##[29]## was employed to conduct searches for optimal MP trees (10 replicate tree bisection-reconnection searches with taxa added randomly) and to assess node support with re-sampling methods (1000 replicate bootstrap and jackknife searches). MrBayes ##REF##12912839##[30]## was used to conduct Bayesian analyses (with default priors and nruns = 3, samplefreq = 1000, nchains = 8). The number of generations and burnin in millions for the mitochondrial datasets were 8 and 4, respectively. As judged by the potential scale reduction factor (PSRF), analyses converged.</p>", "<p>Topologies from ML and Bayesian analyses were quite similar. Topology from MP analysis was also largely similar to others obtained, and most nodes in the MP tree that received bootstrap support appeared as part of the Bayesian analysis with a Bayesian posterior probability greater than 0.95. However, topology of the MP tree differed in one major point, namely the phylogenetic position of the corallimorpharians. The MP tree showed a link between the corallimorpharians and the “complex” corals (as in ##REF##16754865##[8]##), whereas ML and Bayesian trees showed corallimorpharians to be outside the scleractinian corals. In this paper, we show the topology of Bayesian analysis with ML bootstrap values and a Bayesian posterior probability. Three species in the order Actiniaria (<italic>Anemonia</italic> sp., <italic>Stichodactyla</italic> sp., <italic>Metridium senile</italic>\n##REF##9539427##[31]##), one species in the order Anthipatharia (<italic>Cirripathes</italic> sp.) and one species in the order Zoanthidea (<italic>Zoanthus</italic> sp.) were used for outgroups in the mitochondrial analyses (##FIG##0##Fig. 1##).</p>", "<p>In order to further assess various phylogenetic hypotheses not found in our best trees, we tested whether our mitochondrial data significantly contradict these suboptimal hypotheses. Using GARLI ##UREF##18##[28]##, we conducted ten replicate searches for most likely topologies constrained to conform to each of 14 hypotheses (##TAB##0##Table 1##). The program CONSEL ##REF##11751242##[32]## was then used to calculate p-values for several tests – including the Approximately Unbiased (AU) ##REF##12079646##[33]##, Kishino-Hasegawa (KH) ##REF##2509717##[34]##, Shimodaira-Hasegawa (SH); ##UREF##20##[35]##, and the weighted SH (WSH) ##REF##12912839##[30]## tests – aimed at measuring how strongly the various hypotheses are contradicted by the data. Because the SH test is only valid when numerous plausible trees are being compared, all resulting trees from the constrained and unconstrained searches were compared.</p>", "<p>For ß-tubulin, previously published DNA sequences ##REF##14985760##[6]## were retrieved from the database and added to newly obtained sequences for phylogenetic analysis. For the phylogenetic tree construction, only the exon region of ß-tubulin was utilized, since the intron region is too diverse to produce reliable alignments above the generic level ##REF##14985760##[6]##. When all taxa were used for the phylogenetic analyses, Bayesian probability and ML bootstrap values were too low to support the main branches due to the limited number of informative sites, probably because of the phylogenetically distant relationships among species. Therefore, data were separated into two data sets (Tub1 for the “complex” group plus some “robust” corals; Tub2 for most robust corals). The aligned ß-tubulin fragment was 443 bp in length, with 123 and 87 phylogenetically informative sites for Tub1 and Tub2, respectively. As described for the mitochondrial analyses, the most appropriate models of nucleotide evolution for the ML and Bayesian analyses were determined to be HKY+I+G for Tub1 and TrN+I+G for Tub2. PAUP 4.0b10 ##UREF##21##[36]## was used for both Tub1 and Tub2 to reconstruct the ML tree. The robustness of the phylogenies was assessed using the 300 bootstrap option. Bayesian trees for Tub1 and Tub2 were constructed as described for mitochondrial analyses. Four simultaneous Markov chains were run for 1,200,000 (Tub1) or 2,000,000 (Tub2) generations; trees were sampled every 100 generations, with 300,000 (Tub1) or 500,000 (Tub2) initial trees discarded as burn-in, based on visual inspection. In this paper, we show the topology of the Bayesian analysis with ML bootstrap values and Bayesian posterior probabilities. One species in the Antipatharia (<italic>Cirripathes</italic> sp.) was used as an outgroup to the Scleractinia and Corallimorpharia.</p>", "<p>For ribosomal DNA, previously published DNA sequences ##UREF##9##[15]##, ##UREF##17##[26]## were retrieved from the database and added to new sequences for phylogenetic analysis. For the phylogenetic tree construction, only the 3′-end of 18S, 5.8S, and the 5′-end of 28S rDNA were utilized, since the internal transcribed spacers are too divergent to produce reliable alignments above the generic level ##UREF##9##[15]##, ##UREF##15##[21]##. The Acroporidae were also excluded from the analysis due to their extremely high rates of rDNA evolution compared to other scleractinian corals ##UREF##9##[15]##. The aligned rDNA fragment was 554 bp in length, and 70 sites were phylogenetically informative. As described for the mitochondrial analyses, the most appropriate model of nucleotide evolution for the ML and Bayesian analyses was determined to be the TIM+I+G model. PAUP 4.0b10 was used to reconstruct the ML tree. The robustness of the phylogenies was assessed using the 1000 bootstrap option. The Bayesian tree was constructed as described for the mitochondrial analyses. Five simultaneous Markov chains were run for 1,000,000 generations with trees sampled every 10 generations, with 50,000 initial trees discarded as burn-in, based on visual inspection. In this paper, we show the topology of the Bayesian analysis with ML bootstrap values and Bayesian posterior probabilities. One member of the Octocorallia (<italic>Heliopora coerulea</italic>), one member of the Zoanthidea (<italic>Sphenopus marsupialis</italic>) and one member of the Actiniaria (<italic>Condylactis</italic> sp.) were used as outgroups to the Scleractinia and Corallimorpharia.</p>" ]
[ "<title>Results</title>", "<title>Phylogenetic relationships within the Scleractinia</title>", "<p>Our molecular results are shown in ##FIG##0##Figures 1##–\n##FIG##2##3##, and summarized below (see also ##SUPPL##1##Table S2## in Online Supporting Information). Overall, of the 16 traditional families of scleractinian corals with reef-building genera that we analyzed, at least 11 are polyphyletic as currently defined: Mussidae, Faviidae, Pectiniidae, Merulinidae, Siderastreidae, Astrocoeniidae, Euphylliidae, Meandrinidae, Poritidae, Agariciidae, and Oculinidae. Five of these (Oculinidae, Euphylliidae, Meandrinidae, Siderastreidae, Astrocoeniidae) have members placed in both of the highly divergent “complex” and “robust” subgroups ##UREF##0##[2]##–##UREF##2##[5]##. One family, the Trachyphylliidae, is trivially distinct and does not merit recognition at the family level. Traditional members of the four remaining families – Acroporidae, Pocilloporidae, Fungiidae, and Dendrophylliidae – cluster together, but the first three of these now contain genera previously assigned to other families. Several genera or groups of genera are so divergent that they will probably need to be assigned to new families. The azooxanthellate Fungiacyathidae was confirmed to be distinctive at the family level (but not closely related to corallimorpharians, see following section). Below we explore these results in somewhat greater detail, comparing the different sources of information (##FIG##0##Figs. 1##–\n##FIG##2##3##), utilizing formal tests of competing phylogenetic hypotheses (##TAB##0##Table 1##) and exploring implications for needed taxonomic revisions (##TAB##1##Table 2##, see also Online Supporting Information ##SUPPL##1##Table S2##).</p>", "<p>The large family Faviidae Gregory, 1900 is one of the most polyphyletic of all scleractinian families in our analyses, with members scattered throughout the “robust” clade (groups XI, XIII, XIV, XV, XVI, XVII, XXI in ##FIG##0##Fig. 1D##; groups X1-a, XI-b, XIII-a, XIII-b, XIV-b, XVI, XVII-a, XVII-b, XXI in ##FIG##1##Fig. 2##; groups XI, XV, XXI-a in ##FIG##2##Fig. 3##; p&lt;.001 for all four tests of monophyly, ##TAB##0##Table 1##). The type for the family (<italic>Favia fragum</italic>) is in a clade that contains the type for the family Mussidae Ortmann, 1890 (<italic>Mussa angulosa</italic>) (group XXI in ##FIG##0##Figs. 1D##, ##FIG##1##2B##) ##REF##14985760##[6]##; thus the family name Faviidae cannot be retained in the formal taxonomic revisions to come, and the Mussidae will include most of the Atlantic “faviids” with the exception of the polyphyletic genus <italic>Montastraea</italic> (which falls in groups XVI and XVII, ##FIG##0##Figs. 1D## and ##FIG##1##2B##). Two other families when newly revised will include other former members of the Faviidae - the Oculinidae (<italic>Solenastrea</italic>, <italic>Cladocora</italic>) (group XIII ##FIG##0##Figs. 1D##, ##FIG##2##3##; group XIII-b but not XIII-a in ##FIG##1##Fig. 2A##) ##REF##14985760##[6]## and the Fungiidae (<italic>Leptastrea</italic>) (group XI in ##FIG##0##Fig. 1D##, group XI-b in ##FIG##1##Fig. 2A##). <italic>Oulastrea</italic> appears to be a highly distinctive outgroup to the Fungiidae (group XI in ##FIG##0##Figs. 1D##, ##FIG##2##3##) that minimally merits recognition at the subfamilial level. <italic>Plesiastrea</italic> is highly divergent, but with no consistently identified close relatives (group XIV in ##FIG##0##Fig. 1D##, group XIV-b in ##FIG##1##Figs. 2A##, ##FIG##2##3##). <italic>Diploastrea</italic> and <italic>Montastraea cavernosa</italic> are also highly divergent within the robust clade (groups XV and XVI in ##FIG##0##Fig. 1D##, group XVI in ##FIG##1##Fig. 2B##; group XV in ##FIG##2##Fig. 3##). The remaining “faviid” genera form a well defined clade, but it also includes all or some members of three other families: the Merulinidae Verrill, 1866, some of the Pectiniidae Vaughan and Wells, 1943 (<italic>Pectinia</italic>, <italic>Mycedium</italic>; monophyly of Pectiniidae rejected at p&lt;.001 for three of four tests, ##TAB##0##Table 1##), and the Trachyphylliidae Verrill, 1901 (group XVII in ##FIG##0##Fig. 1D##, group XVII-a in ##FIG##1##Fig. 2B##) ##REF##14985760##[6]##. Thus this large group could be redescribed under the family name Merulinidae, although it should be noted that even the four original genera of this family do not form a monophyletic assemblage within the larger group (p&lt; = .001 for three of four tests of monophyly, ##TAB##0##Table 1##).</p>", "<p>The large family Mussidae is also polyphyletic (p&lt;.001 or .028 for the four tests of monophyly, ##TAB##0##Table 1##). It includes Atlantic and Pacific clades, the first of which, as noted above, will retain the family name and also include most of the Caribbean “faviids” (group XXI in ##FIG##0##Figs. 1D##, ##FIG##1##2B##) ##REF##14985760##[6]##. One Pacific clade clearly includes most of the Pacific “mussids” and some of the pectinids (<italic>Oxypora</italic>, <italic>Echinophyllia</italic>) (group XIX in ##FIG##0##Figs. 1D##, ##FIG##1##2B##), a grouping that could be recognized at the family or subfamily level. However, the relationships of <italic>Micromussa</italic> and members the genus <italic>Acanthastrea</italic> (and even the monophyly of <italic>Acanthastrea</italic>) are unstable (groups XVIII and XX in ##FIG##0##Figs. 1D##, ##FIG##1##2B##, ##FIG##2##3##). <italic>Blastomussa</italic> is highly divergent and its phylogenetic placement remains unstable across analyses (group XIV in ##FIG##0##Fig. 1D##, XIV-a in ##FIG##1##Fig. 2A##).</p>", "<p>The family Siderastreidae Vaughan and Wells, 1943 consists of two very distantly related clades (monophyly rejected with p&lt;.001 for all four tests, ##TAB##0##Table 1##). The type genus <italic>Siderastrea</italic> (one of only three monophyletic genera with Pacific and Atlantic species, the others being <italic>Acropora</italic> and <italic>Porites</italic>) has no other close relative in our analyses (group IX in ##FIG##0##Figs. 1B##, ##FIG##1##2A##, ##FIG##2##3##), whereas the Pacific genera <italic>Psammocora</italic> and <italic>Coscinaraea</italic> are probably allied to the Fungiidae (group XI in ##FIG##0##Figs. 1D##, ##FIG##2##3##, but with a different placement as group XI-c in ##FIG##1##Fig. 2A##).</p>", "<p>The family Astrocoeniidae Koby, 1890 also contains two highly divergent clades (monophyly rejected at p&lt;.001 for all four tests) – one represented by the Atlantic <italic>Stephanocoenia</italic> (which has no close relatives in our analyses; group VIII in ##FIG##0##Figs. 1B##, ##FIG##2##3##) and the other represented by <italic>Madracis</italic> and <italic>Stylocoeniella</italic>, which are most closely related to the Pocilloporidae (group X in ##FIG##0##Figs. 1C##, ##FIG##2##3##). The type genus for the family is a fossil genus (<italic>Astrocoenia</italic>) that appears to be close to <italic>Stephanocoenia</italic> in morphology.</p>", "<p>Most other families also have gains and/or losses in their memberships. The Oculinidae Gray, 1847 gains the “faviids” <italic>Cladocora</italic> and <italic>Solenastrea</italic>, as noted above, but loses <italic>Galaxea</italic> to the Euphylliidae Veron, 2000 (group V in ##FIG##0##Figs. 1B##, ##FIG##1##2A##) (monophyly for Oculinidae rejected at p&lt;.001 for all four tests, ##TAB##0##Table 1##). The family Euphylliidae also gains <italic>Ctenella</italic> from the Meandrinidae Gray, 1847 (group V in ##FIG##0##Figs. 1B##, ##FIG##1##2A##), and is allied to at least one representative of the genus <italic>Pachyseris</italic> from the family Agariciidae Gray, 1847 as a sister group (group IV in ##FIG##0##Figs. 1B##, ##FIG##1##2A##) (monophyly of Euphylliidae and Meandrinidae rejected at p&lt;.001 by four of four tests, monophyly of Agariciidae rejected at p&lt;0.02 for two of four tests, ##TAB##0##Table 1##). The former euphylliid <italic>Physogyra</italic> has no close relatives (group XIV in ##FIG##0##Fig. 1D##; group XIV-c in ##FIG##1##Figs. 2A##, ##FIG##2##3##), although the morphologically similar genus <italic>Plerogyra</italic> appears related based on unpublished data. The Meandrinidae and the Agariciidae are otherwise unaffected by these analyses (although two other Pacific genera remain unstudied in the Meandrinidae, which is otherwise Atlantic in distribution). The Fungiidae Dana, 1846, as noted above, gains <italic>Leptastrea</italic>, <italic>Psammocora</italic>, <italic>Coscinaraea</italic> and <italic>Oulastrea</italic>; although our tests did not reject monophyly for this family based on the mitochondrial data (##TAB##0##Table 1##), both mitochondrial and nuclear data support this conclusion (group XI in ##FIG##0##Figs. 1D##, ##FIG##2##3##, group XI-b in ##FIG##1##Fig. 2##). The families Acroporidae Verrill, 1902 and Poritidae Gray, 1842 are largely stable, although the genus <italic>Alveopora</italic> must be transferred from the latter to the former (##FIG##0##Fig. 1B##, ##UREF##9##[15]##) (monophyly of Acroporidae and Poritidae rejected at p&lt;.001 for four of four and three of four tests respectively, ##TAB##0##Table 1##). The family Pocilloporidae Gray, 1842 retains all its conventionally assigned genera (and thus remains monophyletic), but gains new members via a sister clade, as noted above, that contains <italic>Stylocoeniella</italic> and <italic>Madracis</italic> (group X in ##FIG##0##Figs. 1C##, ##FIG##2##3##). Only the Dendrophylliidae Gray, 1847 remains unchanged in composition and without new close relatives.</p>", "<title>Status of the Order Corallimorpharia</title>", "<p>Medina et al. ##REF##16754865##[8]## suggested that corallimorpharians are scleractinian corals which have secondarily lost the skeleton. Our data do not support this hypothesis. Independent analysis of ribosomal DNA and tubulin data both strongly indicate that Corallimorpharia lies outside Scleractinia (##FIG##1##Figs. 2A##, ##FIG##2##3##). Mitochondrial data are more equivocal, in that some trees (e.g. maximum parsimony analysis, see <xref ref-type=\"sec\" rid=\"s4\">methods</xref>) lend support to a link between the complex corals and the corallimorpharians, but our maximum likelihood/Bayesian analyses of the full taxonomic data set using <italic>cox1</italic> and <italic>cob</italic> place the corallimorpharians outside the scleractinians (see also ##UREF##3##[9]##) (although the hypothesis of non-monophyly for the Scleractinia was not rejected by the mitochondrial data, ##TAB##0##Table 1##).</p>" ]
[ "<title>Discussion</title>", "<p>Genetic analyses have transformed our understanding of evolutionary relationships over the last decades. In some cases such as families of angiosperms, molecular data have provided important refinements or helped to resolve long-standing controversies, but left many traditional taxonomic groupings intact ##UREF##10##[16]##. In other cases such as sponges, molecular data have overturned much traditional taxonomy and highlighted many previously unanticipated groupings ##UREF##11##[17]##.</p>", "<p>Although taxonomic problems for corals at the species level have been compared to species ambiguities in angiosperms ##UREF##12##[18]##, at higher taxonomic levels the situation for corals resembles that of many sponges, which also lack organ systems and typically a diversity of complex macro-morphological structures. Almost all the families we analyzed either gained or lost members, and in some cases the changes are very substantial. Studies of families dominated by azooxanthellate corals also indicate extensive polyphyly ##UREF##1##[4]##, ##UREF##6##[12]##, ##UREF##7##[13]##. Most strikingly, at least seven families (five analyzed here plus the Caryophylliidae and the Guyniidae ##REF##16221328##[7]##, ##UREF##7##[13]##) have conventionally defined members in both the complex and robust clades, which all analyses indicate are highly divergent genetically ##UREF##0##[2]##–##UREF##2##[5]##, ##UREF##4##[10]##.</p>", "<p>Although more work remains to be done, our conclusions are robust to a number of possible problems. First, nuclear and mitochondrial data sets give broadly similar results. We tested for family monophyly (##TAB##0##Table 1##) using the largest data set (<italic>cox1</italic>+<italic>cob</italic>, ##FIG##0##Fig. 1##), and in every case where monophyly was rejected using formal tests (##TAB##0##Table 1##), the conclusion is also supported by non-monphyletic topologies in the tubulin data set (##FIG##1##Fig. 2##), the r-DNA data set (##FIG##2##Fig. 3##), or both. This makes mitochondrial pseudogenes a highly unlikely explanation for the extent of non-monophyly of scleractinian families in our mitochondrial analyses. Second, although geographic sampling is limited (that is, most species were collected from a single location), where samples from multiple locations were available (e.g. <italic>Montastraea cavernosa</italic>, <italic>Favia fragum</italic>, and <italic>Scolymia cubensis</italic> from Brazil and Panama; <italic>Plesiastrea versipora</italic> from Palau and Japan, <italic>Siderastrea savignyana</italic> from Taiwan and Oman; Online Supporting Information ##SUPPL##0##Table S1##), sequences were either identical, sister taxa, or grouped with all other members of the genus on the mitochondrial tree (##FIG##0##Fig. 1##). Hybridization is unlikely to contribute to patterns at the level of families or above since it has not been reported between members of different genera.</p>", "<p>Given these results, traditional morphological characters must be plagued by convergence. Several examples emerged from our previous study ##REF##14985760##[6]## and there are numerous others. For example, fenestrate septa are found in both complex corals (Poritidae, Siderastreidae) and robust corals (Fungiidae) and may not be homologous in the two cases. Similarly, synapticulae are found in most but not all complex corals (lacking in the Astrocoeniidae and Euphylliidae) and are absent in most but not all robust corals (present in the Fungiidae and its allies). In addition, some taxa have been included in families because of their overall similar appearance, despite having several characters atypical of the families to which they are currently assigned, including characters noted by previous authors (e.g. <italic>Oulastrea</italic> and <italic>Madracis</italic>\n##UREF##13##[19]##, <italic>Leptastrea</italic> and <italic>Plesiastrea</italic>\n##UREF##14##[20]##). Other examples of morphological support for initially surprising molecular results are summarized in Online Supporting Information (##SUPPL##1##Table S2##). More comprehensive formal morphological analyses in light of emerging molecular data (e.g. ##UREF##15##[21]##, ##UREF##16##[22]##) are clearly needed.</p>", "<p>The results reported here also strengthen the conclusion of Fukami and colleagues ##REF##14985760##[6]## that the distinctiveness of the Atlantic scleractinian taxa has been underappreciated. Several families appear to be now largely or exclusively Atlantic: the newly defined Mussidae, the Meandrinidae, the Oculinidae, and the divergent taxon <italic>Montastraea cavernosa</italic>. <italic>Stephanocoenia</italic> may represent another distinctive Atlantic clade, whose modern members consist of only one genus, but the Pacific astrocoeniid genus <italic>Palauastrea</italic> must be analyzed to confirm this.</p>", "<p>The last decade has brought much change to our understanding of scleractinian relationships, but much still needs to be done. Some zooxanthellate genera remain to be analyzed (Online Supporting Information ##SUPPL##1##Table S2##, plus many azooxanthellates), and firm conclusions about biogeographic distributions and the prevalence of families containing single genera are thus premature. Other genera require additional work either because they are so divergent that phylogenetic analyses are difficult (e.g. long branch attraction ##UREF##9##[15]##) or because the genus itself contains highly divergent species so that conclusions depend on which species are studied. An example of the former is found in the uncertain phylogenetic placement of three small divergent genera [<italic>Blastomussa</italic> (2 spp), <italic>Physogyra</italic> (1 spp), and <italic>Plesiastrea</italic> (2 spp)]. Examples of the latter are several genera that appear to have members which are highly divergent even within ocean basins, for example <italic>Acanthastrea</italic> (<italic>A. hillae</italic> and <italic>A. echinata</italic> are divergent in ##FIG##0##Fig. 1D## but not in ##FIG##1##Fig. 2B##) and <italic>Pachyseris</italic> [one species of which is close to the Euphylliidae (##FIG##0##Figs. 1B##, ##FIG##1##2A##) whereas at least some of the others appear to be good members of the Agariciidae (Hoeksema, unpubl.)]. The need for accurate species identifications (often a challenge in corals) and skeletal vouchers to back up identifications is particularly acute in such cases. Nevertheless, an outline of the family tree based on a diverse array of molecular markers does appear to be emerging for the Scleractinia. With it comes the opportunity to redefine families based on morphological characters, which can then be traced through the fossil record.</p>", "<p>Our results also call into question the hypothesis that the corallimorpharians are “naked” corals that have secondarily lost their skeleton. Three independent analyses yield trees that support the monophyly of the Scleractinia within the Hexacorallia. The mitochondrial tree was rooted by taxa representing the Zoanthidea, Actiniaria and Antipatharia; the tubulin tree was rooted by a member of the Antipatharia, and the rDNA tree was rooted by members of the Actiniaria and the Zoanthidea, as well as by a member of the Octocorallia, so issues of rooting are unlikely to be responsible (see ##UREF##3##[9]## for an extensive analysis of the mitochondrial genome of these groups that also concludes that the Scleractinia are monophyletic). Moreover, Medina et al. ##REF##16754865##[8]## report strong gene order differences between the Corallimorpharia and the Scleractinia, which would be consistent with the idea that the Scleractinia are a monophyletic group that does not include the Corallimorpharia. Thus while the traditional relationships within the Scleractinia are very poorly supported, the group itself appears to be derived from a single evolutionary lineage.</p>", "<p>Finally, accurate understanding of evolutionary relationships has implications for ecology and conservation. For example, the conclusion that members of the Faviidae are resistant to environmental stress because they are over-represented in areas of low diversity ##REF##11375488##[23]## needs to be reexamined in light of the fact that “faviids” appear in at least seven of the 21 molecular clades (##FIG##0##Fig. 1##, ##TAB##1##Table 2##). Our reanalysis of extinction risks for these clades using the recently published listing for all reef-building corals ##REF##18653892##[24]## highlights the vulnerabilities of clades II, V, VI, XV, and XVIII+XX, and the lack of adequate information for clades VI, XII, XIII, XIV, and XXI (##TAB##1##Table 2##). Our ability to protect deep lineages most at risk ##REF##12805539##[25]## depends on knowing what these lineages are.</p>" ]
[]
[ "<p>Conceived and designed the experiments: HF CAC. Performed the experiments: HF CAC YYC CC. Analyzed the data: HF CAC AC YYC. Contributed reagents/materials/analysis tools: HF CAC AC CFD KI CS. Wrote the paper: HF CAC AC NK. Provided financial support and guided the integration of the various contributions from different team members: NK. Provided information on the traditional systematics of corals that contributed to the interpretation of results and data in ##TAB##1##table 2## in supplementary information: AFB CW. Commented on manuscript: AFB CW.</p>", "<p>Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ß-tubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent “robust” and “complex” clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>We thank Bert Hoeksema and Steve Cairns for their advice and assistance, Javier Jara for his help in the field, Eyda Gomez for her help in the laboratory, and Dan Janies for help conducting parsimony analyses of the mitochondrial data set. Permits for new collections were granted by the Kenting National Park, Minister of Interior Affairs and Department of Fishery, Penghu County, Taiwan.</p>" ]
[ "<fig id=\"pone-0003222-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003222.g001</object-id><label>Figure 1</label><caption><title>Phylogenetic relationships among scleractinian (mostly zooxanthellate) corals and outgroups.</title><p>Topology was inferred by Bayesian analysis, based on combined mitochondrial <italic>cox1</italic> and <italic>cob</italic> DNA sequences. Numbers on main branches show percentages of Bayesian probability (&gt;70%) and bootstrap values (&gt;50%) in ML analysis. Dashes mean bootstrap values &lt;50% in ML. Numbers in circles show the connection of trees from A to D; for example, 1′ in circle continues directly from 1 in circle. Bars in black indicate possible new family level groupings (see also Supporting Online Information ##SUPPL##1##Table 2##). Numbers (1, 2) following species names indicate that different colonies of the species had different haplotypes. A. outgroups, B. complex corals and corallimorpharians, C. the family Pocilloporidae, D. robust corals. ACR: Acroporidae, AGA: Agariciidae, AST: Astrocoeniidae, DEN: Dendrophylliidae, EUP: Euphylliidae, FAV: Faviidae, FCY: Fungiacyathidae, FUN: Fungiidae, MEA: Meandrinidae, MER: Merulinidae, MUS: Mussidae, PEC: Pectiniidae, POC: Pocilloporidae, POR: Poritidae, OCU: Oculinidae, SID: Siderastreidae, TRC: Trachyphylliidae.</p></caption></fig>", "<fig id=\"pone-0003222-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003222.g002</object-id><label>Figure 2</label><caption><title>Bayesian tree based on the tubulin gene for a subset of corals shown in ##FIG##0##Fig. 1.##\n</title><p> Letter (a, b) after species names indicates that different alleles were obtained from a single coral sample; see ##FIG##0##Fig. 1## legend for other labeling conventions. A. Phylogenetic relationships among complex corals and some robust corals. B. Phylogenetic relationships within robust corals. Note that the same data for the <italic>Montastraea cavernosa</italic> clade (in box) were used in both trees.</p></caption></fig>", "<fig id=\"pone-0003222-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003222.g003</object-id><label>Figure 3</label><caption><title>Bayesian tree based on the rDNA gene for a subset of the scleractinian corals analyzed in ##FIG##0##Fig. 1##.</title><p>Labeling conventions as in previous figures.</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003222-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003222.t001</object-id><label>Table 1</label><caption><title>Results of hypothesis testing showing p-values for approximately Unbiased (AU), Kishino-Hasegawa (KH), Shimodaira-Hasegawa (SH), and weighted SH (WSH) tests.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">AU</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">KH</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SH</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">WSH</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Acroporidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Agariciidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.014</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.010</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.887</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.143</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Astrocoeniidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Euphylliidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Faviidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fungiidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.215</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.101</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.991</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.804</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Meandrinidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Merulinidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.298</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.001</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mussidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.028</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Oculinidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pectiniidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.288</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Poritidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.087</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Siderastreidae is monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.000</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Scleractinia is not monophyletic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.410</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.234</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.972</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.922</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003222-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003222.t002</object-id><label>Table 2</label><caption><title>Tentative groupings of reef-building scleractinian genera based on molecular clades, with total number of species, % data deficient species, and % threatened species for each clade.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clade</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Members</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"># Spp</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">% DD</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">%&gt; = NT/&gt; = VU</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">II</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">DEN: <italic>Balanophyllia</italic>, <italic>Duncanopsammia</italic>, <italic>Heteropsammia</italic>, <bold><italic>Turbinaria</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71/50</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">III</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">POR: <italic>Calathiscus</italic>, <bold><italic>Goniopora</italic></bold>, <bold><italic>Porites</italic></bold>, <italic>Poritipora</italic>, <italic>Stylaraea</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">87</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50/28</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">IV<xref ref-type=\"table-fn\" rid=\"nt103\">1</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">AGA: <bold><italic>Pachyseris</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">60/40</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">V</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">EUP: <italic>Catalaphyllia</italic>, <bold><italic>Euphyllia</italic></bold>, <italic>Nemenzophyllia</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">90/60</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">MEA: <bold><italic>Ctenella</italic></bold>, <italic>Gyrosmilia</italic>, <italic>Montigyra</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">OCU: <bold><italic>Galaxea</italic></bold>, <italic>Simplastrea</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">FAV: <italic>Parasimplastrea</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">VI</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ACR: <bold><italic>Acropora</italic></bold>, <bold><italic>Anacropora</italic></bold>, <bold><italic>Astreopora</italic></bold>, <italic>Enigmopora</italic>, <bold><italic>Isopora</italic></bold>, <bold><italic>Montipora</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">285</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">73/51</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">POR: <bold><italic>Alveopora</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">VII</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">AGA: <bold><italic>Agaricia</italic></bold>, <italic>Coeloseris</italic>, <bold><italic>Gardinoseris</italic></bold>, <bold><italic>Helioseris</italic></bold>, <bold><italic>Leptoseris</italic></bold>, <bold><italic>Pavona</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35/24</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">VIII</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">AST: <bold><italic>Stephanocoenia</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0/0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">IX<xref ref-type=\"table-fn\" rid=\"nt104\">2</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SID: <bold><italic>Pseudosiderastrea</italic></bold>, <bold><italic>Siderastrea</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40/20</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">X</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">POC: <bold><italic>Pocillopora</italic></bold>, <bold><italic>Seriatopora</italic></bold>, <bold><italic>Stylophora</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41/26</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">AST: <bold><italic>Madracis</italic></bold>, <italic>Palauastrea</italic>, <bold><italic>Stylocoeniella</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XI<xref ref-type=\"table-fn\" rid=\"nt104\">2</xref>,<xref ref-type=\"table-fn\" rid=\"nt105\">3</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">FUN: <italic>Cantharellus</italic>, <bold><italic>Ctenactis</italic></bold>, <bold><italic>Fungia</italic></bold>, <bold><italic>Halomitra</italic></bold>, <bold><italic>Heliofungia</italic></bold>, <bold><italic>Herpolitha</italic></bold>, <italic>Lithophylon</italic>, <bold><italic>Podabacia</italic></bold>, <italic>Polyphyllia</italic>, <bold><italic>Sandalolitha</italic></bold>, <italic>Zoopilus</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34/16</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">SID: <bold><italic>Anomastraea</italic></bold>, <bold><italic>Coscinaraea</italic></bold>, <italic>Craterastrea</italic>, <bold><italic>Horastrea</italic></bold>, <bold><italic>Psammocora</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">FAV: <bold><italic>Leptastrea</italic></bold>, <bold><italic>Oulastrea</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XII</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">MEA: <bold><italic>Dendrogyra</italic></bold>, <bold><italic>Dichocoenia</italic></bold>, <bold><italic>Eusmilia</italic></bold>, <bold><italic>Meandrina</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40/40</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XIII</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">OCU: <bold><italic>Oculina</italic></bold>, <italic>Schizoculina</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17/17</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">FAV: <bold><italic>Cladocora</italic></bold>, <bold><italic>Solenastrea</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XIV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">FAV: <bold><italic>Plesiastrea</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75/25</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">EUP: <bold><italic>Physogyra</italic></bold>, <bold><italic>Plerogyra</italic></bold>\n<xref ref-type=\"table-fn\" rid=\"nt105\">3</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">MUS: <bold><italic>Blastomussa</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">FAV: <bold><italic>Diploastrea</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100/0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XVI</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">FAV: <bold><italic>Montastraea</italic></bold> (<italic>cavernosa</italic> only)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0/0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XVII</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">FAV: <italic>Australogyra</italic>, <bold><italic>Barabattoia</italic></bold>, <bold><italic>Caulastraea</italic></bold>, <bold><italic>Cyphastrea</italic></bold>, <bold><italic>Echinopora</italic></bold>, <italic>Erythrastrea</italic>, <bold><italic>Favia</italic></bold> (Pacific), <bold><italic>Favites</italic></bold>, <bold><italic>Goniastrea</italic></bold>, <bold><italic>Leptoria</italic></bold>, <bold><italic>Montastraea</italic></bold> (except <italic>cavernosa</italic>), <italic>Moseleya</italic>, <bold><italic>Oulophyllia</italic></bold>, <bold><italic>Platygyra</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">135</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">70/22</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">MER: <italic>Boninastrea</italic>, <bold><italic>Hydnophora</italic></bold>, <bold><italic>Merulina</italic></bold>, <italic>Paraclavarina</italic>, <bold><italic>Scapophyllia</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">PEC: <bold><italic>Mycedium</italic></bold>, <bold><italic>Pectinia</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">TRA: <bold><italic>Trachyphyllia</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XVIII+XX<xref ref-type=\"table-fn\" rid=\"nt106\">4</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">MUS: <bold><italic>Acanthastrea</italic></bold>, <bold><italic>Micromussa</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">93/43</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XIX</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">MUS: <italic>Australomussa</italic>, <bold><italic>Cynarina</italic></bold>\n<xref ref-type=\"table-fn\" rid=\"nt107\">5</xref>, <bold><italic>Lobophyllia</italic></bold>, <bold><italic>Scolymia</italic></bold> (Pacific), <bold><italic>Symphyllia</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37/20</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">PEC: <italic>Echinomorpha</italic>, <bold><italic>Echinophyllia</italic></bold>, <bold><italic>Oxypora</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XXI</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">FAV: <bold><italic>Colpophyllia</italic></bold>, <bold><italic>Diploria</italic></bold>, <bold><italic>Favia</italic></bold> (Atlantic), <bold><italic>Manicina</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7/7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">MUS: <italic>Isophyllastrea</italic>, <bold><italic>Isophyllia</italic></bold>, <bold><italic>Mussa</italic></bold>, <bold><italic>Mussismillia</italic></bold>, <bold><italic>Mycetophyllia</italic></bold>, <bold><italic>Scolymia</italic></bold> (Atlantic)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003222.s001\"><label>Table S1</label><caption><p>Species lists, localities, and accession numbers.</p><p>(0.42 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003222.s002\"><label>Table S2</label><caption><p>Summary of possible changes to current taxonomy of reef-building corals [(1) for most corals, (2) for Fungiidae] and evidence supporting those changes. We list provisional placement based on mitochondrial data (from cox1 and cob from ##FIG##0##Fig. 1## unless otherwise noted); sources of additional evidence that supports the mitochondrial data are indicated in footnotes [some of these data also appeared in Fukami et al. (3)]. Note that not all members of speciose genera have been examined; in some cases these genera may ultimately be distributed among families, and we list species names where we know that different species have substantially different phylogenetic placements. This table suggests an outline for a revised taxonomy but does not represent a formal taxonomic revision. Families that are exclusively or almost exclusively azooxanthellate (Rhizangiidae, Caryophylliidae) are not included. In addition, the genera Blastomussa, Micromussa, Physogyra, and Plesiastrea are not included in lists of new affiliations because mitochondrial and nuclear data provide no consistent indication of likely close relatives.</p><p>(0.10 MB DOC)</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"nt101\"><label/><p>Bold indicates rejection at the 95% confidence level. Because the SH test appears to be too conservative ##REF##16754865##[8]##, ##REF##12079647##[37]## and the KH test is only valid for comparing a priori hypotheses, results of the AU test should be emphasized.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt102\"><label/><p>Placement of genera not studied (studied genera in bold) was based on morphological similarity and/or biogeography, and should be regarded as provisional. Names of genera taken from recent analysis of extinction risk ##REF##18653892##[24]##, which categorized species as data deficient (DD), least concern, or levels of increasing threat [near threatened (NT), vulnerable (VU), endangered, and critically endangered]. In this table, percent of species lacking adequate data, percent of species with moderate (NT or above) and high (VU or above) risk are indicated. Family abbreviations (all capitals) and clade roman numerals are as in ##FIG##0##Figure 1##.</p></fn><fn id=\"nt103\"><label>1</label><p>Some <italic>Pachyseris</italic> (particularly <italic>P. gemmae</italic> and <italic>P. rugosa</italic>) may be cluster with remainder of Agariciidae in Clade VII.</p></fn><fn id=\"nt104\"><label>2</label><p>Based also on other analyses ##UREF##15##[21]##.</p></fn><fn id=\"nt105\"><label>3</label><p>Based on unpublished CO1 data.</p></fn><fn id=\"nt106\"><label>4</label><p>Based on ##FIG##1##Fig. 2##.</p></fn><fn id=\"nt107\"><label>5</label><p>Includes <italic>Indophyllia</italic> (pers. obs.).</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>This study was supported through funding by US NSF grants to Nancy Knowlton and Ann Budd (DEB0344310, 0343208), by Academia Sinica Thematic grants (2002–2004, 2005–2007) and a Genomics and Proteinomics grant (2006–2007) to Chaolun Allen Chen, by a Grant-in-Aid for Young Scientists (B) (No. 18770013) to Hironobu Fukami, and by a US NSF Assembling the Tree of Life grant (0531779) to Allen Collins, Daphne Fautin, and Paulyn Cartwright. Yao-Yang Chuang and Chien-Hsun Chen received PhD fellowships from the Biodiversity Research Centre, Academia Sinica.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"pone.0003222.s001.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003222.s002.doc\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["2"], "element-citation": ["\n"], "surname": ["Romano", "Palumbi"], "given-names": ["SL", "SR"], "year": ["1996"], "article-title": ["Evolution of scleractinian corals inferred from molecular systematics."], "source": ["Science"], "volume": ["271"], "fpage": ["640"], "lpage": ["642"]}, {"label": ["4"], "element-citation": ["\n"], "surname": ["Romano", "Cairns"], "given-names": ["SL", "SD"], "year": ["2000"], "article-title": ["Molecular phylogenetic hypotheses for the evolution of scleractinian corals."], "source": ["Bull Mar Sci"], "volume": ["67"], "fpage": ["1043"], "lpage": ["1068"]}, {"label": ["5"], "element-citation": ["\n"], "surname": ["Chen", "Wallace", "Wolstenholme"], "given-names": ["CA", "CC", "J"], "year": ["2002"], "article-title": ["Analysis of mitochondrial 12S RNA gene supports a two-clade hypothesis of the evolutionary history of scleractinian corals."], "source": ["Mol Phyl Evol"], "volume": ["23"], "fpage": ["137"], "lpage": ["149"]}, {"label": ["9"], 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["Wei", "Wallace", "Dai", "Moothien Pillay", "Chen"], "given-names": ["WV", "CC", "CF", "RK", "CA"], "year": ["2006"], "article-title": ["Analyses of the ribosomal internal transcribed spacers (ITS) and the 5.8S gene indicate that extremely high rDNA heterogeneity is a unique feature in the scleractinian coral genus "], "italic": ["Acropora"], "source": ["Zool Stud"], "volume": ["45"], "fpage": ["404"], "lpage": ["418"]}, {"label": ["16"], "element-citation": ["\n"], "surname": ["Chace", "Fay", "Savolainen"], "given-names": ["MW", "MF", "V"], "year": ["2000"], "article-title": ["Higher-level classification in the angiosperms: new insights from the perspective of DNA sequence data."], "source": ["Taxon"], "volume": ["49"], "fpage": ["685"], "lpage": ["704"]}, {"label": ["17"], "element-citation": ["\n"], "surname": ["Boury-Esnault"], "given-names": ["N"], "year": ["2006"], "article-title": ["Systematics and evolution of Demospongiae."], "source": ["Can J Zool"], "volume": ["84"], "fpage": ["205"], "lpage": ["224"]}, {"label": ["18"], "element-citation": ["\n"], "surname": ["Willis", "van Oppen", "Miller", "Vollmer", "Ayre"], "given-names": ["BL", "MJH", "DJ", "SV", "DJ"], "year": ["2006"], "article-title": ["The role of hybridization in the evolution of reef corals."], "source": ["Ann Rev Ecol Evol Syst"], "volume": ["37"], "fpage": ["489"], "lpage": ["517"]}, {"label": ["19"], "element-citation": ["\n"], "surname": ["Vaughan", "Wells"], "given-names": ["TW", "JW"], "year": ["1943"], "article-title": ["Revision of the suborders, families and genera of the Scleratinia."], "source": ["Geol Soc Am Spec Pap"], "volume": ["44"], "fpage": ["1"], "lpage": ["363, pl.1\u201351"]}, {"label": ["20"], "element-citation": ["\n"], "surname": ["Alloiteau"], "given-names": ["J"], "year": ["1957"], "article-title": ["Contribution a la syst\u00e9matique des madr\u00e9poraires fossiles."], "source": ["CNRS, Paris"], "fpage": ["1"], "lpage": ["462, 286 figs, 20 pls"]}, {"label": ["21"], "element-citation": ["\n"], "surname": ["Benzoni", "Stefani", "Stolarski", "Pichon", "Mitta"], "given-names": ["F", "F", "J", "M", "G"], "year": ["2007"], "article-title": ["Debating phylogenetic relationships of the scleractinian "], "italic": ["Psammocora"], "source": ["Contr Zool"], "volume": ["76"], "fpage": ["35"], "lpage": ["54"]}, {"label": ["22"], "element-citation": ["\n"], "surname": ["Budd", "Stolarksi"], "given-names": ["AF", "J"], "year": ["2008"], "article-title": ["Searching for new morphological characters in the systematics of scleractinian reef corals: comparison of septal teeth and granules between Atlantic and Pacific Mussidae."], "source": ["Acta Zool"], "volume": ["89"], "comment": ["(in press)"]}, {"label": ["26"], "element-citation": ["\n"], "surname": ["Chen", "Chang", "Wei", "Chen", "Lein"], "given-names": ["CA", "CC", "NV", "CH", "IT"], "year": ["2004"], "article-title": ["Secondary structure and phylogenetic utility of the ribosomal internal transcribed spacer 2 in the scleractinian corals."], "source": ["Zool Stud"], "volume": ["43"], "fpage": ["759"], "lpage": ["771"]}, {"label": ["28"], "element-citation": ["\n"], "surname": ["Zwickl"], "given-names": ["DJ"], "year": ["2006"], "article-title": ["Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion, ver. 0.95."], "comment": ["Ph.D. ("], "ext-link": ["www.bio.utexas.edu/faculty/antisense/garli/Garli.html"]}, {"label": ["29"], "element-citation": ["\n"], "surname": ["Goloboff", "Farris", "Nixon"], "given-names": ["P", "S", "K"], "year": ["2000"], "source": ["TNT:Tree analysis using New Technology (BETA), ver. 1.0"], "publisher-loc": ["Argentina"], "publisher-name": ["Tucum\u00e1n"]}, {"label": ["35"], "element-citation": ["\n"], "surname": ["Shimodaira", "Hasegawa"], "given-names": ["H", "M"], "year": ["1999"], "article-title": ["Multiple comparisons of log-likelihoods with applications to phylogenetic inference."], "source": ["Mol Biol Evol"], "volume": ["16"], "fpage": ["1114"], "lpage": ["1116"]}, {"label": ["36"], "element-citation": ["\n"], "surname": ["Swofford"], "given-names": ["DL"], "year": ["2002"], "source": ["PAUP*:Phylogenetic Analysis Using Parsimony (and Other Methods), version 4.0b10"], "publisher-loc": ["Sunderland, MA"], "publisher-name": ["Sinauer"]}]
{ "acronym": [], "definition": [] }
37
CC0
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 16; 3(9):e3222
oa_package/a2/26/PMC2528942.tar.gz
PMC2528943
18797504
[ "<title>Introduction</title>", "<p>The nature of spore release mechanisms among fungi has been investigated since the eighteenth century ##UREF##0##[1]##, and contemporary analysis of these extraordinary processes has implications for the fields of plant disease control, terrestrial ecology, indoor air quality, atmospheric sciences, veterinary medicine, and biomimetics ##REF##17784861##[2]##–##REF##16457355##[6]##. Mechanisms include a catapult energized by surface tension that launches mushroom spores, the explosive eversion of a pressurized membrane in the artillery fungus, and the discharge of squirt guns pressurized by osmosis ##UREF##3##[7]##. Squirt gun mechanisms are responsible for launching spores at the highest speeds and are most common in the Ascomycota, including lichenized species, but have also evolved among the Zygomycota ##UREF##4##[8]##. In the so-called “coprophilous” fungi in both phyla, specialized for growth on herbivore dung, these squirt gun mechanisms propel spores over distances of many centimeters or even meters onto fresh vegetation where they may be consumed by their host animals. The range of these mechanisms necessitates very high launch speeds to counteract the otherwise overwhelming influence of viscous drag on the flight of microscopic projectiles ##REF##15886452##[9]##.</p>", "<p>In the absence of highspeed photographic records of these processes, estimates of launch speeds have been based on spore capture on discs spinning at known angular velocity ##UREF##5##[10]##, ##UREF##6##[11]## and from the interruption of light beams ##UREF##7##[12]##. Other studies have relied on models that could only infer velocity from measured distances of discharge ##REF##15275665##[13]##, ##REF##15878295##[14]##. However, typical spore launches involve initial velocities that are characterized by intermediate Reynolds numbers (Re≈10–100). Thus, the validity of these inferred estimates of launch speed is limited by the assumptions of the drag modeling. In this paper, we provide unambiguous measurements of launch speeds and acceleration using ultra-high-speed video recordings of discharge processes in four coprophilous fungi. These data allow the experimental evaluation of different drag models. In addition to these ballistic questions, there is considerable uncertainty about the magnitude of the pressures that power spore discharge and the identity of the compounds responsible for generating these pressures. Previous authors have suggested that enormous turgor pressures might be required to discharge spores, requiring the accumulation of very high concentrations of ions and sugars ##REF##17784861##[2]##, ##REF##15878295##[14]##. We address this using a pair of complementary spectroscopic methods to provide an inventory of inorganic ions and sugars. The spectroscopic data show that spore discharge mechanisms are driven by very modest levels of pressure that are characteristic of the majority of fungal cells.</p>", "<p>The fungi chosen for analysis were: <italic>Ascobolus immersus</italic>, <italic>Podospora anserina</italic>, <italic>Pilobolus kleinii</italic>, and <italic>Basidiobolus ranarum</italic>. <italic>A. immersus</italic> is a coprophilous ascomycete that discharges eight spores from each of its multiple asci that are exposed on the surface of a gelatinous fruiting body or ascoma. A second ascomycete, <italic>P. anserina</italic>, produces its asci within a flask-shaped ascoma or perithecium; its spores are harnessed to one another by means of mucilaginous appendages. The zygomycete <italic>P. kleinii</italic> produces a bulbous, fluid-filled stalk or sporangiophore, that squirts a spore-filled sporangium from the dung on which the fungus thrives. Finally, the zygomycete <italic>B. ranarum</italic> flourishes in the dung of amphibians and reptiles and causes rare infections in mammalian hosts, including humans ##REF##16978407##[15]##. Its spore-producing structure is reminiscent of the <italic>Pilobolus</italic> sporangium, but discharges a single spore, or conidium, rather than a sporangium.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Culture methods</title>", "<p>\n<italic>Ascobolus immersus</italic> strains 18558 (+) and 18559 (−) obtained from the American Type Culture Collection (ATCC, Manassas, VA), were crossed on horse dung agar ##REF##15275665##[13]##, incubated at room temperature in the dark until ascomata developed, then exposed to continuous illumination during the experiments. <italic>Podospora anserina</italic> strains F7300 (S<sup>−</sup>) and F7301 (S<sup>+</sup>), kindly supplied by A. Hamann (University of Frankfurt), were treated in the same way. <italic>Pilobolus kleinii</italic> strain 14499 (ATCC) was cultured on rabbit dung agar, incubated in the dark for 5–7 d, then exposed to 12 h dark/12 h light to induce sporangiophore formation. <italic>Basidiobolus ranarum</italic> strain Br02 was isolated from frog dung and grown on Czapek-Dox agar under 12 h dark/12 h light to induce conidiophore formation.</p>", "<title>Ultra-high speed video microscopy</title>", "<p>Video recordings were made with FASTCAM-ultima APX and APX-RS cameras (Photron, San Diego, CA) attached to a binocular dissecting microscope and to an inverted compound microscope fitted with long-working distance objectives (Olympus, Tokyo). Each video clip was compiled from ≤100 image files extracted from recordings consisting up to 1 million images captured in ≤4 s (e.g., 1 million image files captured with 2 µs shutter at 250,000 fps in 4 s). Analysis of digital images was performed with VideoPoint v.2.5 (Lenox Softworks, Lenox MA), Image-Pro Plus 6.2 (Media Cybernetics, Bethesda, MD), and proprietary software from Photron.</p>", "<title>Measuring ascus range</title>", "<p>Discharge distances were measured by attaching culture plates in a vertical orientation at one end of an acrylic box (42×42×6 cm) using Velcro® tape. The inner walls of the box were covered with black paper to exclude light, with the exception of a 5×5 cm widow cut into the far end of the box through which a light source was directed. Wet paper towels were placed in the box to maintain high humidity. Numbered microscope slides were placed in straight paths beneath the culture plates to catch spores after horizontal discharge from their phototropic asci ##UREF##6##[11]##. Spores on each slide were counted to produce spore density versus distance plots.</p>", "<title>Analysis of sap composition</title>", "<p>Sap expelled from asci and sporangiophores was collected on the underside of Petri dish lids above sporulating cultures. The lids were air dried and the number of spores captured on each lid was counted under a dissecting microscope. Measurements of the mean sap volume shot from the asci or sporangiophores of each species were made from light microscopic images of mature asci; these values were multiplied by the number of spore clusters (12.5% of total spore count in <italic>A. immersus</italic>, 25% of spore count in <italic>P. anserina</italic>) or sporangia (of <italic>P. kleinii</italic>) to provide estimates of sap volume deposited on each Petri dish lid. Water soluble ions and organic compounds in the sap were harvested by swirling 1 mL of sterile distilled water in the inverted lids. Aqueous extracts were then transferred to microfuge tubes. Most of the spores remained attached to the lids, but spores that were transferred with the aqueous extracts were removed by centrifuging at 10,000 <italic>g</italic> for 5 min. The supernatants from the microfuge tubes were then stored at –20°C. Sugars and sugar alcohols contributing to sap osmolality were identified and quantified using GC/MS. Samples were derivatized to produce alditol acetates of the sugar alcohols ##UREF##13##[21]##. The dried samples were resuspended in 10 µL of chloroform and multiple injections of 1 µL, separated by blank runs of chloroform, were analyzed on a Varian CP-3800 GC/Saturn 2000 MS. Osmolytes were identified by comparison to GC/MS of purified samples of alditol acetates, and mass spectra of alditol acetates obtained from the NIST/EPA/NIH Mass Spectral Library (NIST, Gaithersburg, MD). The concentrations of the major osmolytes were determined from standard curves produced by plotting the log of the concentration of standards versus the log of the ion intensity of a characteristic ion fragment for each osmolyte (103 for glycerol, and 139 for mannitol). Metals analysis was carried out using a Varian 800 series ICP-MS (Mulgrave, Victoria, Australia) controlled with Varian ICP-MS Expert software. Samples harvested from Petri dish lids were diluted to 10 mL and ion concentrations were determined from standard curves produced from dilutions of ion standards in nitric acid (Inorganic Ventures, Inc., Lakewood, NJ).</p>", "<title>Mathematical model</title>", "<p>Spore flight trajectories were modeled in two ways. Stokes' law describes the drag force on a spherical particle moving through a viscous fluid in the laminar flow, low Reynolds number (Re&lt;1) regime in which viscous forces dominate over inertial forces. In this model, where is the force due to viscous drag, <italic>r</italic> is the aerodynamic radius of the projectile, <italic>η</italic> is the viscosity of the air, and is the projectile velocity. This vector force, combined with Newton's second law, can be analytically integrated to yield expressions for the <italic>x</italic>- and <italic>y</italic>-positions of the spore as functions of time which can be plotted parametrically to determine the spore trajectory. An analytical expression for the range of the projectile can also be derived.</p>", "<p>Spore trajectories were also calculated using a more complicated, quasi-empirical model for the drag which has been proposed for particles moving through fluids at the onset of turbulence, a regime characterized by Reynolds numbers between 1 and 1,000 ##UREF##8##[16]##. In this model,where <italic>ρ<sub>gas</sub></italic> is the viscosity of the air. This expression can be combined with Newton's second law but cannot be integrated analytically. Instead, it must be numerically integrated using any standard numerical integration algorithm. In some cases, acceleration was computed from the position of the projectiles in multiple video frames, but in others, the accelerations were accomplished so swiftly that we estimated acceleration from the observed change in velocity during the time interval between two successive frames.</p>" ]
[ "<title>Results and Discussion</title>", "<p>High speed videos reveal that the octet of spores of <italic>A. immersus</italic> is propelled as a single mass, embedded in mucilage and fluid from the ascus, so the drag upon the projectile is not determined by the shape of the individual spores (##FIG##0##Fig. 1a##, ##SUPPL##0##Video S1##). In some cases, the spores remain in an elongated form during flight; in others, surface tension pulls the spores together after launch, forming a spherical projectile. The ballistics of <italic>P. anserina</italic> ascospores are similar, with the spores moving as an irregularly-shaped projectile held together by appendages and embedded in sap squirted from the ascus (##FIG##0##Fig. 1b##, ##SUPPL##1##Video S2##). In <italic>P. kleinii</italic> the sporangium is propelled from the tip of the sporangiophore by a stream of ejected fluid (##FIG##0##Fig. 1c##, ##SUPPL##2##Video S3##). The spore of <italic>B. ranarum</italic> is launched when the wall of the subtending conidiophore ruptures around its circumference, discharging its tip with the spore (##FIG##0##Fig. 1d##, ##SUPPL##3##Video S4##). In half of the video sequences obtained from this species, the conical conidiophore tip separates from the spore during flight. Median launch speeds in the four species varied from 4 m s<sup>−1</sup> (in <italic>B. ranarum</italic>) to 21 m s<sup>−1</sup> (in <italic>P. anserina</italic>), with a maximum measured acceleration of 1.8×10<sup>6</sup> m s<sup>−2</sup> in <italic>A. immersus</italic> (##TAB##0##Table 1##). In terms of acceleration, these are the fastest recorded flights in nature.</p>", "<p>There have been previous estimates of very high accelerations of fungal spores, but these were based on drag models that appear to be unsuited for the speeds characteristic of spores. Trail et al. ##REF##15878295##[14]## estimated initial accelerations of 8.5×10<sup>6</sup> m s<sup>−2</sup> during ascospore discharge in the wheat pathogen <italic>Gibberella zeae</italic>. This estimate was derived from a semi-empirical equation for the drag coefficient ##UREF##8##[16]##, the dimensions of the discharged spores, and an ascus range of 9 mm. The new video data from our study provide a unique opportunity to test different models for the effect of viscous drag on the motion of microscopic projectiles. Although the fast movement of the spores falls beyond the regime where Stokes Law is known to apply ##UREF##8##[16]##, this model nonetheless correctly predicts the measured ranges from the speeds determined from our video recordings (##TAB##0##Table 1##). The more complex drag model ##REF##15878295##[14]## underestimates these ranges by a factor of two or more (e.g., only 0.7 m for <italic>P. klenii</italic>). In order to reproduce the observed ranges, this model would require launch speeds and pressures an order of magnitude larger than those measured (e.g., 180 m s<sup>−1</sup> and a non-physiological pressure of 2.3 MPa for <italic>P. kleinii</italic>).</p>", "<p>A possible explanation for this discrepancy is that while the generally accepted correlation between drag coefficient and Reynolds number was experimentally determined via constant-velocity sedimentation studies ##UREF##9##[17]##, fungal spores show exceedingly rapid deceleration after launch. In a computational fluid dynamics paper, Wakaba and Balachandar showed that decelerating spheres are overtaken by a wake created in the surrounding medium ##UREF##10##[18]##. Under these conditions, the moving object behaves as if it had additional mass (added mass force) and experiences less drag than a sphere moving at constant velocity. Initial calculations suggest that this effect accounts for a small proportion of the apparent reduction in drag in our study. It is clear that the non-equilibrium conditions experienced by decelerating spores complicates the drag modeling.</p>", "<p>The agreement between the discharge distances predicted from our velocity data using Stokes drag and the measured ranges is remarkably good, given that the video recordings are limited to the initial launch events (##FIG##1##Fig. 2##). In the case of <italic>P. klenii</italic>, for example, the image in ##FIG##0##Fig. 1c## shows the sporangium 0.8 ms after separation from its sporangiophore when it has traveled 8 mm, which is less than 1% of the maximum range of this species.</p>", "<p>To enrich our picture of the discharge processes in these fungi, we used quantitative spectroscopic methods to determine the chemical composition of the ascus sap in <italic>A. immersus</italic> and <italic>P. anserina</italic>, and the sporangiophore sap in <italic>P. kleinii</italic>. The minuscule quantity of fluid ejected with the discharged spores of <italic>B. ranarum</italic> precluded chemical analysis of this species. In the three species examined, hydrostatic pressure was generated by the combined osmolality of sugar alcohols and inorganic ions. The dominant sugars were mannitol, glycerol, erythritol, and pinitol, but the relative concentrations varied between species. In the ascus sap of <italic>A. immersus</italic>, the combined concentration of mannitol and glycerol was 80 mM, and potassium and counter ions boosted the osmolyte concentration by 100 mM. The combined osmolality of these compounds will generate a turgor pressure of 0.44 MPa or 4.4 atm at maximum ascus hydration, which is consistent with a published measurement of the mean ascus turgor pressure of 0.31 MPa using a miniature strain gauge device ##REF##15275665##[13]##. The most abundant sugars in <italic>P. anserina</italic> were erythritol and pinitol, accounting for a combined concentration of 70 mM, which is very similar to the sugar content of the asci of <italic>A. immersus</italic>. In <italic>P. kleinii</italic>, the dominant sugars were mannitol, pinitol, and erythritol, but in this species, ions accounted for 95% of the total osmolality in the clear sporangiophore fluid. The corresponding turgor pressure estimate from the spectroscopic data was 0.50 MPa, which is in excellent agreement with published data ##UREF##11##[19]##. Finally, published pressure measurements and estimates from spectroscopic data for <italic>A. immersus</italic>, <italic>P. anserina</italic>, and <italic>P. kleinii</italic> were consistent with the necessary pressures for the various launch mechanisms predicted from the simple Stokes model for drag (##TAB##0##Table 1##).</p>", "<p>In this paper we have documented a series of remarkable feats of natural engineering, based on universal aspects of fungal structure and metabolism. The turgor pressures of &lt;1.0 MPa (10 atm) that power these supremely fast movements are no higher than those measured from fungal hyphae ##UREF##12##[20]##, suggesting that explosive mechanisms of spore discharge do not require any extraordinary mechanisms of osmolyte accumulation, nor the elaboration of any specialized cell wall structures to maintain this pressure prior to discharge. Unusual features of these mechanisms include the controlled and rapid rupture of the pressurized squirt guns that allow the nearly instantaneous release of energy and discharge of the spores and sporangia. The match between predicted and measured flights also suggest that very little of this energy is lost to friction during the earliest phases of spore release. The launch speeds of the species in this study are likely to be among the fastest among any fungi because their coprophilous ecology has demanded much longer ranges than those necessary for the dispersal of species that need only escape boundary layers.</p>" ]
[ "<title>Results and Discussion</title>", "<p>High speed videos reveal that the octet of spores of <italic>A. immersus</italic> is propelled as a single mass, embedded in mucilage and fluid from the ascus, so the drag upon the projectile is not determined by the shape of the individual spores (##FIG##0##Fig. 1a##, ##SUPPL##0##Video S1##). In some cases, the spores remain in an elongated form during flight; in others, surface tension pulls the spores together after launch, forming a spherical projectile. The ballistics of <italic>P. anserina</italic> ascospores are similar, with the spores moving as an irregularly-shaped projectile held together by appendages and embedded in sap squirted from the ascus (##FIG##0##Fig. 1b##, ##SUPPL##1##Video S2##). In <italic>P. kleinii</italic> the sporangium is propelled from the tip of the sporangiophore by a stream of ejected fluid (##FIG##0##Fig. 1c##, ##SUPPL##2##Video S3##). The spore of <italic>B. ranarum</italic> is launched when the wall of the subtending conidiophore ruptures around its circumference, discharging its tip with the spore (##FIG##0##Fig. 1d##, ##SUPPL##3##Video S4##). In half of the video sequences obtained from this species, the conical conidiophore tip separates from the spore during flight. Median launch speeds in the four species varied from 4 m s<sup>−1</sup> (in <italic>B. ranarum</italic>) to 21 m s<sup>−1</sup> (in <italic>P. anserina</italic>), with a maximum measured acceleration of 1.8×10<sup>6</sup> m s<sup>−2</sup> in <italic>A. immersus</italic> (##TAB##0##Table 1##). In terms of acceleration, these are the fastest recorded flights in nature.</p>", "<p>There have been previous estimates of very high accelerations of fungal spores, but these were based on drag models that appear to be unsuited for the speeds characteristic of spores. Trail et al. ##REF##15878295##[14]## estimated initial accelerations of 8.5×10<sup>6</sup> m s<sup>−2</sup> during ascospore discharge in the wheat pathogen <italic>Gibberella zeae</italic>. This estimate was derived from a semi-empirical equation for the drag coefficient ##UREF##8##[16]##, the dimensions of the discharged spores, and an ascus range of 9 mm. The new video data from our study provide a unique opportunity to test different models for the effect of viscous drag on the motion of microscopic projectiles. Although the fast movement of the spores falls beyond the regime where Stokes Law is known to apply ##UREF##8##[16]##, this model nonetheless correctly predicts the measured ranges from the speeds determined from our video recordings (##TAB##0##Table 1##). The more complex drag model ##REF##15878295##[14]## underestimates these ranges by a factor of two or more (e.g., only 0.7 m for <italic>P. klenii</italic>). In order to reproduce the observed ranges, this model would require launch speeds and pressures an order of magnitude larger than those measured (e.g., 180 m s<sup>−1</sup> and a non-physiological pressure of 2.3 MPa for <italic>P. kleinii</italic>).</p>", "<p>A possible explanation for this discrepancy is that while the generally accepted correlation between drag coefficient and Reynolds number was experimentally determined via constant-velocity sedimentation studies ##UREF##9##[17]##, fungal spores show exceedingly rapid deceleration after launch. In a computational fluid dynamics paper, Wakaba and Balachandar showed that decelerating spheres are overtaken by a wake created in the surrounding medium ##UREF##10##[18]##. Under these conditions, the moving object behaves as if it had additional mass (added mass force) and experiences less drag than a sphere moving at constant velocity. Initial calculations suggest that this effect accounts for a small proportion of the apparent reduction in drag in our study. It is clear that the non-equilibrium conditions experienced by decelerating spores complicates the drag modeling.</p>", "<p>The agreement between the discharge distances predicted from our velocity data using Stokes drag and the measured ranges is remarkably good, given that the video recordings are limited to the initial launch events (##FIG##1##Fig. 2##). In the case of <italic>P. klenii</italic>, for example, the image in ##FIG##0##Fig. 1c## shows the sporangium 0.8 ms after separation from its sporangiophore when it has traveled 8 mm, which is less than 1% of the maximum range of this species.</p>", "<p>To enrich our picture of the discharge processes in these fungi, we used quantitative spectroscopic methods to determine the chemical composition of the ascus sap in <italic>A. immersus</italic> and <italic>P. anserina</italic>, and the sporangiophore sap in <italic>P. kleinii</italic>. The minuscule quantity of fluid ejected with the discharged spores of <italic>B. ranarum</italic> precluded chemical analysis of this species. In the three species examined, hydrostatic pressure was generated by the combined osmolality of sugar alcohols and inorganic ions. The dominant sugars were mannitol, glycerol, erythritol, and pinitol, but the relative concentrations varied between species. In the ascus sap of <italic>A. immersus</italic>, the combined concentration of mannitol and glycerol was 80 mM, and potassium and counter ions boosted the osmolyte concentration by 100 mM. The combined osmolality of these compounds will generate a turgor pressure of 0.44 MPa or 4.4 atm at maximum ascus hydration, which is consistent with a published measurement of the mean ascus turgor pressure of 0.31 MPa using a miniature strain gauge device ##REF##15275665##[13]##. The most abundant sugars in <italic>P. anserina</italic> were erythritol and pinitol, accounting for a combined concentration of 70 mM, which is very similar to the sugar content of the asci of <italic>A. immersus</italic>. In <italic>P. kleinii</italic>, the dominant sugars were mannitol, pinitol, and erythritol, but in this species, ions accounted for 95% of the total osmolality in the clear sporangiophore fluid. The corresponding turgor pressure estimate from the spectroscopic data was 0.50 MPa, which is in excellent agreement with published data ##UREF##11##[19]##. Finally, published pressure measurements and estimates from spectroscopic data for <italic>A. immersus</italic>, <italic>P. anserina</italic>, and <italic>P. kleinii</italic> were consistent with the necessary pressures for the various launch mechanisms predicted from the simple Stokes model for drag (##TAB##0##Table 1##).</p>", "<p>In this paper we have documented a series of remarkable feats of natural engineering, based on universal aspects of fungal structure and metabolism. The turgor pressures of &lt;1.0 MPa (10 atm) that power these supremely fast movements are no higher than those measured from fungal hyphae ##UREF##12##[20]##, suggesting that explosive mechanisms of spore discharge do not require any extraordinary mechanisms of osmolyte accumulation, nor the elaboration of any specialized cell wall structures to maintain this pressure prior to discharge. Unusual features of these mechanisms include the controlled and rapid rupture of the pressurized squirt guns that allow the nearly instantaneous release of energy and discharge of the spores and sporangia. The match between predicted and measured flights also suggest that very little of this energy is lost to friction during the earliest phases of spore release. The launch speeds of the species in this study are likely to be among the fastest among any fungi because their coprophilous ecology has demanded much longer ranges than those necessary for the dispersal of species that need only escape boundary layers.</p>" ]
[]
[ "<p>Conceived and designed the experiments: NPM. Performed the experiments: LY LC YC DD AH JK HK JS JSR ZS. Analyzed the data: DD MF JSR. Wrote the paper: DD MF NPM.</p>", "<title>Background</title>", "<p>A variety of spore discharge processes have evolved among the fungi. Those with the longest ranges are powered by hydrostatic pressure and include “squirt guns” that are most common in the Ascomycota and Zygomycota. In these fungi, fluid-filled stalks that support single spores or spore-filled sporangia, or cells called asci that contain multiple spores, are pressurized by osmosis. Because spores are discharged at such high speeds, most of the information on launch processes from previous studies has been inferred from mathematical models and is subject to a number of errors.</p>", "<title>Methodology/Principal Findings</title>", "<p>In this study, we have used ultra-high-speed video cameras running at maximum frame rates of 250,000 fps to analyze the entire launch process in four species of fungi that grow on the dung of herbivores. For the first time we have direct measurements of launch speeds and empirical estimates of acceleration in these fungi. Launch speeds ranged from 2 to 25 m s<sup>−1</sup> and corresponding accelerations of 20,000 to 180,000 <italic>g</italic> propelled spores over distances of up to 2.5 meters. In addition, quantitative spectroscopic methods were used to identify the organic and inorganic osmolytes responsible for generating the turgor pressures that drive spore discharge.</p>", "<title>Conclusions/Significance</title>", "<p>The new video data allowed us to test different models for the effect of viscous drag and identify errors in the previous approaches to modeling spore motion. The spectroscopic data show that high speed spore discharge mechanisms in fungi are powered by the same levels of turgor pressure that are characteristic of fungal hyphae and do not require any special mechanisms of osmolyte accumulation.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>The authors thank R. Taylor and I. Peat for assistance with the spectroscopy.</p>" ]
[ "<fig id=\"pone-0003237-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003237.g001</object-id><label>Figure 1</label><caption><title>Single frames from high speed video recordings of spore discharge in four coprophilous fungi.</title><p>a, <italic>Ascobolus immersus</italic>, with 8 ascospores discharged from ascus tip, 60 µs into the launch. b, <italic>Podospora anserina</italic>, with 4 ascospores harnessed by mucilaginous appendages, 96 µs after release from apex of fruiting body at left of frame. c, <italic>Pilobolus kleinii</italic>, sporangium with sap trailing behind, 0.8 ms after beginning of discharge. Undischarged sporangium at top of frame. d, <italic>Basidiobolus ranarum</italic>, single spore carrying portion of dehisced conidiophore, 24 µs into the launch. Scale bars, a, b, d, 50 µm, c, 1 mm. Frame rates a, 100,000 fps, b, 250,000 fps, c, 50,000 fps, d, 210,000 fps.</p></caption></fig>", "<fig id=\"pone-0003237-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003237.g002</object-id><label>Figure 2</label><caption><title>Predicted trajectories of spores and sporangia of four fungi based on launch data obtained by high speed video microscopy.</title><p>Trajectories of spores and sporangia of <italic>Ascobolus immersus</italic> (<italic>A.i.</italic>, blue), <italic>Podospora anserina</italic> (<italic>P.a.</italic>, red), <italic>Basidiobolus ranarum</italic> (<italic>B.r.</italic>, green), and <italic>Pilobolus kleinii</italic> (<italic>P.k.</italic>, blue in inset). Points indicate projectile positions at 10 ms intervals. The truncated trajectories of <italic>A. immersus</italic>, <italic>P. anserina</italic>, and <italic>B. ranarum</italic> are indicative of the dominance of viscous forces over inertial forces in the motional regimes for these launches. Inertia is more significant for the flight of the larger sporangia of <italic>P. kleinii</italic>. Launch angles of 40° (<italic>A.i.</italic>), 30° (<italic>P.a., P.k.</italic>), and 20° (<italic>B.r.</italic>), were chosen to separate the trajectories from one another but also reflect the phototropic orientation of these asci, sporangiophores, and conidiophores in nature.</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003237-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003237.t001</object-id><label>Table 1</label><caption><title>Ballistics of spore and sporangium discharge in four coprophilous fungi based upon high speed video analyses and different models for the effects of viscous drag on particle flight.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Ascobolus immersus</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Podospora anserina</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Pilobolus kleinii</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Basidiobolus ranarum</italic>\n</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Measured launch speed (range, median, sample size)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5–18, 14 (12)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10–25, 21 (17)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2–13, 9 (14)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2–9, 4 (10)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Measured maximum acceleration (m s<sup>−2</sup>)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1,800,000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1,500,000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">210,000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1,500,000</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Measured maximum range (m)<xref ref-type=\"table-fn\" rid=\"nt101\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.02</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Estimated maximum range (m) using Stokes drag</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Measured turgor pressure (MPa)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.30 (strain gauge##REF##15275665##[13]##)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.40 (spectroscopy)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.55 (osmometry ##UREF##11##[19]##)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.40 (spectroscopy)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.55 (spectroscopy)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Estimated pressure (MPa)<xref ref-type=\"table-fn\" rid=\"nt102\">**</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.30–1.00</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.11–0.29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03–0.17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.01–0.72</td></tr></tbody></table></alternatives></table-wrap>" ]
[ "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>", "<disp-formula></disp-formula>" ]
[]
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[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003237.s001\"><label>Video S1</label><caption><p>Ascobolus immersus, 1000,000 fps.</p><p>(0.33 MB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003237.s002\"><label>Video S2</label><caption><p>Podospora anserina, 250,000 fps.</p><p>(0.33 MB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003237.s003\"><label>Video S3</label><caption><p>Pilobolus kleinii, 50,000 fps.</p><p>(4.92 MB AVI)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003237.s004\"><label>Video S4</label><caption><p>Basidiobolus ranarum, 210,000 fps.</p><p>(0.19 MB MPG)</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"nt101\"><label>*</label><p>Maximum range measurements by authors with exception of <italic>P. klenii</italic> data published by Buller ##UREF##11##[19]##.</p></fn><fn id=\"nt102\"><label>**</label><p>Pressure estimates (for measured range of launch speeds) were obtained by calculating the force needed to cause the observed projectile accelerations via Newton's second law and the area over which that force was applied.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>The authors' studies on spore discharge are funded by the National Science Foundation and National Institutes of Health.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"pone.0003237.s001.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003237.s002.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003237.s003.avi\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003237.s004.mpg\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["1"], "element-citation": ["\n"], "surname": ["Micheli"], "given-names": ["PA"], "year": ["1729"], "source": ["Nova plantarum genera"], "publisher-loc": ["Florence"], "publisher-name": ["Bernardi Paperinii"]}, {"label": ["4"], "element-citation": ["\n"], "surname": ["Elbert", "Taylor", "Andreae", "P\u00f6schl"], "given-names": ["W", "PE", "MO", "U"], "year": ["2006"], "article-title": ["Contribution of fungi to primary biogenic aerosols in the atmosphere: active discharge of spores, carbohydrates, and ionorganic ions by Asco- and Basidiomycota."], "source": ["Atmos Chem Phys Discuss"], "volume": ["6"], "fpage": ["11317"], "lpage": ["11355"]}, {"label": ["5"], "element-citation": ["\n"], "surname": ["Robinson"], "given-names": ["J"], "year": ["1962"], "article-title": ["\n"], "italic": ["Pilobolus", "Dictyocaulus viviparus"], "source": ["Nature"], "volume": ["193"], "fpage": ["353"], "lpage": ["354"]}, {"label": ["7"], "element-citation": ["\n"], "surname": ["Ingold"], "given-names": ["CT"], "year": ["1971"], "source": ["Fungal spores: their liberation and dispersal"], "publisher-loc": ["Oxford"], "publisher-name": ["Oxford University Press"]}, {"label": ["8"], "element-citation": ["\n"], "surname": ["Webster", "Weber"], "given-names": ["J", "RWS"], "year": ["2007"], "source": ["Introduction to fungi, 3rd edition"], "publisher-loc": ["Cambridge"], "publisher-name": ["Cambridge University Press"]}, {"label": ["10"], "element-citation": ["\n"], "surname": ["Pringsheim", "Czurda"], "given-names": ["EG", "V"], "year": ["1927"], "article-title": ["Phototropische und ballistische Probleme bei "], "italic": ["Pilobolus"], "source": ["Jahrb Wiss Bot"], "volume": ["66"], "fpage": ["863"], "lpage": ["901"]}, {"label": ["11"], "element-citation": ["\n"], "surname": ["Ingold", "Hadland"], "given-names": ["CT", "SA"], "year": ["1959"], "article-title": ["The ballistics of "], "italic": ["Sordaria"], "source": ["New Phytol"], "volume": ["58"], "fpage": ["46"], "lpage": ["57"]}, {"label": ["12"], "element-citation": ["\n"], "surname": ["Page", "Kennedy"], "given-names": ["RM", "D"], "year": ["1964"], "article-title": ["Studies on the velocity of discharged sporangia of "], "italic": ["Pilobolus kleinii"], "source": ["Mycologia"], "volume": ["56"], "fpage": ["363"], "lpage": ["368"]}, {"label": ["16"], "element-citation": ["\n"], "surname": ["White"], "given-names": ["FM"], "year": ["1974"], "source": ["Viscous fluid flow"], "publisher-loc": ["New York"], "publisher-name": ["McGraw Hill"]}, {"label": ["17"], "element-citation": ["\n"], "surname": ["Lapple", "Shepherd"], "given-names": ["CE", "CB"], "year": ["1940"], "article-title": ["Calculation of particle trajectories."], "source": ["Ind Eng Chem"], "volume": ["32"], "fpage": ["605"], "lpage": ["617"]}, {"label": ["18"], "element-citation": ["\n"], "surname": ["Wakaba", "Balachandar"], "given-names": ["L", "S"], "year": ["2007"], "article-title": ["On the added mass force at finite Reynolds and acceleration numbers."], "source": ["Theor Comput Fluid Dyn"], "volume": ["21"], "fpage": ["147"], "lpage": ["153"]}, {"label": ["19"], "element-citation": ["\n"], "surname": ["Buller"], "given-names": ["AHR"], "year": ["1934"], "source": ["Researches on fungi, vol. 6"], "publisher-loc": ["London"], "publisher-name": ["Longmans, Green & Co"]}, {"label": ["20"], "element-citation": ["\n"], "surname": ["Money", "Howard", "Gow"], "given-names": ["NP", "RJ", "NAR"], "year": ["2007"], "article-title": ["Biomechanics of invasive hyphal growth."], "source": ["The Mycota, Volume 8, Biology of the Fungal Cell, 2nd edition"], "publisher-loc": ["New York"], "publisher-name": ["Springer Verlag"], "fpage": ["237"], "lpage": ["249"]}, {"label": ["21"], "element-citation": ["\n"], "surname": ["Higgins", "Bly", "Morgan", "Fox"], "given-names": ["MK", "R", "SL", "A"], "year": ["1994"], "article-title": ["Differentiation of isomeric alditol hexaacetates and identification of aldohexoses by electron-impact mass-spectrometry."], "source": ["Anal Chem"], "volume": ["66"], "fpage": ["2625"], "lpage": ["2668"]}]
{ "acronym": [], "definition": [] }
21
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 17; 3(9):e3237
oa_package/d5/08/PMC2528943.tar.gz
PMC2528944
18795099
[ "<title>Introduction</title>", "<p>The recent discovery of miRNAs introduced a new mechanism of gene expression regulation ##REF##15372042##[1]##, ##REF##14744438##[2]##. Despite the fact that biological functions have been assigned to only a few dozen miRNAs, it is becoming apparent that miRNAs participate in the regulation of a variety of developmental and physiological processes ##REF##16510870##[3]##. Not surprisingly, recent studies have shown that miRNAs play important roles in the regulation of muscle development. The functional characterization of miR-1 and miR-133 has been an important step in our understanding of miRNA-mediated muscle development. miR-1-1 and miR-1-2 were first found to be specifically expressed in mouse cardiac and skeletal muscle precursor cells and were found to be transcriptionally regulated by the myogenic differentiation factors MyoD, Mef2, and SRF ##REF##12968692##[4]##. Overexpression of miRNA-1 in the mouse developing heart has a negative effect on muscle proliferation as it targets the transcription factor that promotes ventricular cardiomyocyte expansion, Hand2 ##REF##12968692##[4]##. In <italic>Drosophila</italic>, the expression of miR-1 is controlled by the Twist and Mef2 transcription factors ##REF##16166373##[5]##. Investigation of a loss-of-function phenotype of <italic>Drosophila</italic> miR-1 showed that miR-1 is not required for the formation or physiological function of the larval musculature, but is required for the post-mitotic growth of larval muscle ##REF##16166373##[5]##. Recent studies showed that miR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4), a transcriptional repressor of muscle gene expression, and that miR-133 enhances myoblast proliferation by repressing serum response factor (SRF) ##REF##16314309##[6]##, both examples of new molecular mechanisms to regulate skeletal muscle gene expression and embryonic developmental ##REF##16314309##[6]##.</p>", "<p>Another miRNA, miR-206, has also been characterized as a muscle regulator in recent studies. In co-operation with miR-133, miR-206 can repress myoblast fusion by targeting the connexin 43 (Cx43) gap junction channels without altering the Cx43 mRNA level ##REF##17062625##[7]##. These findings have generated more detailed insights into the mechanisms underlying the myogenesis process and have uncovered different pathways that lead to myofiber proliferation and differentiation. However, the complete roles of miRNAs in muscle growth &amp; development still remain to be elucidated.</p>", "<p>In mammals, muscle mass is mainly determined by the number and size of muscle fibers. In the pig, for example, the number of muscle fibers is prenatally determined during primary and secondary muscle fiber formation, while the postnatal hypertrophy process then increases the length and diameter of these fibers. Primary muscle fiber formation begins at approximately 30 days following gestation. Secondary muscle fiber formation begins at about 50 to 60 days post-gestation, when myoblasts align and fuse to form secondary muscle fibers at the surface of existing primary muscle fibers ##REF##12968692##[4]##. Identification of genes governing these processes will provide insights into the regulation of muscle growth. Currently, numerous genes, including growth factors, regulatory proteins, receptors, and transcription factors have been identified as participating in the regulation of the myogenesis. However, the underlying molecular pathway elements, such as the decisive secondary regulatory factors of the major genes responsible for controlling prenatal muscle growth, remains poorly understood.</p>", "<p>We hypothesized that there were more miRNAs associated with muscle growth and development in prenatal pigs yet to be discovered. Profiling of transcriptome changes of mature miRNAs isolated from key developmental stages is a promising technique to use in uncovering these miRNAs. In the present study, we identify miRNAs whose expression has not previously been reported in pigs. Our results also identify a number of differentially expressed miRNAs that could represent new regulatory elements in muscle growth and development.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Homolog search for miRNA candidates</title>", "<p>Analysis of the current porcine genomic draft sequences (August 2007) was performed by comparing porcine genomic sequences with both experimentally confirmed and predicted data sets from other species using BLAST (Basic Local Alignment Search Tool). The alignments, requiring at least 90% pre-miRNA similarity and 100% mature miRNA similarity, were reserved for further study. The predicted miRNA secondary structure was generated by the RNAfold software package (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.tbi.univie.ac.at/ivo/RNA/RNAfold.html\">http://www.tbi.univie.ac.at/ivo/RNA/RNAfold.html</ext-link>). We also checked the phylogenetic shadowing profile of these sequence pairs as characterized by the miRNA coding arm, which suffered the highest selective pressure, and then in succession the non-coding arm, the stem region, the loop region, and the flanking sequence. The candidates not following these rules were removed from the datasets. After these steps, we ended up with hundreds of miRNA candidates.</p>", "<title>Samples and RNA preparation</title>", "<p>Our experiments included three RNA samples isolated from three independent fetal or adult pigs. Sample collection was approved by the ethics committee of Huazhong agricultural university. The longissimus tissues were dissected after removing the epimysium coverings. These samples were snap-frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated using a Trizol protocol (Invitrogen). After quantification, the RNA was isolated using PEG (polyethylene glycol) and labeled by RNA ligase according to the method of Thomson et al. ##REF##15782152##[34]##.</p>", "<title>Microarray hybridization and data analysis</title>", "<p>The microarrays used in this study were bought from CapitalBio Company (NO. 225011). The hybridization was done by the CapitalBio Company service. In brief, labeled RNA was dissolved in 16 ul hybridization mixture (15% formamide; 0.2% SDS; 3×SSC; 50×Denhardt's) and hybridized overnight. The slides were washed in 0.2% SDS, 2×SSC for four minutes at 42°C, and in 0.2×SSC for four minutes. The slides were scanned using the LuxScan 10 K/A scanner (CapitalBio Company) and the raw pixel intensities were extracted using the LuxScan3.0 software (CapitalBio Company). The median pixel intensities were background subtracted. Hybridization signals that failed to exceed the average background value by more than two standard deviations (Signal&gt;Mean+2SD) were excluded from analysis. In all of the three duplicate slides, probe signal&gt;Mean+2SD was classified as detected (for E33.p and E33.f, no duplicate experiments were performed, thus signal exceeding 2×Mean+2SD were defined as detected.). The data were normalized between slides from different ages groups using the quantile normalization method proposed by Bolstad et al ##REF##12538238##[35]##. The differentially expressed genes, classified as those with Fold changes&gt;2, P value&lt;0.001 and FDR&lt;0.001, were selected using the SAM software, version 2.1 (Significance Analysis of Microarrays, <ext-link ext-link-type=\"uri\" xlink:href=\"http://www-stat.stanford.edu/tibs/SAM/\">http://www-stat.stanford.edu/tibs/SAM/</ext-link>). The subsequent analysis of miRNA targets prediction and target gene functional annotation was performed using the TargetScan software (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.targetscan.org/\">http://www.targetscan.org/</ext-link>) and the DAVID gene annotation tool (<ext-link ext-link-type=\"uri\" xlink:href=\"http://david.abcc.ncifcrf.gov/\">http://david.abcc.ncifcrf.gov/</ext-link>), respectively.</p>", "<title>Stem-Loop Real-time RT-PCR</title>", "<p>A miRNA quantification method similar to that described by Chen et al. ##REF##16314309##[6]## was used to validate the microarray data. Three independent samples from each time point were analyzed. In brief, the assay was performed using Stem–loop RT followed by SYBR Green Real-time PCR analysis. Firstly, 1 µg total RNA was reverse transcribed using 200 U M-MLV Reverse Transcriptase (Takara: 02640A) and 1 µl Stem-loop RT primer in an Applied Biosystems 9700 Thermocycler with incubation at 30°C for 15 min, 42°C for 60 min and 85°C for 5 min. Importantly, all reverse transcriptase reactions were run along with “no-template controls”. The no-template controls gave non-detectable signals in all samples, confirming the high specificity of the miRNA quantification assay. Real-time PCR was performed using a standard SYBR Green PCR kit (Toyobo: QPK-201) on the BIO-RAD iQ5 Real-Time PCR Detection System. Porcine Met-tRNA was used as an internal control and all reactions were run in triplicate. The ΔΔCt method was used to determine the expression level differences between surveyed stages ##REF##16845603##[36]##. The significant level was set to 0.05.</p>" ]
[ "<title>Results and Discussion</title>", "<title>Identification of porcine miRNA candidates</title>", "<title>\n<italic>In silico</italic> porcine miRNA prediction by homolog searches</title>", "<p>We made use of the property of miRNAs to be highly conserved between closely related species in order to predict novel porcine miRNA candidates ##REF##17868480##[8]##. Pair-wise comparison of the porcine genomic sequences (August 2007) to hairpin sequences collected from mirBase (Version 10.0) resulted in 12,048 alignments. After removing the redundant alignments, we ended up with a total of 775 unique porcine miRNA candidates (##SUPPL##1##Table S1##). All candidates were found to have the potential for the hairpin-loop secondary structures typical to known miRNA transcripts. Among these candidates, 49 had been reported while the remaining were new.</p>", "<p>Homolog search and <italic>de novo</italic> prediction are two typical approaches widely used in miRNA prediction. The homolog search approach is essential in our study since the porcine genome is not yet available for a direct prediction. Although the approach is limited by its inability to detect less conserved miRNAs, it is a nonetheless efficient and cost-effective.</p>", "<title>Detection of expressed porcine miRNAs by microarray hybridization</title>", "<p>To validate these miRNA candidates, a recently developed mammalian miRNA microarray was used to evaluate the expression of porcine miRNAs. At the design time of the microarray, there were 576 human miRNAs, 238 rat miRNAs and 358 mouse miRNAs reported. After removing the redundant sequences, there remained 743 unique mature miRNA sequences. The microarray was designed to contain 743 probes complementary to these sequences (See probe list of the microarray in ##SUPPL##2##Table S2##). The <italic>in silico</italic> prediction mentioned above was based on the alignment of the reported miRNAs of human, mouse and rat to the porcine genomic sequences. As expected, the microarray covered all of the candidates found by this method, and thus can be used to detect their expression.</p>", "<p>Microarray hybridization with RNA samples prepared from the 33-day post-gestation stage porcine whole embryo (E33.f) and placenta (P33.p) detected expression of 296 miRNAs (230 in E33.f and 275 in E33.p, signal&gt;2Mean+2SD. See full list in ##SUPPL##3##Table S3.1##). For the 49 porcine miRNAs deposited in miRbase, 41 of them were detected (35 in E33.f and 39 in E33.p). The six porcine miRNAs identified by Kim et al. were also detected ##REF##17213910##[9]##. The remaining 255 miRNAs have not been previously reported to be expressed in pig. We also found a large number of probes that showed strong signals but were not included in our candidate list, such as the miR-13 and miR-557. The failure to detect these candidates by the homolog search method is possibly due to the fact that only part of the porcine genome (60%) was available at the time.</p>", "<p>The first reported porcine miRNA was the identification of the mir17-92 cluster using the homolog search method ##REF##16180141##[10]##. A more extensive homology search has since been performed by Kim et al. ##REF##17213910##[9]##. They identified 58 candidates and validated six of them by northern blot. Other miRNA entries in miRBase are predictions found by genomic comparisons with other model organisms such as human, mouse and rat without proof of expression ##REF##15885146##[11]##. There are 49 miRNAs reported so far. Our experiments expanded the number of porcine miRNAs (with identified sequence and confirmed expression) to 116 (##TAB##0##Table 1## lists the top 20 highly expressed new miRNAs. See the full list in ##SUPPL##4##Table S4.1## and the predicted secondary structures in ##SUPPL##4##Table S4.2##).</p>", "<title>Global miRNA expression profiling of porcine skeletal muscle tissues</title>", "<title>An overview of the expression profile</title>", "<p>To identify the miRNAs that might be involved in muscle development and to discriminate these from the miRNAs possibly involved in promoting or repressing muscle myogenesis and differentiation, we carried out a comparative miRNA expression profile across skeletal muscle samples collected from pigs of 33-days post-gestation (E33), 65-days post-gestation (E65) and adult age (Adu). Samples from each age group were collected independently and the analysis performed in triplicate to ensure reliability. Comparisons between each of the replicates showed that the replicates have good reproducibility (##FIG##0##Figure 1##).</p>", "<p>The use of short RNA probes antisense to the mature miRNA sequence has not proven to be an effective approach to reliably quantify the expression differences between miRNAs that have only one mismatch or a few mismatches ##REF##15574827##[12]##. Luo et al. previously performed a sensitivity test of the microarray using the artificially transcribed miRNA of let-7a to hybridize to the let-7 probe set (let-7a to let-7g, let7-i). Their results showed that the microarray utilized in this study was able to distinguish between the mismatched sequences, but was unable to distinguish between the highly similar sequences ##UREF##0##[13]##. Therefore microarray results for closely related miRNAs should be interpreted with caution, as expression differences of a given miRNA could be exaggerated or diminished by the expression of their paralogs.</p>", "<p>Of the 576 miRNAs on the microarray, 256 (44%) were expressed in the muscle samples. Of those expressed, 227 were in E33 and 228 in E65, while only 163 were expressed in Adu (see ##SUPPL##3##Table S3.2##). Taking into account the fact that miRNAs are negative regulators of coding genes that act by either inhibiting translation or inducing mRNA degradation of the target gene ##REF##16510870##[3]##, ##REF##15211354##[14]##, ##REF##16314451##[15]##, these results suggest lower expression levels of the coding genes regulated by the miRNAs in the prenatal stages. The modulation of muscle development processes is triggered by sequential events of gene activation and inhibition. The differences in miRNA expression between the ages detected in this study support the complexity of their roles in muscle development.</p>", "<title>Differentially expressed miRNAs detected by the microarray</title>", "<p>Of the 256 miRNAs detected by the microarray, expression levels of 140 of them changed significantly between the developmental stages investigated (Fold change&gt;2, p&lt;0.001, FDR&lt;0.001, see ##SUPPL##5##Table S5##) and 51 changed more than ten-fold (##TAB##1##Table 2##). For example, the average increase of miR-486 signal from E33 to E65 was 3.3-fold, and 13.4-fold from E65 to Adu; the average increase of miR-376b signal from E33 to E65 was 4.6-fold, but decreased 54.7-fold from E65 to Adu, and therefore in Adu it appeared 11.9-fold lower than in E33; miR-422a signal increased more than 6.9-fold from E33 to E65, after which it remained stable; miR-495 signal was strong in E33 and E65, but nearly undetectable in the Adu stage. Interestingly, we found that three miRNAs (miR-363, miR-365 and miR-422b) were differentially expressed between E33 and Adu, despite their expression not being significantly different when comparing either E33 to E65 or E65 to Adu. This may represent a type of long term regulation.</p>", "<p>Pair-wise comparisons showed that large numbers of miRNAs are differentially expressed between any given two ages. In addition, the number of differentially expressed miRNAs as well as the value of the average fold changes varied between the three developmental ages investigated. As shown in ##TAB##2##Table 3##, the number of differentially expressed miRNAs between E33 and E65 is much smaller than between E65 and Adu, and the value of the average fold change between E33 and Adu is much lower than between E65 and Adu. These findings show that the expression patterns of the three ages are unique.</p>", "<p>Of the three miRNAs reported as regulators of development in skeletal and cardiac muscle, miR-206 was found to be up-regulated 2.9-fold in Adu compared to E65, but the expression variance of miR-1 and miR-133 failed to reach statistically significant levels. These two miRNAs showed a high level of expression in the microarray analysis, thus technical error could be ruled out. It should be noted that the functional discovery of these miRNAs was made mostly in cell culture systems, which may differ from the in vivo system.</p>", "<p>Several of the differentially expressed miRNAs identified here were shown to play roles in growth and development related processes in recent studies. These include miR-214, miR-140, miR-150, miR-10, as well as miR-181. In the zebrafish, miR-214 can modulate hedgehog signaling, thus changing muscle cell fate ##REF##17220889##[18]##, and miR-10 was shown to represses HoxB1a and HoxB3a, which are involved in patterning the anterior-posterior axis ##REF##18167555##[19]##. In mouse cells, the cartilage specific miRNA, miR-140, targets the histone deacetylase 4 (HDAC4), suggestive of a role in long bone development ##REF##16828749##[20]##. In mature B and T cells, the miR-150 was found to block early B cell development when expressed prematurely, and also found to control B cell differentiation by targeting the transcription factor of c-Myb ##REF##17923094##[21]##. Furthermore, miR-181 was found to be involved in the process of mammalian skeletal-muscle differentiation, by targeting the homeobox protein Hox-A11 during mammalian myoblast differentiation ##REF##16489342##[22]##. These findings suggest that identifying differentially expressed miRNAs may lead to the discovery of miRNAs related to muscle growth and development.</p>", "<title>Validation of the microarray results by direct quantification</title>", "<p>Five representative differentially expressed miRNAs (miR-150, miR-193b, miR-196a, miR-187b and miR-495) were chosen for validation by the stem–loop RT–PCR based real-time PCR method ##REF##16314309##[6]## using three independent samples (The primer sequences are available in ##SUPPL##6##Table S6##). The expression levels for miR-150, miR-193b, miR-187b and miR-196a, as determined by RT-PCR, were in concordance with the normalized microarray data (Pearson correlation coefficient &gt;0.9, q value&lt;0.001, ##FIG##1##Figure 2##). In general, the results of qPCR validated the microarray results. An exception was miR-495, for which the expression levels in E33 and E65 varied dramatically. Although we have not verified the exact cause, the variance may come from biological differences between the samples. Furthermore, it should be noted that the purification process of the stem–loop RT–PCR assay is unable to completely remove long RNA nucleotides, thus we cannot exclude the possibility that the precursors are also quantified ##REF##16314309##[6]##.</p>", "<title>Distinctive miRNA expression patterns during muscle development</title>", "<p>To visually illustrate the expression type of the miRNAs being expressed during different developmental stages, a hierarchical cluster analysis was performed for the differentially expressed miRNAs. The results show that the miRNA expression patterns fall into seven typical categories: A) prenatally expressed, expression level increased between E33 and E65; B) universally expressed, expression level decreased between E33 and E65; C) universally expressed, expression level increased through the three ages; D) moderately expressed in E65, expression levels in E33 and Adu nearly undetectable; E) moderately expressed in E33, expression levels in E65 and Adu nearly undetectable; F) postnatally expressed, expression nearly undetectable in prenatal ages; G) moderately expressed, expression level increase through the three ages. The expression patterns described above are clearly reflected by the formation of several big clusters in the tree map of the clustering results (##FIG##2##Figure 3##).</p>", "<p>The myogenesis process takes place mostly in the prenatal stage and becomes almost inhibited in the postnatal stage ##REF##6630038##[16]##. It has been demonstrated in the pig that primary muscle fiber formation begins at approximately 30 days post-gestation and the secondary muscle fiber formation begins at about 50 to 60 days post-gestation ##REF##7647372##[17]##. The categories of miRNA expression patterns described above provide a sensible basis for generating specific hypotheses of how miRNAs function in the biological context of the developmental ages investigated. The prenatally expressed miRNA clusters may include miRNAs that play roles in the promotion of myogenesis (##FIG##2##Figure 3 A and B##). In contrast, the postnatally expressed miRNAs clusters may include miRNAs that act as inhibitors of myogenesis (##FIG##2##Figure 3 C and F##). The E33 and E65 highly expressed miRNA clusters may include miRNAs that play roles in the process of primary and secondary muscle fiber formation, respectively (##FIG##2##Figure 3 E and D##). In previous studies, we found that although the secondary muscle fiber formation took place later than the primary muscle fiber formation process, the two temporally overlapped at the beginning of secondary muscle fiber formation ##REF##7647372##[17]##. This may be the primary reason why a large number of miRNAs are expressed both at the E33 stage and the E65 stage (##FIG##2##Figure 3 A, B and F##).</p>", "<title>Differentially expressed miRNAs may play important roles in porcine muscular development</title>", "<p>A major challenge to understanding the function of these developmentally regulated miRNAs is the question of target identification. It is commonly recognized that the miRNA and its targets must be located in the same subcellular position to make the biological interactions operable, thus the spatial and temporal information of miRNA expression may narrow the search for miRNA target candidates.</p>", "<p>The differentially expressed miRNAs detected by this microarray analysis provide a valuable candidate list of muscle growth and development related miRNAs. In this analysis, we used a well established miRNA-target dataset generated by TargetScan to investigate the possible functions of these miRNAs and to provide evidence for their involvement in the muscle development process ##REF##15652477##[23]##, ##REF##14697198##[24]##. The relevant mRNA sequences used in this analysis are orthologous genes. In addition, the target sites were characterized as evolutionarily conserved in five species (human, mouse, rat, dog and chicken), a criterion that also acted as a good filter for false positive assignments of miRNAs to genes ##REF##14697198##[24]##, ##REF##16337999##[25]##. Altogether we analyzed 6,654 genes that have at least one predicted miRNA binding site in their 3′ UTR, and a total of 84 miRNA families in the TargetScan datasets. As a result, we obtained a total of 24,555 predicted miRNA-mRNA interaction sites (##SUPPL##7##Table S7##). As we expected, most of the miRNAs investigated targeted hundreds of genes and over 65% of the targets were regulated by more than one miRNA (##SUPPL##8##Tables S8.1 and S8.2##). The high degree of connectivity between the miRNA-mRNA pairs supports the idea that these miRNAs have extensive and complicated roles during the muscle development process. Three genes, NFIB, TNRC6B and ZNF148 assigned the highest number of miRNA interaction sites. The NFIB gene was previously identified as an activator of the differentiation-specific genes, such as MCSFR ##REF##18056638##[26]##. TNRC6B was co-purified with a cytoplasmic HeLa cell protein complex containing AGO2, DICER, and MOV10, and thus is implicated in mediating miRNA-guided mRNA cleavage in HeLa cells ##REF##16289642##[27]##. ZNF148 (alias ZBP89) was originally reported as a gastrin gene expression repressor ##REF##8943318##[28]##, ##REF##9457682##[29]## and recently, studies of mice expressing only ZBP89-delta-N showed significant growth delay and a reduction of viability ##REF##16517939##[30]##. GO terms and KEGG pathway annotation of the miRNA targets using the DAVID gene annotation tool (<ext-link ext-link-type=\"uri\" xlink:href=\"http://david.abcc.ncifcrf.gov/\">http://david.abcc.ncifcrf.gov/</ext-link>) further illustrate the possible roles and mechanisms of these differentially expressed miRNAs in muscle development (##SUPPL##0##Document S1##).</p>", "<p>The above analyses provide an overview investigation on the possible functions of differentially expressed miRNAs based on computationally predicted target datasets. Although the accuracy of the computational approaches for identification of mammalian miRNA targets is still limited ##REF##17060911##[33]##, these results will definitely advance the hypothesis-driven functional studies of these miRNAs.</p>" ]
[ "<title>Results and Discussion</title>", "<title>Identification of porcine miRNA candidates</title>", "<title>\n<italic>In silico</italic> porcine miRNA prediction by homolog searches</title>", "<p>We made use of the property of miRNAs to be highly conserved between closely related species in order to predict novel porcine miRNA candidates ##REF##17868480##[8]##. Pair-wise comparison of the porcine genomic sequences (August 2007) to hairpin sequences collected from mirBase (Version 10.0) resulted in 12,048 alignments. After removing the redundant alignments, we ended up with a total of 775 unique porcine miRNA candidates (##SUPPL##1##Table S1##). All candidates were found to have the potential for the hairpin-loop secondary structures typical to known miRNA transcripts. Among these candidates, 49 had been reported while the remaining were new.</p>", "<p>Homolog search and <italic>de novo</italic> prediction are two typical approaches widely used in miRNA prediction. The homolog search approach is essential in our study since the porcine genome is not yet available for a direct prediction. Although the approach is limited by its inability to detect less conserved miRNAs, it is a nonetheless efficient and cost-effective.</p>", "<title>Detection of expressed porcine miRNAs by microarray hybridization</title>", "<p>To validate these miRNA candidates, a recently developed mammalian miRNA microarray was used to evaluate the expression of porcine miRNAs. At the design time of the microarray, there were 576 human miRNAs, 238 rat miRNAs and 358 mouse miRNAs reported. After removing the redundant sequences, there remained 743 unique mature miRNA sequences. The microarray was designed to contain 743 probes complementary to these sequences (See probe list of the microarray in ##SUPPL##2##Table S2##). The <italic>in silico</italic> prediction mentioned above was based on the alignment of the reported miRNAs of human, mouse and rat to the porcine genomic sequences. As expected, the microarray covered all of the candidates found by this method, and thus can be used to detect their expression.</p>", "<p>Microarray hybridization with RNA samples prepared from the 33-day post-gestation stage porcine whole embryo (E33.f) and placenta (P33.p) detected expression of 296 miRNAs (230 in E33.f and 275 in E33.p, signal&gt;2Mean+2SD. See full list in ##SUPPL##3##Table S3.1##). For the 49 porcine miRNAs deposited in miRbase, 41 of them were detected (35 in E33.f and 39 in E33.p). The six porcine miRNAs identified by Kim et al. were also detected ##REF##17213910##[9]##. The remaining 255 miRNAs have not been previously reported to be expressed in pig. We also found a large number of probes that showed strong signals but were not included in our candidate list, such as the miR-13 and miR-557. The failure to detect these candidates by the homolog search method is possibly due to the fact that only part of the porcine genome (60%) was available at the time.</p>", "<p>The first reported porcine miRNA was the identification of the mir17-92 cluster using the homolog search method ##REF##16180141##[10]##. A more extensive homology search has since been performed by Kim et al. ##REF##17213910##[9]##. They identified 58 candidates and validated six of them by northern blot. Other miRNA entries in miRBase are predictions found by genomic comparisons with other model organisms such as human, mouse and rat without proof of expression ##REF##15885146##[11]##. There are 49 miRNAs reported so far. Our experiments expanded the number of porcine miRNAs (with identified sequence and confirmed expression) to 116 (##TAB##0##Table 1## lists the top 20 highly expressed new miRNAs. See the full list in ##SUPPL##4##Table S4.1## and the predicted secondary structures in ##SUPPL##4##Table S4.2##).</p>", "<title>Global miRNA expression profiling of porcine skeletal muscle tissues</title>", "<title>An overview of the expression profile</title>", "<p>To identify the miRNAs that might be involved in muscle development and to discriminate these from the miRNAs possibly involved in promoting or repressing muscle myogenesis and differentiation, we carried out a comparative miRNA expression profile across skeletal muscle samples collected from pigs of 33-days post-gestation (E33), 65-days post-gestation (E65) and adult age (Adu). Samples from each age group were collected independently and the analysis performed in triplicate to ensure reliability. Comparisons between each of the replicates showed that the replicates have good reproducibility (##FIG##0##Figure 1##).</p>", "<p>The use of short RNA probes antisense to the mature miRNA sequence has not proven to be an effective approach to reliably quantify the expression differences between miRNAs that have only one mismatch or a few mismatches ##REF##15574827##[12]##. Luo et al. previously performed a sensitivity test of the microarray using the artificially transcribed miRNA of let-7a to hybridize to the let-7 probe set (let-7a to let-7g, let7-i). Their results showed that the microarray utilized in this study was able to distinguish between the mismatched sequences, but was unable to distinguish between the highly similar sequences ##UREF##0##[13]##. Therefore microarray results for closely related miRNAs should be interpreted with caution, as expression differences of a given miRNA could be exaggerated or diminished by the expression of their paralogs.</p>", "<p>Of the 576 miRNAs on the microarray, 256 (44%) were expressed in the muscle samples. Of those expressed, 227 were in E33 and 228 in E65, while only 163 were expressed in Adu (see ##SUPPL##3##Table S3.2##). Taking into account the fact that miRNAs are negative regulators of coding genes that act by either inhibiting translation or inducing mRNA degradation of the target gene ##REF##16510870##[3]##, ##REF##15211354##[14]##, ##REF##16314451##[15]##, these results suggest lower expression levels of the coding genes regulated by the miRNAs in the prenatal stages. The modulation of muscle development processes is triggered by sequential events of gene activation and inhibition. The differences in miRNA expression between the ages detected in this study support the complexity of their roles in muscle development.</p>", "<title>Differentially expressed miRNAs detected by the microarray</title>", "<p>Of the 256 miRNAs detected by the microarray, expression levels of 140 of them changed significantly between the developmental stages investigated (Fold change&gt;2, p&lt;0.001, FDR&lt;0.001, see ##SUPPL##5##Table S5##) and 51 changed more than ten-fold (##TAB##1##Table 2##). For example, the average increase of miR-486 signal from E33 to E65 was 3.3-fold, and 13.4-fold from E65 to Adu; the average increase of miR-376b signal from E33 to E65 was 4.6-fold, but decreased 54.7-fold from E65 to Adu, and therefore in Adu it appeared 11.9-fold lower than in E33; miR-422a signal increased more than 6.9-fold from E33 to E65, after which it remained stable; miR-495 signal was strong in E33 and E65, but nearly undetectable in the Adu stage. Interestingly, we found that three miRNAs (miR-363, miR-365 and miR-422b) were differentially expressed between E33 and Adu, despite their expression not being significantly different when comparing either E33 to E65 or E65 to Adu. This may represent a type of long term regulation.</p>", "<p>Pair-wise comparisons showed that large numbers of miRNAs are differentially expressed between any given two ages. In addition, the number of differentially expressed miRNAs as well as the value of the average fold changes varied between the three developmental ages investigated. As shown in ##TAB##2##Table 3##, the number of differentially expressed miRNAs between E33 and E65 is much smaller than between E65 and Adu, and the value of the average fold change between E33 and Adu is much lower than between E65 and Adu. These findings show that the expression patterns of the three ages are unique.</p>", "<p>Of the three miRNAs reported as regulators of development in skeletal and cardiac muscle, miR-206 was found to be up-regulated 2.9-fold in Adu compared to E65, but the expression variance of miR-1 and miR-133 failed to reach statistically significant levels. These two miRNAs showed a high level of expression in the microarray analysis, thus technical error could be ruled out. It should be noted that the functional discovery of these miRNAs was made mostly in cell culture systems, which may differ from the in vivo system.</p>", "<p>Several of the differentially expressed miRNAs identified here were shown to play roles in growth and development related processes in recent studies. These include miR-214, miR-140, miR-150, miR-10, as well as miR-181. In the zebrafish, miR-214 can modulate hedgehog signaling, thus changing muscle cell fate ##REF##17220889##[18]##, and miR-10 was shown to represses HoxB1a and HoxB3a, which are involved in patterning the anterior-posterior axis ##REF##18167555##[19]##. In mouse cells, the cartilage specific miRNA, miR-140, targets the histone deacetylase 4 (HDAC4), suggestive of a role in long bone development ##REF##16828749##[20]##. In mature B and T cells, the miR-150 was found to block early B cell development when expressed prematurely, and also found to control B cell differentiation by targeting the transcription factor of c-Myb ##REF##17923094##[21]##. Furthermore, miR-181 was found to be involved in the process of mammalian skeletal-muscle differentiation, by targeting the homeobox protein Hox-A11 during mammalian myoblast differentiation ##REF##16489342##[22]##. These findings suggest that identifying differentially expressed miRNAs may lead to the discovery of miRNAs related to muscle growth and development.</p>", "<title>Validation of the microarray results by direct quantification</title>", "<p>Five representative differentially expressed miRNAs (miR-150, miR-193b, miR-196a, miR-187b and miR-495) were chosen for validation by the stem–loop RT–PCR based real-time PCR method ##REF##16314309##[6]## using three independent samples (The primer sequences are available in ##SUPPL##6##Table S6##). The expression levels for miR-150, miR-193b, miR-187b and miR-196a, as determined by RT-PCR, were in concordance with the normalized microarray data (Pearson correlation coefficient &gt;0.9, q value&lt;0.001, ##FIG##1##Figure 2##). In general, the results of qPCR validated the microarray results. An exception was miR-495, for which the expression levels in E33 and E65 varied dramatically. Although we have not verified the exact cause, the variance may come from biological differences between the samples. Furthermore, it should be noted that the purification process of the stem–loop RT–PCR assay is unable to completely remove long RNA nucleotides, thus we cannot exclude the possibility that the precursors are also quantified ##REF##16314309##[6]##.</p>", "<title>Distinctive miRNA expression patterns during muscle development</title>", "<p>To visually illustrate the expression type of the miRNAs being expressed during different developmental stages, a hierarchical cluster analysis was performed for the differentially expressed miRNAs. The results show that the miRNA expression patterns fall into seven typical categories: A) prenatally expressed, expression level increased between E33 and E65; B) universally expressed, expression level decreased between E33 and E65; C) universally expressed, expression level increased through the three ages; D) moderately expressed in E65, expression levels in E33 and Adu nearly undetectable; E) moderately expressed in E33, expression levels in E65 and Adu nearly undetectable; F) postnatally expressed, expression nearly undetectable in prenatal ages; G) moderately expressed, expression level increase through the three ages. The expression patterns described above are clearly reflected by the formation of several big clusters in the tree map of the clustering results (##FIG##2##Figure 3##).</p>", "<p>The myogenesis process takes place mostly in the prenatal stage and becomes almost inhibited in the postnatal stage ##REF##6630038##[16]##. It has been demonstrated in the pig that primary muscle fiber formation begins at approximately 30 days post-gestation and the secondary muscle fiber formation begins at about 50 to 60 days post-gestation ##REF##7647372##[17]##. The categories of miRNA expression patterns described above provide a sensible basis for generating specific hypotheses of how miRNAs function in the biological context of the developmental ages investigated. The prenatally expressed miRNA clusters may include miRNAs that play roles in the promotion of myogenesis (##FIG##2##Figure 3 A and B##). In contrast, the postnatally expressed miRNAs clusters may include miRNAs that act as inhibitors of myogenesis (##FIG##2##Figure 3 C and F##). The E33 and E65 highly expressed miRNA clusters may include miRNAs that play roles in the process of primary and secondary muscle fiber formation, respectively (##FIG##2##Figure 3 E and D##). In previous studies, we found that although the secondary muscle fiber formation took place later than the primary muscle fiber formation process, the two temporally overlapped at the beginning of secondary muscle fiber formation ##REF##7647372##[17]##. This may be the primary reason why a large number of miRNAs are expressed both at the E33 stage and the E65 stage (##FIG##2##Figure 3 A, B and F##).</p>", "<title>Differentially expressed miRNAs may play important roles in porcine muscular development</title>", "<p>A major challenge to understanding the function of these developmentally regulated miRNAs is the question of target identification. It is commonly recognized that the miRNA and its targets must be located in the same subcellular position to make the biological interactions operable, thus the spatial and temporal information of miRNA expression may narrow the search for miRNA target candidates.</p>", "<p>The differentially expressed miRNAs detected by this microarray analysis provide a valuable candidate list of muscle growth and development related miRNAs. In this analysis, we used a well established miRNA-target dataset generated by TargetScan to investigate the possible functions of these miRNAs and to provide evidence for their involvement in the muscle development process ##REF##15652477##[23]##, ##REF##14697198##[24]##. The relevant mRNA sequences used in this analysis are orthologous genes. In addition, the target sites were characterized as evolutionarily conserved in five species (human, mouse, rat, dog and chicken), a criterion that also acted as a good filter for false positive assignments of miRNAs to genes ##REF##14697198##[24]##, ##REF##16337999##[25]##. Altogether we analyzed 6,654 genes that have at least one predicted miRNA binding site in their 3′ UTR, and a total of 84 miRNA families in the TargetScan datasets. As a result, we obtained a total of 24,555 predicted miRNA-mRNA interaction sites (##SUPPL##7##Table S7##). As we expected, most of the miRNAs investigated targeted hundreds of genes and over 65% of the targets were regulated by more than one miRNA (##SUPPL##8##Tables S8.1 and S8.2##). The high degree of connectivity between the miRNA-mRNA pairs supports the idea that these miRNAs have extensive and complicated roles during the muscle development process. Three genes, NFIB, TNRC6B and ZNF148 assigned the highest number of miRNA interaction sites. The NFIB gene was previously identified as an activator of the differentiation-specific genes, such as MCSFR ##REF##18056638##[26]##. TNRC6B was co-purified with a cytoplasmic HeLa cell protein complex containing AGO2, DICER, and MOV10, and thus is implicated in mediating miRNA-guided mRNA cleavage in HeLa cells ##REF##16289642##[27]##. ZNF148 (alias ZBP89) was originally reported as a gastrin gene expression repressor ##REF##8943318##[28]##, ##REF##9457682##[29]## and recently, studies of mice expressing only ZBP89-delta-N showed significant growth delay and a reduction of viability ##REF##16517939##[30]##. GO terms and KEGG pathway annotation of the miRNA targets using the DAVID gene annotation tool (<ext-link ext-link-type=\"uri\" xlink:href=\"http://david.abcc.ncifcrf.gov/\">http://david.abcc.ncifcrf.gov/</ext-link>) further illustrate the possible roles and mechanisms of these differentially expressed miRNAs in muscle development (##SUPPL##0##Document S1##).</p>", "<p>The above analyses provide an overview investigation on the possible functions of differentially expressed miRNAs based on computationally predicted target datasets. Although the accuracy of the computational approaches for identification of mammalian miRNA targets is still limited ##REF##17060911##[33]##, these results will definitely advance the hypothesis-driven functional studies of these miRNAs.</p>" ]
[]
[ "<p>Conceived and designed the experiments: THH MJZ XYL SHZ. Performed the experiments: THH. Analyzed the data: THH. Contributed reagents/materials/analysis tools: THH. Wrote the paper: THH SHZ.</p>", "<p>MiRNAs (microRNAs) play critical roles in many important biological processes such as growth and development in mammals. In this study, we identified hundreds of porcine miRNA candidates through <italic>in silico</italic> prediction and analyzed their expression in developing skeletal muscle using microarray. Microarray screening using RNA samples prepared from a 33-day whole embryo and an extra embryo membrane validated 296 of the predicted candidates. Comparative expression profiling across samples of longissimus muscle collected from 33-day and 65-day post-gestation fetuses, as well as adult pigs, identified 140 differentially expressed miRNAs amongst the age groups investigated. The differentially expressed miRNAs showed seven distinctive types of expression patterns, suggesting possible involvement in certain biological processes. Five of the differentially expressed miRNAs were validated using real-time PCR. <italic>In silico</italic> analysis of the miRNA-mRNA interaction sites suggested that the potential mRNA targets of the differentially expressed miRNAs may play important roles in muscle growth and development.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>We thank Dr. Min Yao for assistance in developing of the Loop-primer Real-time PCR method and Dr Zhiliang Hu from Iowa State University for suggestions and paper editing.</p>" ]
[ "<fig id=\"pone-0003225-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003225.g001</object-id><label>Figure 1</label><caption><title>Reproducibility of the microarray experiments.</title><p>We examined the miRNA expression in three developmental stages of skeletal muscle (E33, E65 and Adu). Samples from each stage were isolated in triplicate and hybridized to the microarray. Scatter plots demonstrate the pair-wise comparison between each two sets of triplicates. The R represents the Spearman correlation coefficient.</p></caption></fig>", "<fig id=\"pone-0003225-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003225.g002</object-id><label>Figure 2</label><caption><title>Validation of the microarray results using Real-time PCR method.</title><p>Expression levels of five miRNAs (miR-150, miR-193b, miR-196a, miR-187b and miR-495) were detected by Real time PCR (right) and microarray (left). We have made a scaling to the raw data of Real time PCR to make it comparable to the microarray data. R represents the Pearson correlation coefficient.</p></caption></fig>", "<fig id=\"pone-0003225-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003225.g003</object-id><label>Figure 3</label><caption><title>Hierarchical cluster analysis.</title><p>We performed a data adjustment (median center and normalization) in the cluster analysis. The color codes of red, white, black and dark green represents expression levels of high, average, low and absent respectively. A detailed view of the miRNA expression levels in clustering patterns is shown in the plot areas from A to G.</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003225-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003225.t001</object-id><label>Table 1</label><caption><title>New porcine miRNAs identified in 33 day post-gestation samples of whole embryo (E33.f) and placenta (E33.p).</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">MiRNA Name</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Microarray Probe Sequence</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Porcine Trace Sequence</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Normalized Expression Level</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">E33.p</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">E33.f</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-let-7d</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">ACTATGCAACCTACTACCTCT</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1380820092</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7274.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25372.67</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-let-7e</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">ACTATACAACCTCCTACCTCA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1577748346</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16353.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18411.33</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-10b</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">ACAAATTCGGTTCTACAGGGTA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|2022730406</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16918.67</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7983.33</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-124a-1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">TGGCATTCACCGCGTGCCTTAA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1420670121</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21323</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">373</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-15b</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">TGTAAACCATGATGTGCTGCTA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|2020963538</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9361</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12985</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-16-1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">CGCCAATATTTACGTGCTGCTA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1579971821</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22503.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26688</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">ACTACCTGCACTGTAAGCACTTTG</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1579909832</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32828.67</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9736.67</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-191</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">AGCTGCTTTTGGGATTCCGTTG</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|2025394035</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9408.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14004.33</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-199(a/b)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">AACCAATGTGCAGACTACTGTA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|(2019854499/1377265104)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40134.67</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">42470</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-19b-1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">TCAGTTTTGCATGGATTTGCACA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1579909838</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30333.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8530</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">ACAGTTCTTCAACTGGCAGCTT</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1377639070</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5877.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27348.67</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-29a</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">AACCGATTTCAGATGGTGCTA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|860609555</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5892.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6809.67</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-30b</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">AGCTGAGTGTAGGATGTTTACA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1574275341</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6958</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17706.67</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-30d</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">CTTCCAGTCGGGGATGTTTACA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1008617003</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14482.67</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19839.33</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-320</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">TTCGCCCTCTCAACCCAGCTTTT</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|2027985691</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9252.67</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21585.67</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-376a-1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">ACGTGGATTTTCCTCTATGAT</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1008637782</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11473</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10293</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-382</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">CGAATCCACCACGAACAACTTC</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|775596795</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3858.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10386.33</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-487b</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">AAGTGGATGACCCTGTACGATT</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|851619303</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7105.67</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13520</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-99a</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">CACAAGATCGGATCTACGGGTT</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|2020960585</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33488</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35344</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ssc-mir-185</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<named-content content-type=\"gene\">GAACTGCCTTTCTCTCCA</named-content>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">gnl|ti|1575367821</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6678.67</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10275.67</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003225-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003225.t002</object-id><label>Table 2</label><caption><title>MiRNAs differentially expressed between E33, E65 and Adu stages (Fold change &gt;10.0, p&lt;0.001 and FDR&lt;0.001).</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">MiRNA Name</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">E65/E33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adu/E65</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adu/E33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">MiRNA Name</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">E65/E33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adu/E65</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adu/E33</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-214</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-493</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-422a</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.93</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.01</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-409-5p</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.35</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.19</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-503</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-379</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.21</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-497</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.80</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-95</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.23</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16.38</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-721</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.00</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.07</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-369-5p</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.85</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.16</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-189</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.79</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.37</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-557</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30.97</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-378</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.58</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-655</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-487a</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.92</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.59</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-656</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-680</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.98</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-182</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.13</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-127</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-376a</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.65</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.26</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-495</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.01</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.02</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-365</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.39</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-411</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.01</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-486</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.26</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.44</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">43.75</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-487b</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.41</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-323</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.09</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-29a</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.92</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16.78</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-660</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.17</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-193b</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22.41</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22.68</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-409-3p</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-29b</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.49</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.19</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">MIR-202</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.74</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.13</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-376b</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.59</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.02</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-382</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.11</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-29c</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17.44</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17.45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-503</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.07</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-376a</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-431</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.90</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.15</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-335</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-410</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-411</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-150</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.74</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48.39</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-532</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-380-3p</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.19</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-299-5p</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.73</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-432</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.70</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-362</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-196a</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-455-3p</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">miR-329</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.35</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.19</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003225-t003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003225.t003</object-id><label>Table 3</label><caption><title>Summarization of differentially expressed miRNAs.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Comparison Groups</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Change Direction</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">Number of Regulated miRNAs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Average FC (fold change)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;2-fold</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;5-fold</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;10-fold</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">E33 VS E65</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">up</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">down</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">all</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">E65 VS Adu</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">up</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">down</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">all</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">110</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">65</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">E33 VS Adu</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">up</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">51</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">down</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">all</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">101</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">56</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td></tr></tbody></table></alternatives></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003225.s001\"><label>Document S1</label><caption><p>(0.08 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003225.s002\"><label>Table S1</label><caption><p>(0.16 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003225.s003\"><label>Table S2</label><caption><p>(0.12 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003225.s004\"><label>Table S3</label><caption><p>(0.19 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003225.s005\"><label>Table S4</label><caption><p>(0.08 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003225.s006\"><label>Table S5</label><caption><p>(0.04 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003225.s007\"><label>Table S6</label><caption><p>(0.02 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003225.s008\"><label>Table S7</label><caption><p>(1.87 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003225.s009\"><label>Table S8</label><caption><p>(0.51 MB XLS)</p></caption></supplementary-material>" ]
[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>Financial support was provided by the Key Project of National Basic Research and Developmental Plan (2006CB102105) and the National Natural Science Foundation (30671138) of China.</p></fn></fn-group>" ]
[ "<graphic id=\"pone-0003225-t001-1\" xlink:href=\"pone.0003225.t001\"/>", "<graphic xlink:href=\"pone.0003225.g001\"/>", "<graphic id=\"pone-0003225-t002-2\" xlink:href=\"pone.0003225.t002\"/>", "<graphic id=\"pone-0003225-t003-3\" xlink:href=\"pone.0003225.t003\"/>", "<graphic xlink:href=\"pone.0003225.g002\"/>", "<graphic xlink:href=\"pone.0003225.g003\"/>" ]
[ "<media xlink:href=\"pone.0003225.s001.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003225.s002.xls\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003225.s003.xls\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003225.s004.xls\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003225.s005.xls\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003225.s006.xls\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003225.s007.xls\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003225.s008.xls\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003225.s009.xls\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["13"], "element-citation": ["\n"], "surname": ["Luo", "Tian", "Xu", "Zhang", "Wang"], "given-names": ["MY", "ZG", "Z", "L", "YX"], "year": ["2007"], "article-title": ["Construction and Application of a Microarray for Profiling MicroRNA Expression."], "source": ["Progress in Biochemistry and Biophysics"], "volume": ["34"], "fpage": ["31"], "lpage": ["41"]}]
{ "acronym": [], "definition": [] }
36
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 16; 3(9):e3225
oa_package/e2/1a/PMC2528944.tar.gz
PMC2528945
18802458
[ "<title>Introduction</title>", "<p>\n<italic>Streptococcus pneumoniae</italic> (pneumococcus) is an “extracellular” pathogen, generally considered to be killed by phagocytic ingestion, which is facilitated by opsonic antibodies. The success of anti-pneumococcal serum therapy using passive transfer of serotype-specific antibodies ##UREF##0##[1]## and of vaccinations based on purified or conjugated capsular antigens ##REF##10749457##[2]##,##REF##11920317##[3]## clearly shows that anticapsular antibodies protect humans against pneumococcal colonization and disease. There is good epidemiologic evidence for the importance of such immunity in certain common serotypes ##REF##15995951##[4]##,##REF##18471062##[5]##. However, we and others have found that factors other than anticapsular antibodies may play a role in the natural development of protection against pneumococcal colonization and disease. First, the reduction in pneumococcal disease incidence after the first year of life occurs simultaneously for both rare and common serotypes, suggesting the acquisition of one rather than many individual immune responses ##REF##15696204##[6]##. Similarly, the duration of carriage of many serotypes declines steeply between the first and second birthdays for many serotypes ##REF##17202280##[7]##. Since experience with conjugate vaccines has suggested that anticapsular antibodies reduce incidence of carriage but leave duration unaffected ##UREF##1##[8]##, this observation also suggests a mechanism of acquired immunity other than anticapsular antibodies. Moreover, the declines in carriage duration and invasive disease incidence precede by several years the detection of naturally-acquired anticapsular antibody in most children ##REF##15696204##[6]##,##REF##17202280##[7]##. Experimental ##REF##14500493##[9]##,##REF##15385481##[10]## and observational ##REF##15995951##[4]##,##REF##17703425##[11]## studies in adults have found little or no evidence that higher anticapsular antibody concentrations are associated with greater protection from colonization. Pneumococci also express non-capsular antigens common among serotypes, and certain of these have been found to elicit antibodies with protective potential in animal models. The role of such antibodies in human immunity has been evaluated ##REF##12870114##[12]##,##REF##10769009##[13]##,##REF##10689153##[14]##,##REF##16928889##[15]##,##REF##12922089##[16]##,##REF##11237805##[17]##.</p>", "<p>Surprisingly however, recent studies have shown that immunity in mice to pneumococcal colonization acquired from prior exposure to live bacteria ##REF##16177389##[18]## or a killed, whole-cell vaccine [WCV, consisting of killed pneumococcal whole cell antigen (WCA) with cholera toxin (CT) as an adjuvant] ##REF##15781870##[19]## is independent of antibodies of any specificity, and clearance of longstanding carriage in previously unexposed animals can likewise be antibody-independent ##REF##16239576##[20]##. Immunity had been shown to be dependent on the presence of CD4+ T cells at the time of challenge ##REF##16177389##[18]##,##REF##15781870##[19]##, but the co-participation of specific immune factors other than antibody was not ruled out.</p>", "<p>Here we show that intranasal immunization with the WCV confers protection against experimental pneumococcal colonization via the chemoattractant and neutrophil activating cytokine IL-17A, in a neutrophil-dependent fashion. Methods were devised to assay expression of IL-17A <italic>in vitro</italic> using peripheral blood samples. IL-17A expression by peripheral blood of WCV-immunized mice is highly correlated with subsequent protection against colonization, and expression by human cells, including those from adults and children, can be shown as well. Finally, we developed a surface phagocytosis assay with which we show that IL-17A enhances pneumococcal killing by human polymorphonuclear cells in the absence as well as presence of opsonins.</p>", "<p>The data indicate the possibility that IL-17A responses play a role in naturally-acquired immunity to pneumococcus in humans and that assay of this cytokine <italic>in vitro</italic> may assist in the evaluation of certain candidate pneumococcal vaccines that target mucosal colonization.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Bacterial strains and immunogens</title>", "<p>\n<italic>S. pneumoniae</italic> strain 0603 is a serotype 6B clinical strain ##REF##11447162##[49]##. Frozen mid-log phase aliquots were diluted to ∼10<sup>6</sup> cfu/10 µl of intranasal inoculum for challenge. The whole cell antigen (WCA) was derived from strain Rx1AL-, a capsule- and autolysin-negative mutant and prepared as described ##REF##11447162##[49]##. A pneumolysin-negative WCA (WCA(ply-)) was derived from an isogenic, pneumolysin-negative strain of Rx1AL- using methods previously described ##REF##16177389##[18]##. The final vaccine mixture (whole cell antigen WCA + adjuvant CT) for routine immunization contained 100 µg (dry weight) of WCA plus 1 µg of CT (List Biological Laboratories, Campbell, CA) per 10 µl dose. For potency comparisons, lower amounts of WCA were used (ranging from 0.1 µg to 10 µg). For all experiments, control mice were immunized nasally with 1 µg of CT in 10 µl saline.</p>", "<title>Animal models</title>", "<p>The animal model used in these experiments has been previously described ##REF##11447162##[49]##. C57BL/6J mice or mutants in the same background (female, age 6 weeks, Jackson Laboratories, Bar Harbor, ME) were randomized to receive 10 µl of whole cell vaccine or adjuvant alone intranasally twice at one week interval. Three weeks following the last inoculation, mice were anesthetized for retro-orbital blood sampling. One week later, mice were challenged intranasally with ∼10<sup>6</sup> cfu of strain 0603. At 1 week after challenge, the mice were euthanized by CO<sub>2</sub> inhalation; an upper respiratory wash was done by instilling sterile, nonbacteriostatic saline retrograde through the transected trachea and collecting the first 6 drops (about 0.1 ml) from the nostrils. Following collection of nasopharyngeal samples, the heads of WCV- and CT-immunized mice were removed and placed in formalin prior to histopathological preparation with hematoxylin and eosin (H&amp;E) staining.</p>", "<p>To evaluate the time to eradication of carriage, in a separate experiment, 4 mice from groups of 16 each were sacrificed at 1, 2, 4 and 6 days post inoculation and sequential dilutions of nasal washes were plated. To test whether CD4+ T cells are sufficient for protection, adoptive transfer experiments were performed. Splenocytes from wild type C57Bl/6 mice immunized with WCV or CT alone were harvested 2 months after the last immunization and CD4+ T cells were purified by magnetic bead positive selection (Miltenyi Biotec, Auburn, CA). A total of 3×10<sup>6</sup> CD4+ T cells were injected retro-orbitally in naïve RAG1<sup>−/−</sup> mice (B6.129S7-Rag1<sup>tm1Mom</sup>/J) that lack both B and T cells. The following day, these mice were challenged intranasally with strain 0603; one week later, density of colonization was determined as described above.</p>", "<p>To determine which T cell subset is responsible for protection, mice in the C57BL/6 background and deficient in IFN-γ (B6.129S7-Ifng<sup>tm1Ts</sup>/J, Jackson Laboratories, Bar Harbor, ME) IL-4 (B6.129P2-Il4<sup>tm1Cgn</sup>/J, Jackson Laboratories) or the IL-17 receptor (B6.129 IL17Ra<sup>−/−</sup>\n##REF##11514607##[50]##) were immunized and challenged as described above. For neutrophil depletion experiments, mice were immunized as described above; on days −1, +1 and +4 relative to challenge, mice were injected intraperitoneally with 100 µg of antineutrophil monoclonal antibody RB6-8C5 (purified from myeloma cell line by Bio Express, Lebanon, NH), a rat anti-mouse IgG2b directed against Ly-6G on the surface of murine myeloid (and limited subpopulations of lymphoid) lineage cells; in pilot experiments, this regimen resulted in &gt;90% depletion of neutrophils in most mice, although variability was observed. Because of this variability, peripheral neutrophil counts were determined at the time of euthanasia and correlated with the number of recovered pneumococci from that animal.</p>", "<title>Measurement of IL-17A secretion by splenocytes</title>", "<p>Cellular suspensions of splenocytes were obtained by passing spleens from immunized or control mice through a 70-µm cell strainer (BD Biosciences, Bedford, MA). After washing and removal of red blood cells by hemolysis, cells were plated into 24-well tissue culture plates at a concentration of 5×10<sup>6</sup> cells/well in 500 µl of DMEM/F12 with L-glutamine supplemented with 10% fetal calf-serum, 50 µM 2-mercaptoethanol (Sigma), and 10 µg/ml ciprofloxacin. Following 72-hour stimulation with concanavalin A (5 µg/ml, Sigma) or WCA (equivalent to 10<sup>6</sup> cfu/ml), supernatants were collected following centrifugation and stored at −80°C until analyzed by ELISA for IL-17A concentration (R&amp;D Systems, Minneapolis, MN). Supernatants were analyzed in duplicate and read against a standard, following directions provided by the manufacturer.</p>", "<p>For CD4+ T cell depletion, splenocytes were harvested as described above. CD4+ T cells were depleted from half of each spleen by magnetic bead selection (Miltenyi Biotec, Auburn, CA) following instructions by the manufacturer. Flow cytometry confirmed removal of &gt;95% CD4+ T cells (data not shown). Cells were seeded at the same concentration as described above (5×10<sup>6</sup> cells/well). In some cases, we repleted CD4+ T cells from depleted splenocytes, by adding 10<sup>6</sup> CD4+ T cells in the relevant wells.</p>", "<title>Intracellular staining for IL-17A</title>", "<p>Splenocytes were harvested, seeded, and stimulated with medium or WCA (10 µg/ml) as described above. Twenty-four later, monensin (BD GolgiStop, BD Biosciences) was added as per the manufacturer's instructions and cells were harvested 12 hours later. Cells were washed, stained with anti-CD4+ antibody (antiCD4+-PE, BD Biosciences) in the presence of Fc block, permeabilized with Perm/Wash buffer (BD Biosciences), and incubated with antimouse IL17A Alexa Fluor-647 (eBioscience) for 30 minutes. Intracellular cytokine staining for IL-17A was compared in CD4- or CD4+ cells in medium alone or following stimulation with WCA. Samples were analyzed on a Cytomation MoFlo (Beckman Coulter, Fullerton, CA), and results analyzed with Summit Version 4.3 (Dako, Fort Collins, CO).</p>", "<title>Measurement of IL-17A secretion by NALT</title>", "<p>NALT was harvested from immunized and control mice as described ##REF##9393634##[51]##. Mice were euthanized humanely, bled via intracardiac puncture to avoid blood contamination, and placed on a dissection board. The mouth was opened wide to expose the palate, which was cut carefully, so that the strips of NALT could be easily peeled off. These strips of cells were collected in medium (DMEM/F12 with L-glutamine supplemented with 10% fetal calf-serum, 50 µM 2-mercaptoethanol (Sigma), and 10 µg/ml ciprofloxacin) on ice. Cells were passed through a 70 µm strainer as described above and plated at 3×10<sup>5</sup> cells/well in a 96-well tissue culture plate in a total volume of 100 µl. Cells were stimulated with medium with or without added WCA (10 µg/ml) for a total of 3 days, after which supernatants were collected and assayed for IL-17A concentration by ELISA as above.</p>", "<title>Measurement of IL-17A secretion by whole blood</title>", "<p>For whole blood assays, blood of mice or humans at a final concentration of 10% was incubated in DMEM/F12 with L-glutamine supplemented with 10% fetal calf-serum, 50 µM 2-mercaptoethanol (Sigma), and 10 µg/ml ciprofloxacin in the absence or presence of killed pneumococcal antigen (corresponding to 10<sup>7</sup> cfu/ml for mice and 10<sup>6</sup> cfu/ml for human samples). Supernatants were collected after 6 days and the concentration of IL-17A measured as above for mice and, for human samples, by IL-17A ELISA (eBioscience Inc, San Diego, CA).</p>", "<title>Human subjects and samples</title>", "<p>For peripheral blood, samples were obtained at Children's Hospital Boston (for healthy adult volunteers) or from Cambridge Health Alliance, Cambridge, MA (for parturient women or umbilical cord) after written informed consent had been obtained. The studies were approved by the Children's Hospital Boston and Cambridge Health Alliance research ethics committees. For tonsillar specimens, tonsils were obtained from children who were 2 to 12 years old (median age, 5 years), were undergoing tonsillectomy for hypertrophy, and were otherwise healthy at Bristol Royal Hospital for Children, Bristol, United Kingdom. Patients who were immunized against pneumococcus previously, who had received antibiotics within 2 weeks of the operation or steroids, or who had an immunodeficiency or serious infection were excluded. The study was approved by the South Bristol local research ethics committee and written informed consent was obtained in all cases.</p>", "<title>Agar surface phagocytic killing without opsonins</title>", "<p>This assay approximates the “surface phagocytosis” described by Smith and Wood ##REF##13481251##[52]##. Neutrophils were isolated from heparinized blood by density gradient centrifugation (Histopaque, Sigma) following manufacturer's instructions. Neutrophils were washed extensively then resuspended in Hanks' Balanced Solution (+ Ca<sup>2+</sup> and Mg<sup>2+</sup>) with 0.2% bovine serum albumin (Sigma), then co-incubated for 30 minutes at 37°C with recombinant human IL-17A (R&amp;D Biosystems) at different concentrations. In some experiments, the cells were harvested by centrifugation and the supernatant collected, to examine whether the potentiating effect of IL-17A could be detected with the supernatant alone. Between 8–10 replicates of 10 µl of a bacterial suspension containing on average 100 cfu of strain 0603 were plated onto blood agar and the fluid allowed to adsorb into the agar for 15 min; 15 µl of the neutrophil suspension was overlaid and allowed to adsorb; the plates were incubated at 37°C with 5% C02 overnight after which colonies were counted.</p>", "<title>Phagocytic killing in suspension with suboptimal opsonization</title>", "<p>Neutrophils were isolated from whole blood as described above, washed twice with cold Hanks Balanced Salt Solution (HBSS-) (Mediatech, Herndon, VA), and resuspended to a final concentration of 6×10<sup>6</sup> cells/ml in cold HBSS containing calcium and magnesium (HBSS+) (Cellgro Mediatech, Herndon, VA) then held on ice until used. Cell counts were determined on a standard hemocytometer by counting viable cells (as determined by an absence of blue staining in the presence of Trypan Blue (Cellgro Mediatech, Herndon, VA)). <italic>S. pneumoniae</italic> (strain 0603 ##REF##11447162##[49]##) was diluted in HBSS+ to a final concentration of 5×10<sup>4</sup> bacteria/ml and incubated with antibodies to pneumococcal polysaccharide (Bacterial Polysaccharide Immune Globulin, BPIG-8, a kind gift of Dr. George Siber, consisting of concentrated IgG obtained from serum of adult volunteers immunized with pneumococcal, <italic>Haemophilus</italic> and meningococcal polysaccharide vaccines ##REF##1588146##[27]##) diluted in HBSS+. The reaction was incubated at 37°C for 15 minutes rotating at 200 RPM to promote bacterial opsonization. After bacterial opsonization, the opsonophagocytic killing reaction was initiated with the addition of baby rabbit complement (Pelfreez Biologicals, Rogers, AR) and neutrophils (ratio of 1∶200 bacteria∶cells) with or without recombinant human IL-17A (R&amp;D Systems, Minneapolis, MN) at 0.01, 0.1 or 1 µg/ml. A 1∶1600 dilution of BPIG was chosen to give sub-optimal bacterial killing (&lt;50% killing when compared to the same conditions without BPIG) in the presence of complement and neutrophils. The opsonophagocytic killing assay was performed in a 96-well round-bottom plate (Thermo Fisher Scientific, Waltham, MA) at 37°C for 90 minutes rotating at 200 RPM. After incubation, the opsonophagocytic reaction was diluted two fold and aliquots of each reaction were plated on blood agar plates then incubated at 37°C with 5% CO2 overnight.</p>", "<title>Isolation and culture of tonsillar mononuclear cells</title>", "<p>Mononuclear cells were isolated by using methods described previously ##REF##15071283##[53]##,##REF##16342325##[54]##. Tonsillar MNC were washed in sterile phosphate-buffered saline (PBS) and resuspended at a concentration of 4×10<sup>6</sup> cells/ml in RPMI medium containing HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Sigma, Dorset, United Kingdom). Cells were cultured in 96-well culture plates (Corning Inc, Corning, NY), and cell culture supernatants were collected at predetermined times and stored at −70°C until assays for human IL-17A were performed by sandwich ELISA (R&amp;D Biosystems).</p>", "<title>Statistical analysis</title>", "<p>Incidence of carriage was compared by Fisher's exact test and colonization density in challenged mice was compared by the Mann-Whitney <italic>U</italic> test. Statistical significance of the difference between time-to-clearance curves was assessed as follows. For each group <italic>i</italic> (<italic>i</italic> = WCV, CT, live, or naïve), the proportion of mice cleared at each time point <italic>t</italic>, <italic>p<sub>i</sub></italic>(<italic>t</italic>), was calculated. Using the max-min formula for isotonic regression ##UREF##3##[55]##, these proportions were smoothed to assure they were nondecreasing in <italic>t</italic>, yielding smoothed proportions <italic>q<sub>i</sub></italic>(<italic>t</italic>). Then, a test statistic was calculated to quantify the distance between the smoothed curves for two groups (e.g., WCV vs. CT): . The significance level of this test statistic was estimated by permuting the group identifiers of the cleared mice at each time point, fixing the total number of mice in each group and the total number cleared at each time point. 100,000 replicates of the permuted data were obtained, and <italic>T</italic> was calculated for each. The p value was calculated as the fraction of these 100,000 permutations having a test statistic strictly less than that calculated for the data. The correlation between neutrophil count or IL-17A concentration and colonization density was determined by Spearman rank correlation. The effect of increasing IL-17A concentrations on enhancing killing of pneumococcus was assessed by Wilcoxon matched pairs test. For all comparisons, P&lt;0.05 was considered to represent a significant difference.</p>" ]
[ "<title>Results</title>", "<title>Prior exposure of mice to killed or live pneumococci reduces the duration of experimental pneumococcal carriage</title>", "<p>The duration of carriage was followed after intranasal challenge with serotype 6B pneumococci 4 weeks post-exposure to WCV. Both WCV-vaccinated and control mice immunized with CT alone were colonized one day after challenge. In mice immunized with WCV however, carriage became significantly reduced after 4 days compared to controls given cholera toxin (CT) adjuvant alone (median density of colonization on day 4 in WCV- vs. CT-immunized mice 251 vs. 3720 cfu/nasal wash, P = 0.029 by Mann-Whitney U test) and was undetectable by day 6 (0/4 WCV-immunized mice had detectable colonies on day 6 vs. 4/4 mice that received CT, P = 0.029 by Fisher's Exact test, ##FIG##0##Figure 1A##). A similar differential was observed in mice that had been repeatedly exposed to live pneumococci vs. saline controls: the density of colonization became significantly different by day 4 after inoculation (##FIG##0##Figure 1A##). By day 6, similar to what we observed in WCV-immunized mice, 0/4 mice exposed to live pneumococci had detectable colonies compared to 4/4 saline controls (P = 0.029 by Fisher's exact test). When data from all time points were compared, mice immunized with WCV or exposed to live pneumococci had a significantly shorter time to clearance compared to their respective CT or saline controls (P = 0.0001 for comparison of WCV vs. CT and P = 0.004 for comparison of live exposure vs. saline). Thus the protection by prior pneumococcal exposure involves not immediate blockage of colonization but rather an accelerated clearance over days. Subsequent studies compared WCV-vaccinated with control animals 7 days after the intranasal challenge.</p>", "<title>CD4+ T cells transfer acquired immunity to pneumococcal colonization to RAG1<sup>−/−</sup> mice</title>", "<p>Several previous studies showed that acquired immunity to pneumococcal colonization in mice can be antibody-independent and dependent on CD4+ T cells ##REF##15781870##[19]##,##REF##16239576##[20]##,##REF##16552049##[21]##,##REF##17698570##[22]##. Here, adoptive transfer showed that CD4+ T cells are not only necessary but also sufficient for the accelerated clearance induced by WCV: unimmunized Rag1<sup>−/−</sup> mice (lacking both B and T cells) were infused with CD4+ T cells from mice immunized with WCV or CT alone. The RAG1<sup>−/−</sup> mice that received CD4+ T cells from WCV-immunized wild-type mice had significantly reduced density of colonization by day 7 compared to mice infused with CD4+ T cells from mice immunized with CT alone (P = 0.0001 by Mann-Whitney U, ##FIG##0##Figure 1B##).</p>", "<title>Acquired immunity to pneumococcal colonization is associated with the T<sub>H</sub>17 subset of CD4+ T cells</title>", "<p>To evaluate which CD4+ T cell subset is responsible for protection, IFN-γ, IL-4 or IL-17A receptor (IL-17AR) knockout mice were immunized with WCV vs. CT alone. IFN- γ- and IL-4-deficient mice immunized with WCV were significantly protected against colonization both with respect to proportion of colonized mice (P&lt;0.001 by Fisher's Exact test for comparison of % of colonization in WCV- and CT-immunized IFN-γ- or IL-4-deficient mice) and density of colonization (P≤0.001 compared to their respective CT controls, ##FIG##1##Figure 2A##). In contrast, mice with a targeted deletion of the IL-17A receptor were not protected (P&gt;0.5 vs. CT controls for % colonized mice or density of colonization, ##FIG##1##Figure 2A##). It is noteworthy that IL-17AR-knockout mice in the CT control group had, on average, a ten-fold greater density of colonization than the corresponding IFN-γ or IL-4 deficient mice, suggesting that IL-17A may also be involved in resistance to colonization in naïve mice.</p>", "<p>Splenocytes from mice immunized with WCV expressed significantly more IL-17A in response to WCA <italic>in vitro</italic> than cells from CT control animals (##FIG##1##Figure 2B##). We have previously shown that immunization with WCV confers protection against NP colonization in a CD4+ T cell dependent fashion ##REF##15781870##[19]##; additional experiments were performed here to confirm that IL-17A is produced by CD4+ T cells. Depletion of CD4+ T cells effectively abrogated IL-17A expression from WCA-stimulated splenocytes, such that there was no detectable increase in IL-17A production compared to splenocytes stimulated with medium alone; repletion of CD4+ T cells restored the IL-17A response seen with nondepleted splenocytes stimulated with WCA (##FIG##1##Figure 2C##). Moreover, intracellular cytokine staining experiments confirmed that IL-17A production originates primarily from CD4+ cells (##FIG##1##Figure 2D##). Splenocytes from WCV-immunized animals were stimulated with WCA and analyzed by flow cytometry. The frequency of IL-17A positive cells increased 2.3 fold among CD4+ T cells (3.37% in WCA stimulated cells vs. 1.49% in cells stimulated with medium alone, P = 0.008 by Mann-Whitney U) whereas the non-CD4+ population was unaffected (1.5% vs. 1.7% for stimulation with WCA vs. medium alone in CD4- cells, P = 0.5). Similar analyses were performed using splenocytes from mice immunized with CT alone; no increase in IL-17A positive cells was noted, either in the CD4+ or CD4- population (data not shown). Collectively, these results indicate that IL-17A is produced by CD4+ T cells.</p>", "<p>Next, we harvested nasal associated lymphoid tissue (NALT) from WCV- and CT-immunized mice. Cells were incubated for 3 days in the presence of medium alone or with WCA, after which IL-17A expression was measured by ELISA. NALT cells from WCV-immunized mice showed significantly greater IL-17A production than CT controls in response to stimulation with WCA (##FIG##1##Figure 2E##).</p>", "<p>To test whether the capacity to produce IL-17A predicted an individual mouse's protection, a total of 90 mice were intranasally immunized with CT (1 µg) plus a range of doses of WCA ranging from 1 to 100 µg and blood samples were taken 7 days before challenge and stimulated with WCA <italic>in vitro</italic> for IL-17A production. The IL-17A concentrations following 6 days of culture <italic>in vitro</italic> varied from undetectable (&lt;0.02 ng/ml) to about 6 ng/ml, and the cfu of pneumococci/nasal wash 7 days post-challenge varied from undetectable (&lt;1.6 cfu/nasal wash) to about 3000; there was a strong inverse correlation (Spearman ρ = −0.62, P &lt;0.0001, ##FIG##2##Figure 3##); 95% of mice with pre-challenge IL-17A concentrations above 0.3 ng/ml were free of pneumococcal colonization.</p>", "<title>Neutrophil-like cells are required for acquired immunity to pneumococcal carriage</title>", "<p>Because IL-17 A induces neutrophil recruitment and activation ##REF##15485625##[23]##, we evaluated whether neutrophils were required for protection against colonization. Mice intranasally immunized with WCV (or CT alone) were challenged, with or without administration of monoclonal antibody RB6-8C5 (which targets neutrophil-like cells) at the time of challenge. Several experiments were performed to ensure that treatment with this antibody did not affect CD4+ T cell number or function. Evaluation of splenocytes of antibody-treated animals showed no reduction in the CD4+ T cell population (data not shown). Furthermore, we confirmed that IL-17A production from the peripheral blood or from NALT of WCV-immunized mice was not affected by treatment with RB6-8C5 antibody. The peripheral blood IL-17A expression from immunized, neutrophil-depleted mice was similar to that of immunized, nondepleted mice (median IL-17A whole blood expression in neutrophil depleted vs. non-depleted mice: 1059 vs. 1290 pg/ml, P = 0.7 by Mann-Whitney U test); similarly, there was no reduction in NALT IL-17A expression from immunized mice following neutrophil depletion (median IL-17A expression from NALT in depleted vs. nondepleted mice 23.6 vs. 19.8 pg/ml, P = 0.69 by Mann-Whitney U test).</p>", "<p>Neutrophil depletion significantly diminished protection by immunization (##FIG##3##Figure 4A##). WCV-immunized and neutrophil depleted mice had both higher proportion of colonized mice (9/14 vs. 3/15 colonized mice for neutrophil-depleted vs. non-depleted WCV-immunized mice respectively, P = 0.025 by Fisher's Exact test) and density of colonization (median 12.8 cfu/nasal wash vs. 0.8 cfu/nasal wash respectively, P = 0.05 by Mann-Whitney U). While WCV-immunized, neutrophil-depleted mice had reduced colonization density compared to mice that received CT alone (median colonization density 453 cfu/nasal wash, P = 0.006 by Mann-Whitney U), the percentage of remaining neutrophils was strongly negatively correlated with recovered cfu from challenged mice (Spearman ρ = −0.77, P = 0.001, ##FIG##3##Figure 4B##), suggesting that residual protection was accounted for in large part by incomplete neutrophil depletion.</p>", "<p>Consistent with these results, blinded review of histopathology of nasopharyngeal tissue of 6/8 WCV-immunized mice seven days after challenge with pneumococcus showed a distinct neutrophilic infiltrate in the submucosa at the junction of the olfactory and respiratory epithelium (##FIG##3##Figure 4C, right panel##), which is not seen in CT-immunized subsequently challenged with pneumococci (left panel) (presence of infiltrate in 6/8 WCV immunized mice vs. 0/7 CT controls, P = 0.007). Thus the data support a role for IL-17A acting upon neutrophils in protection against pneumococcal colonization in mice.</p>", "<title>IL-17A expression in human samples following pneumococcal stimulation</title>", "<p>Next we determined whether IL-17A responses to pneumococcus could be measured in humans. Tonsillar mononuclear cells (from 8 donors) were stimulated with medium alone, WCA obtained from a pneumolysin-negative strain (WCA(ply-)) or WCA from the wild-type strain. IL-17A expression measured at 72 hours was significantly higher following stimulation with WCA than with medium alone (##FIG##4##Fig 5A##); this increase was abrogated when a pneumolysin-negative WCA was used as stimulus, consistent with prior findings in humans and in mice regarding the association between T cell-mediated responses to this toxin and prevention of pneumococcal colonization (##REF##17357058##[24]## and unpublished data). Furthermore, whole blood from unimmunized adult human volunteers, presumed to have been naturally exposed to pneumococcus, produced IL-17A in response to WCA <italic>in vitro</italic> (##FIG##4##Fig 5B##). Eighteen subjects produced a range of IL-17A concentrations from about 4 to 200 pg/ml, with a geometric mean of 20 pg/ml. Of these volunteers, 11 were parturient women, whose geometric mean IL-17A expression was 18 pg/ml. Umbilical cord blood was available in each of these cases; IL-17A in these samples was at the lower limit of detection of the assay (4 pg/ml) in all but one case, significantly lower than that of all adult subjects or parturient women (P&lt;0.001 and P&lt;0.01, respectively by Mann-Whitney U).</p>", "<title>IL-17A enhances in vitro phagocytic killing of pneumococci</title>", "<p>We evaluated whether IL-17A enhances <italic>in vitro</italic> killing of pneumococci by human neutrophils in different assays. Having reported previously that WCV induced protection in antibody-deficient mice, we developed a surface phagocytic killing assay to evaluate whether -17A could potentiate killing of non-opsonized pneumococci. Neutrophils isolated from healthy volunteers were pre-incubated with recombinant human IL-17A, then overlaid on pneumococci that had been plated onto blood agar. The overlay of IL-17A in the absence of neutrophils did not result in any killing, consistent with studies in which the addition of IL-17A to culture medium did not affect growth of pneumococci and arguing against any direct killing effect of the cytokine or contaminant present in the preparation (as shown in ##FIG##5##Figure 6B##). In the presence of neutrophils, IL-17A induced dose-dependent killing of pneumococci (##FIG##5##Figure 6A##). Thus IL-17A potentiated <italic>in vitro</italic> neutrophil killing of pneumococcus, in the absence of antibodies or complement.</p>", "<p>To evaluate whether the potentiating effect of IL-17A is the result of lysis or degranulation of neutrophils, we performed trypan blue exclusion studies of neutrophils with and without preincubation with IL-17A at the highest dose studied (10 µg/ml). There was no difference in viability when IL-17A was added; over 95% of cells remained viable after 30 minutes of incubation. Furthermore, neutrophils preincubated with IL-17A then washed showed the same enhancement of pneumococcal killing as incubated neutrophils that were not washed. In contrast, the supernatant obtained after incubation of neutrophils and IL-17A had no detectable antipneumococcal activity (##FIG##5##Figure 6B##). Taken together, these data argue against a toxic or degranulating effect of IL-17A on neutrophils and are more consistent with enhancement of phagocytic activity of neutrophils by this cytokine.</p>", "<p>Because capsular and noncapsular antipneumococcal antibodies gradually increase with age in children ##REF##15696204##[6]##,##REF##10979911##[25]##,##REF##15155637##[26]##, we also evaluated whether IL-17A would potentiate opsonophagocytic killing of <italic>S. pneumoniae</italic> in the presence of limiting amounts of antibody. Bacterial polysaccharide immune globulin (BPIG) is a plasma concentrate from volunteers immunized with bacterial polysaccharides including pneumococcal serotype 6B ##REF##1588146##[27]##. To evaluate whether IL-17A would synergize with antipneumococcal antibodies, we added IL-17A to an opsonophagocytic assay using a suboptimal dose of BPIG. When IL-17A was added to the reaction, killing of pneumococcus was enhanced in a dose-dependent fashion in all individuals tested (##FIG##5##Figure 6C##). Killing was significantly increased when either 1 µg or 10 µg/ml IL-17A were added (P = 0.016 by Wilcoxon matched pairs test). There was no significant pneumococcal killing in the assay in the absence of any of the principal components, including PMNs, antibody (BPIG) or complement (data not shown). Additionally, as in the surface phagocytosis assay, IL-17A alone had no effect on growth of bacteria <italic>in vitro</italic> or survival in our assay, implying that direct killing of pneumococci by IL-17A is very unlikely.</p>" ]
[ "<title>Discussion</title>", "<p>The importance of CD4+ T cells in protection against pulmonary infections became clearly evident with the advent of the HIV epidemic. Infected individuals have a dramatically increased risk of infections with opportunistic pulmonary pathogens such as <italic>Mycobacterium tuberculosis</italic> or <italic>Pneumocystis jiroveci</italic> and this risk is inversely related to the number of circulating CD4+ T cells ##REF##7902536##[28]##,##REF##1967190##[29]##. For <italic>S. pneumoniae</italic>, HIV infection confers a 50-fold increased risk of infection, which is also inversely related to CD4+ T cell count ##REF##8602001##[30]##,##REF##11229848##[31]##. Most recently, a study in Zambian mothers has demonstrated that HIV infection is associated with a significantly increased risk of colonization and reduced time to new colonization ##REF##18419536##[32]##. Various hypotheses have been advanced to explain why CD4+ T cell-deficiency is associated with such a high risk of infection with pneumococcus, a primarily extracellular encapsulated bacterium; these include reduced opsonic activity of anticapsular antibodies ##REF##14614677##[33]##, loss of memory B cells ##REF##16645169##[34]##, and alteration of innate pulmonary immunity ##REF##16170759##[35]##. To date, however, it is fair to state that the paradox remains unexplained. The data presented here suggest that a loss of T<sub>H</sub>17 cells may also contribute to this increased susceptibility.</p>", "<p>While the ability of pneumococcal conjugate vaccine-induced anticapsular antibodies to protect against pneumococcal colonization is clear, less is known about the natural development of immunity to pneumococcal colonization. There appear to be both antibody-dependent and antibody-independent mechanisms that reduce the likelihood or duration of carriage. Several studies have documented a homotypic anticapsular serum antibody response to colonizing pneumococcal serotypes ##REF##15995951##[4]##,##REF##3183428##[36]##,##REF##9114197##[37]##,##REF##15995949##[38]##. In a longitudinal study examining the relationship between antibodies and carriage in adults, Goldblatt et al. showed that, among six serotypes tested, anticapsular antibody concentration to serotype 14 was significantly associated with reduced odds of carriage ##REF##15995951##[4]##. More recently, by analyzing longitudinal carriage data from Israeli toddlers in daycare, we found a lower risk of colonization with type 6A, 14, and 23F after previous exposure to the homologous type ##REF##18471062##[5]##. For types 14 and 23F, this specific protection correlated with increased serotype-specific antibody concentration. On the other hand, as in the previous study ##REF##15995951##[4]##, there was no evidence for such a correlation with several other serotypes. Several studies have argued against the role of anticapsular antibody. For example, the risk of acquisition of new pneumococcal strains in adults with chronic obstructive pulmonary disease was associated with <italic>higher</italic> preacquisition concentrations of anticapsular and noncapsular pneumococcal antibodies ##REF##17703425##[11]##. These results suggest that in this population, antipneumococcal antibodies are markers of prior exposure and perhaps <italic>greater</italic> susceptibility rather than predictors of protection. Finally, in the sole published example of experimental pneumococcal colonization of humans, antibodies to the capsular polysaccharide did not predict protection against colonization ##REF##11828011##[39]##.</p>", "<p>We, and others, have presented data supporting a role of CD4+ T cells, independently of antibody, in reducing pneumococcal colonization in mouse models ##REF##16177389##[18]##,##REF##15781870##[19]##,##REF##16239576##[20]##,##REF##17698570##[22]##,##UREF##2##[40]##. In studies involving immunization with the pneumococcal zwitterionic conserved cell wall polysaccharide, we showed that neutralization of IL-17A significantly reduced protection, albeit not completely ##REF##16552049##[21]##. Zhang et al. then showed that reduced CD4+ T cell proliferative responses to the pneumococcal toxin pneumolysin were associated with nasopharyngeal pneumococcal carriage in children ##REF##17357058##[24]##. We subsequently showed that immunization with a mixture of a pneumolysin nontoxic mutant and two other pneumococcal proteins elicits T<sub>H</sub>17 cells and confers protection against colonization in a CD4+ T cell-dependent, antibody-independent fashion ##REF##17698570##[22]##. Others have argued against this possibility, proposing instead a T<sub>H</sub>1, IFN-γ mediated mechanism, based on the finding that IL-12p40-deficient mice cleared pneumococcal colonization as well as wild-type mice ##REF##16239576##[20]##. Since that report, it has become clear that IL-23, which is lacking in IL-12p40 deficient mice, is not absolutely required for the generation of T<sub>H</sub>17 cells as once was thought, but instead participates in their maintenance or expansion ##REF##16473830##[41]##,##REF##16648838##[42]##.</p>", "<p>Here we present evidence that acquired CD4+ T<sub>H</sub>17 cells reduce the duration of experimental colonization with <italic>S. pneumoniae</italic> in a manner reminiscent of the age-dependent decline in duration of carriage ##REF##17202280##[7]## and that this mechanism occurs independently of key T<sub>H</sub>1 or T<sub>H</sub>2 cytokines, IFN- γ and IL-4 respectively. We show that CD4+ T cells are sufficient to provide the protection against colonization, which is abrogated in the absence of the IL-17A receptor and highly dependent on neutrophils, one of the main targets of this cytokine. Recombinant human IL-17A enhances both antibody-independent and –dependent killing of <italic>S. pneumoniae in vitro</italic>. Importantly, IL-17A expression can be induced by exposure to pneumococcal antigens of tonsillar cells from children and peripheral blood from healthy adult volunteers, but not in umbilical cord blood, consistent with the view that this responsiveness may be the result of prior exposure to the pathogen.</p>", "<p>IL-17A signaling been shown to participate in host defense against extracellular pathogens, such as <italic>Klebsiella</italic> and <italic>Candida</italic> in naïve mice ##REF##11588011##[43]##,##REF##15243941##[44]##. Prior to this report, there have been two demonstrations of a role of T<sub>H</sub>17 cells in vaccine-induced immunity and in both cases, whole organisms, killed or live, were used. Higgins et al. showed that protection against <italic>Bordetella pertusssis</italic> with a whole cell vaccine induced T<sub>H</sub>17-dependent protection and Khader et al. presented similar findings with the mycobacterial protein ESAT6-induced protection against <italic>M. tuberculosis</italic>\n##REF##17114471##[45]##,##REF##16002675##[46]##,##REF##17351619##[47]##. The data derived from both mouse and human studies in the present report thus add to the growing evidence that T<sub>H</sub>17 cells contribute to immunity to respiratory pathogens.</p>", "<p>Numerous attempts have been made to define correlates of protection against pneumococcal carriage and have focused on the humoral response to pneumococcal capsular or noncapsular antigens ##REF##15995951##[4]##,##REF##16928889##[15]##,##REF##18008233##[48]##; although associations between levels of antibodies in saliva and reduced risk of otitis media have been reported ##REF##18008233##[48]##, no reliable correlate has been identified. Here we show that the IL-17A response in immunized mice is highly correlated with reduced carriage; in particular above a certain concentration, colonization beyond 7 days is very unlikely. Naturally-exposed humans have low, but measurable IL-17A responses, which could be evaluated in response to immunization with candidate pneumococcal vaccines, such as the whole cell vaccine currently under development. The demonstrated association between carriage and reduced T cell proliferative responses to pneumolysin in childhood ##REF##17357058##[24]## provides further support for a functional T cell assay such as the one proposed here.</p>" ]
[]
[ "<p>Co-senior authors.</p>", "<p>Conceived and designed the experiments: YJL JG DB AF LB QZ JKK AS AL CMT KFH PWA ML RM. Performed the experiments: YJL JG DB AF LB QZ AS AL SF CMT PWA ML RM. Analyzed the data: YJL JG DB AF LB QZ JKK AS AL SF CMT PWA ML RM. Contributed reagents/materials/analysis tools: YJL DB JKK KFH RM. Wrote the paper: PWA ML RM.</p>", "<p>Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell–dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to naïve RAG1<sup>−/−</sup> mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-γ or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. <italic>In vitro</italic> expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection <italic>in vivo</italic>. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation <italic>in vitro</italic> could help in the development of candidate pneumococcal vaccines.</p>", "<title>Author Summary</title>", "<p>The bacterium <italic>Streptococcus pneumoniae</italic> (pneumococcus) causes serious disease in children and the elderly, including pneumonia and meningitis (inflammation of the brain). Carriage of pneumococcus in the nose is a necessary first step for most infections. As children age, they carry pneumococcus for shorter periods of time and their risk of disease decreases also. The mechanisms underlying this age-related decrease of carriage are not well understood. A deeper understanding of resistance to colonization would enable us to develop better pneumococcal vaccines. Using experimental mouse models, we show that repeated exposure to pneumococci leads to a subsequent reduction in duration of pneumococcal carriage, similar to what is observed in humans. We identify the immune cells that are responsible for this process, so-called T<sub>H</sub>17 cells, which release a factor that enables human blood cells to kill pneumococcus more efficiently. We show that these T<sub>H</sub>17 cells exist in adults and children, but not in newborn babies, which suggests that they may arise as a consequence of humans being exposed to pneumococcus. We describe an assay for the measurement of these cells in humans. Such an assay could facilitate the development of novel vaccines directed against pneumococcal carriage.</p>" ]
[]
[ "<p>The authors thank Michael Wessels for helpful discussions, Jay Mizgerd and Bryanne Robson for their assistance with neutrophil depletion studies, Rod Bronson for assistance with histopathological studies, Eric Tchetgen for statistical advice, Jessica Hartman Jacobs for programming assistance and Elizabeth Boush for advice and assistance with flow cytometry.</p>" ]
[ "<fig id=\"ppat-1000159-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000159.g001</object-id><label>Figure 1</label><caption><title>Duration of carriage and effect of adoptive transfer following immunization with killed or live pneumococci.</title><p>A. Effect of intranasal immunization with WCV or live exposure upon density of pneumococcal colonization in C57BL/6 mice. Density of colonization in mice immunized with WCV vs. CT alone or repeatedly exposed to live pneumococcal strain 0603 vs. saline alone at various time points (n = 4 per time point) following challenge. By day 4, both the incidence and density of carriage were significantly lower in mice immunized with WCV or exposed to live pneumococcus compared to mice immunized with CT or saline, respectively. * P&lt;0.05. The dashed line represents the lower limit of detection of bacterial colonization. B. Effect of adoptive transfer of CD4+ T cells from WCV-immunized mice on pneumococcal colonization of recipient, unimmunized RAG1<sup>−/−</sup> mice. Each data point represents the density of nasopharyngeal colonization in cfu/ nasal wash for each mouse one week post-challenge. The horizontal bar shows the geometric mean cfu/nasal wash for each group and the dashed line represents the lower limit of detection of bacterial colonization. C57BL/6 mice were immunized with WCV or CT as indicated. Four weeks after the last immunization, CD4+ T cells were harvested from splenocytes of mice and infused into naïve, unimmunized RAG1<sup>−/−</sup> mice; challenge of these mice and quantification of colonization was then performed. RAG1<sup>−/−</sup> mice that received CD4+ T cells from WCV-immunized mice had significantly lower density of colonization than mice that received cells from CT-immunized mice (*** P&lt;0.0001 by Mann-Whitney U).</p></caption></fig>", "<fig id=\"ppat-1000159-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000159.g002</object-id><label>Figure 2</label><caption><title>Role of T-helper-subset-associated cytokines in protection from nasopharyngeal colonization.</title><p>A. Mice defective in IFN-γ, IL-4 or IL-17A receptor were immunized as described, then challenged with pneumococcal strain 0603. Mice with IFN-γ or IL-4 deficiency were significantly protected by WCV (P&lt;0.001 vs. respective CT controls) whereas IL-17A receptor deficient mice were not protected (P&gt;0.5 vs. CT). Dashed line represents the lower limit of detection of bacterial colonization. B. Expression of IL-17A from splenocytes of WCV-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone, Concanavalin A (5 µg/ml), WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice, although response to concanavalin A was similar. C. Effect of CD4+ T cell depletion upon IL-17A expression from splenocytes. Splenocytes (without or with CD4+ T cell depletion) from mice immunized with WCV were stimulated for 72 hours with medium alone or WCA after which IL-17A was measured by ELISA. IL-17A expression in splenocytes following WCA stimulation was significantly higher in the presence of CD4+ T cells compared to stimulation with medium alone or when CD4+ T cells were depleted. Repletion of CD4+ T cells restored the response. ** P&lt;0.01 compared to cells stimulated with medium alone. D. IL-17A intracellular staining of splenocytes from WCV immunized mice. Splenocytes from WCV immunized mice were stimulated with WCA, blocked with monensin, harvested and stained for CD4+ and intracellular IL-17A as described. There is a statistically significant increase in CD4+ IL-17A positive cells following stimulation with WCA, which is not observed in the CD4- population. No increase in IL-17A positive cells could be detected in cells from unimmunized mice (data not shown). **P = 0.008 for comparison of frequency of IL-17A-positive cells in absence and presence of WCA stimulation among CD4+ cells. Data shown here are representative of three experiments, including at least 5 mice per experiment. E. Expression of IL-17A from NALT of WCV- vs. CT-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone or with WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice. **P&lt;0.01 for comparison of IL-17A in WCV vs. CT-immunized mice following stimulation with WCA.</p></caption></fig>", "<fig id=\"ppat-1000159-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000159.g003</object-id><label>Figure 3</label><caption><title>Correlation of IL-17A expression and density of nasopharyngeal colonization in mice.</title><p>Three weeks after immunization of mice (n = 90) with CT with doses of WCA ranging from 1 to 100 µg, and one week before pneumococcal challenge, blood samples were obtained and stimulated with WCA (10 µg) for 6 days, after which supernatants were collected and assayed for IL-17A concentration. The correlation between density of colonization (cfu/nasal wash) 7 days after challenge and pre-challenge IL-17A expression was evaluated. IL-17A expression was significantly correlated with density of colonization.</p></caption></fig>", "<fig id=\"ppat-1000159-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000159.g004</object-id><label>Figure 4</label><caption><title>Effect of neutrophils on WCV-induced protection against pneumococcal colonization.</title><p>A. Effect of neutrophil depletion on WCV-induced protection from nasopharyngeal colonization. Each data point represents the density of nasopharyngeal colonization in cfu/nasal wash for each mouse. The horizontal bar shows the geometric mean cfu/nasal wash for each group and the dashed line shows the lower limit of detection of bacterial colonization. Mice were immunized with CT or WCV as indicated; just prior to the time of challenge, mice were randomized to receive antineutrophil antibody vs. saline. Proportion of colonized mice and density of colonization was determined 7 days post challenge. WCV-immunized mice that received saline treatment were significantly better protected than WCV-immunized mice that received antineutrophil antibody, with a lower proportion of colonized mice (P = 0.025 by Fisher's Exact) and density of colonization (P = 0.05 by Mann-Whitney U). B. Correlation between neutrophil count and density of pneumococcal colonization. Neutrophil counts following neutrophil depletion were assayed at the time of sacrifice and plotted against density of colonization. There was a strong negative association between neutrophil counts and colonization density (Spearman <italic>ρ</italic> = −0.75). C. Histopathology of nasopharyngeal tissue following nasopharyngeal challenge of CT- (left panel) and WCV-immunized (right panel) mice. Seven days post pneumococcal challenge, mice were euthanized, heads stored in formalin, and H&amp;E sections of nasopharyngeal tissue prepared and examined under light microscopy at 60× magnification. The presence of a dense neutrophilic infiltrate in the submucosa at the junction of the olfactory and respiratory epithelium was noted in WCV-immunized mice following pneumococcal nasopharyngeal challenge but not in CT-immunized mice. The two slides shown are representative of a total of 15 examined specimens (8 WCV-immunized and 7 CT controls, all at day 7 post pneumococcal challenge). Lesions like those represented here were observed in 6/8 immunized mice and 0/7 controls.</p></caption></fig>", "<fig id=\"ppat-1000159-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000159.g005</object-id><label>Figure 5</label><caption><title>Effect of exposure to pneumococcus on IL-17A expression from human tissues and cells.</title><p>A. Expression of IL-17A from tonsillar mononuclear cells from children. Tonsillar cells (n = 8) were cultured as described and stimulated with WCA or WCA derived from an isogenic, pneumolysin-negative strain (WCAply-). Stimulation with WCA was associated with significantly increased IL-17A expression compared to exposure to medium alone (P = 0.008 by Wilcoxon signed rank test), whereas stimulation with WCAply- did not increase IL-17A production. B. Expression of IL-17A from peripheral blood of adults and umbilical cord blood. Peripheral blood samples from adults (healthy adult volunteers (n = 7), parturient women (n = 11) and umbilical cord blood (n = 11) were stimulated with WCA for 6 days after which IL-17A concentration was assayed by ELISA. IL-17A production was significantly greater in adults than cord blood (P&lt;0.001 by Mann-Whitney U test).</p></caption></fig>", "<fig id=\"ppat-1000159-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000159.g006</object-id><label>Figure 6</label><caption><title>IL-17A-mediated enhanced killing of <italic>S. pneumoniae</italic>.</title><p>A and B. Effect of human IL-17A on surface phagocytic killing of <italic>S. pneumoniae</italic>. A. Isolated neutrophils from healthy adult volunteers were incubated with recombinant human IL-17A at the indicated concentrations and evaluated in a surface phagocytic killing assay with pneumococcal strain 0603; colonies were counted after overnight incubation at 37°C with 5% CO<sub>2</sub>. IL-17A induces a dose-dependent enhancement of neutrophil killing of <italic>S. pneumoniae</italic> (P = 0.01 for 1 or 10 µg of IL-17A vs. no added IL-17A). B. Supernatant obtained from neutrophils after incubation with IL-17A did not have any demonstrable antipneumococcal effect, whereas washed neutrophils after incubation with IL-17A demonstrated enhanced killing. C. Effect of human IL-17A on opsonophagocytic killing of <italic>S. pneumoniae</italic>. Neutrophils purified from the peripheral blood of healthy adult volunteers were incubated with pneumococci anticapsular antibodies, complement, and a range of concentrations of IL-17A as indicated for 90 minutes, following which viable counts were obtained by plating on blood agar plates. Each line represents a different volunteer. IL-17A enhanced killing of pneumococci in a dose-dependent fashion in 6/6 subjects. *P = 0.016 by Wilcoxon matched pairs test.</p></caption></fig>" ]
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[ "<fn-group><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p>The authors gratefully acknowledge support from the Pamela and Jack Egan Fund. RM is supported by grants from the National Institutes of Health (AI067737-01 and AI51526-01) and PATH. CMT and ML's work was supported by grant R01 AI048935 from the National Institutes of Health. JG was supported by the U.S. Army; DB was supported by the Ter Meulen Fund, Royal Netherlands Academy of Arts and Sciences; LB was supported by a fellowship from the European Society for Paediatric Infectious Diseases. AF and QZ are supported by a grant from SPARKS, UK. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"ppat.1000159.g001\"/>", "<graphic xlink:href=\"ppat.1000159.g002\"/>", "<graphic xlink:href=\"ppat.1000159.g003\"/>", "<graphic xlink:href=\"ppat.1000159.g004\"/>", "<graphic xlink:href=\"ppat.1000159.g005\"/>", "<graphic xlink:href=\"ppat.1000159.g006\"/>", "<inline-graphic xlink:href=\"ppat.1000159.e001.jpg\" mimetype=\"image\"/>" ]
[]
[{"label": ["1"], "element-citation": ["\n"], "surname": ["Lord", "Heffron"], "given-names": ["F", "R"], "year": ["1938"], "source": ["Pneumonia and Serum Therapy"], "publisher-loc": ["London"], "publisher-name": ["Commonwealth Fund"]}, {"label": ["8"], "element-citation": ["\n"], "surname": ["Dagan", "Lipsitch", "Tuomanen"], "given-names": ["R", "M", "E"], "year": ["2004"], "article-title": ["Changing the Ecology of Pneumococci with Antibiotics and Vaccines."], "source": ["The Pneumococcus"], "publisher-loc": ["Washington, D.C."], "publisher-name": ["ASM Press"], "fpage": ["283"], "lpage": ["313"]}, {"label": ["40"], "element-citation": ["\n"], "surname": ["Trzcinski", "Thompson", "Srivastava", "Basset", "Malley"], "given-names": ["K", "CM", "A", "A", "R"], "year": ["2008"], "article-title": ["Protection against Nasopharyngeal Colonization by Streptococcus pneumoniae is Mediated by Antigen-Specific CD4+ T Cells."], "source": ["Infect Immun"]}, {"label": ["55"], "element-citation": ["\n"], "surname": ["Robertson", "Wright", "Dykstra"], "given-names": ["T", "FT", "RL"], "year": ["1988"], "source": ["Order Restricted Statistical Inference"], "publisher-loc": ["Chichester"], "publisher-name": ["Wiley"]}]
{ "acronym": [], "definition": [] }
55
CC BY
no
2022-01-13 03:40:34
PLoS Pathog. 2008 Sep 19; 4(9):e1000159
oa_package/c3/7c/PMC2528945.tar.gz
PMC2528946
18797505
[ "<title>Introduction</title>", "<p>Demand for intensive care unit (ICU) services is increasing ##UREF##0##[1]##, and at a rate that is higher than the average for all health care services ##REF##7988140##[2]##. Increase in treatment and monitoring technology, patients' expectations, and ageing population all contribute to this increased demand for intensive care services ##UREF##0##[1]##. Indeed, intensive care is increasingly being provided to older and sicker patients, whom in the past were not treated in intensive care ##REF##12185433##[3]##. Intensive care services accounted for 10% of the US$2.1 trillion total health expenditures on health care in the United States in 2006 ##REF##17314105##[4]## and has been estimated to cost more than £700 million in the United Kingdom in 1999 ##UREF##1##[5]##. The cost of intensive care services coupled with increasing demand provides the rationale for improved modelling of outcomes of critically ill patients.</p>", "<p>Long-term survival after critical illness is increasingly being recognized as an important outcome in assessing effectiveness of new therapy ##REF##18191684##[6]##. In order to control for confounding and bias in assessing long-term survival of critically ill patients in a clinical trial, a risk adjustment tool that can objectively estimate long-term survival is essential. From a clinical perspective, many patients and clinicians are also interested in knowing the long-term survival outcome after critical illness, in addition to other information such as burden of treatment and quality of life after recovery, when making treatment decisions in the ICU. Although many clinicians may foretell patient hospital survival outcome more accurately than some objective prognostic models ##REF##16505667##[7]##, treatment decisions made by clinicians do vary considerably with their practice style and work experience ##REF##7853627##[8]##–##REF##18095808##[10]##. The strategy of continuing intensive treatment for all patients until death will reduce the need for patients and clinicians to make difficult treatment decisions and may improve the survival time of some. This strategy is, however, expensive and undesirable by imposing excessive burden of treatment on those who have a very poor prognosis ##REF##9934872##[11]##. For example, initiating acute renal replacement therapy in critically ill patients with less than 10% probability of 6-month survival was estimated to cost US$274,000 (£137,000) per quality-adjusted life year saved ##REF##9245224##[12]##.</p>", "<p>The SUPPORT investigators from the United States and Wright et al. from the United Kingdom published two prognostic models that were based on age, severity of acute illness and admission diagnosis to estimate 6-month and 5-year survival of critically ill patients, respectively ##REF##7810938##[13]##, ##REF##12790812##[14]##. The utility of latter model is, however, limited by its ability to classify 5-year survival probabilities only into three risk categories when the calculated risk score is either &lt;70, 70–80, or &gt;80 ##REF##12790812##[14]##. This model also did not consider the potential effect of detailed co-morbidity data on long-term survival of critically ill patients beyond the usual assessment included in the Acute Physiology and Chronic Health Evaluation (APACHE) score ##REF##12790812##[14]##, ##REF##3928249##[15]##. There is currently no prognostic model that is available to estimate the survival of critically ill patients beyond 5 years after the onset of critical illness. Furthermore, the relative importance of age, co-morbidity, and severity of acute illness in determining long-term prognosis of critically ill patients also remains unknown. In this study we examined the long-term survival of 11,930 critically ill adult patients who survived at least 5 days and developed a new prognostic model (<bold>P</bold>redicted <bold>R</bold>isk, <bold>E</bold>xisting <bold>D</bold>iseases, and <bold>I</bold>ntensive <bold>C</bold>are <bold>T</bold>herapy: the <bold>PREDICT</bold> model) to estimate their median survival time and long-term survival probabilities.</p>" ]
[ "<title>Methods</title>", "<title>The characteristics of the cohort</title>", "<p>This cohort study utilized the clinical database of the ICU at Royal Perth Hospital (RPH) in Western Australia. RPH is the largest tertiary university teaching hospital in Western Australia and the 22-bed ICU admits patients of all specialties except liver transplantation and captures over 40% of all critically ill patients in Western Australia. The database analyzed in this study includes details of all ICU admissions between 1989 and 2002, including demographic factors, admission diagnosis, admission source, severity of acute illness as measured by APACHE II scores based on the worst first 24-hour ICU data ##REF##3928249##[15]##, daily organ failure assessment and supportive therapy required ##REF##16802482##[16]##, and ICU and hospital survival outcome.</p>", "<p>In this study the patients with a diagnosis excluded from the original APACHE II cohort (e.g. coronary artery graft surgery, burns, snake bite)##REF##3928249##[15]##, those who resided outside Western Australia at the time of ICU admission (who could not be followed for survival), readmissions after the first ICU readmission, patients who were younger than 16 years old, and patients who did not survive more than 5 days during their hospitalization of the index ICU admission were excluded. The data were reviewed for internal consistency annually, and there were no patients with missing hospital mortality data. Some of the other details of this cohort have been described in our previous publications ##REF##16802482##[16]##–##REF##18434893##[18]##.</p>", "<p>The ICU clinical database was linked to the Western Australian hospital morbidity and mortality databases using probabilistic matching ##REF##16802482##[16]##, providing information on patients' co-morbidities as recorded in all private and public hospital admissions including any prior ICU admissions up to 5 years before the index ICU admission. A relatively long five-year ‘look back’ period was chosen in this study to capture all existing co-morbidities of each patient. We ascertained the presence of co-morbidities in the Charlson co-morbidity index (##TAB##0##\nTable 1\n##) using the published ICD-9-CM and ICD-10-AM coding algorithms ##REF##16802482##[16]##, ##REF##3558716##[19]##. We did not assign a co-morbidity to a patient if that condition was diagnosed during the hospitalization of the index ICU admission. The proportions of invalid (false positive) and missed links (false negatives) between Western Australian hospital morbidity and mortality databases were evaluated several years ago, and both false positives and negatives were estimated to be 0.11% ##UREF##2##[20]##.</p>", "<p>The survival status of the patients was assessed on 31 December 2003 and the length of follow up ranged from 1 year to 15 years with an average of 6 years. Western Australia is geographically isolated and has a very low rate of emigration (&lt;0.03% in 2002)##REF##16802482##[16]##, and as such, lost to long-term survival follow-up by the Western Australian mortality database is likely to be very low. The data analyzed had the patient name and address removed and the study was approved by the RPH Ethics Committee and the Western Australian Confidentiality of Health Information Committee (CHIC).</p>", "<title>Development of the model</title>", "<p>The prognostic model was fitted using Cox proportional hazards regression ##UREF##3##[21]##, restricting predictors to factors that were likely to be important predictors of long-term survival of hospitalized patients ##REF##7810938##[13]##, ##REF##12790812##[14]##, ##REF##16478903##[22]##, ##REF##1940999##[23]##. These pre-selected factors included age ##REF##12790812##[14]##, ##REF##16478903##[22]##, gender ##REF##16478903##[22]##, APACHE II predicted mortality risk ##REF##7810938##[13]##–##REF##3928249##[15]##, Charlson co-morbidity index ##REF##3558716##[19]##, ##REF##1940999##[23]##, days of mechanical ventilation, acute renal replacement therapy or hemofiltration, and vasopressor or inotropic therapy during the first 5 days of the index ICU admission ##REF##7810938##[13]##. The APACHE II predicted mortality was chosen as a measure of severity of acute illness because it is widely used and summarizes the diagnosis, acute physiologic derangement within the first 24 hours of ICU admission, severe co-morbidities, and whether the ICU admission is after elective or emergency surgery. Our previous study also showed that the APACHE II predicted mortality has a very stable performance in this cohort over the past 10–15 years ##REF##16356207##[17]##. Although age and severe co-morbidities are already used to calculate the APACHE II predicted mortality ##REF##3928249##[15]##, these two factors may still have a profound effect on long-term survival over and beyond the weightings used in the APACHE II predicted mortality ##REF##12790812##[14]##, ##REF##16478903##[22]##, ##REF##1940999##[23]##. As such, both age and Charlson co-morbidity index were used as separate predictors in additional to the APACHE II predicted mortality in this prognostic model. These seven predictors were also chosen because they are often recorded in the administrative databases of many ICUs, and as such, it is possible for other ICUs to validate this model using their data ##UREF##4##[24]##.</p>", "<p>The proportional hazards assumption of the continuous predictors in the Cox model was assessed and found to be acceptable (##FIG##0##\nFigure 1a, 1b, 1c\n##). During the modelling process, we avoided categorizing continuous predictors ##UREF##4##[24]##, ##UREF##5##[25]## and allowed a non-linear relationship with hazard of death using a 6-knot restricted cubic spline function ##UREF##5##[25]##. The relative contribution of each predictor was assessed using the chi-square statistic minus the degrees of freedom ##UREF##5##[25]##. The discrimination performance of the model was assessed with the c-index, which is a generalization of the c-statistic or the area under the receiver-operating characteristic curve, allowing for censored data ##UREF##5##[25]##, ##REF##7063747##[26]##. In this study, a c-index between 0.70 and 0.80 was regarded as acceptable and a c-index above 0.80 was regarded as excellent ##REF##18071171##[27]##. Using the Design library in S-PLUS software (version 8.0, 2007. Insightful Corp., Seattle, Washington, USA), the c-index was computed and adjusted for optimism (arising from using the same data to develop the model and assess its performance) by a bootstrap technique to penalise for possible over-fitting, with 200 re-samples and at least 200 patients per risk group ##UREF##5##[25]##, ##UREF##6##[28]##. The bootstrapping technique was used in this study instead of splitting the data into development and validation data set because this method is regarded as most data ‘efficient’ and accurate in developing a prognostic model ##UREF##5##[25]##. Model calibration (similarity of predicted risks and proportions actually dying) was assessed graphically and used a bootstrap re-sampling to construct a bias-corrected calibration curve and its slope ##UREF##5##[25]##, ##REF##17573992##[29]##. Nagelkerke's R<sup>2</sup> (a generalized measure of the percentage of the variance in survival accounted for by the model) was computed to assess the overall performance of the model ##UREF##5##[25]##, ##UREF##7##[30]##. The performance of the model was assessed over the full 15 years of follow-up, when follow-up was restricted to a maximum of 5 years for each patient, and also when only patients admitted after 1997 were considered.</p>", "<p>A nomogram was developed for the model that generates the median survival time and selected annual survival probabilities by adding up the scores for each of the seven predictors ##UREF##5##[25]##. The use of the nomogram and how each predictor may affect a patient's long-term prognosis is described for a selection of typical patient scenarios. In particular, these scenarios were selected to illustrate how the long-term prognosis of a patient can be different from the short-term prognosis. Nevertheless, the results of the nomogram should only be considered as an average estimate of patients with similar characteristics and not be used for individual patients.</p>" ]
[ "<title>Results</title>", "<title>Characteristics of the cohort</title>", "<p>The study cohort consisted of a heterogeneous group of critically ill patients, with elective surgery including heart valve surgery, urology, gastrointestinal and spinal surgery accounting for 36.2% of all ICU admissions. The emergency admissions consisted of patients with multiple trauma (8.5%), isolated head trauma (2.5%), acute myocardial infarction, congestive heart failure, cardiac arrhythmias, or cardiogenic shock (7.4%), hypovolemic or hemorrhagic shock (0.8%), drug overdoses (7.2%), subarachnoid or intracranial hemorrhage (5.1%), sepsis (4.3%), pneumonia or aspiration (3.7%), obstructive airway diseases (2.1%), cardiorespiratory arrest (4.0%), gastrointestinal hemorrhage, perforation or obstruction (2.4%), and other medical and surgical emergencies. Details of this cohort including demographic factors, co-morbidities, severity of acute illness, and the length of ICU and hospital stay are described in ##TAB##1##\nTable 2\n##.</p>", "<title>Effect of the Predictors on Hazard of Death</title>", "<p>Among all the seven pre-selected predictors in the model, age (50%), co-morbidity as measured by Charlson co-morbidity index (27%), and severity of acute illness as measured by the APACHE II predicted mortality (20%) made the strongest contributions in predicting survival time (##FIG##1##\nFigure 2\n##). After adjusting for other predictors, the log hazard of death increased linearly with age, Charlson co-morbidity index, and the number of days of vasopressor or inotropic therapy, mechanical ventilation, or hemofiltration therapy (##FIG##2##\nFigure 3\n##). The relationship between the APACHE II predicted mortality and log hazard of death was non-linear with a steep effect when the APACHE II predicted mortality was less than 10% and a smaller effect when it was more than 10%. The estimated (adjusted) hazard ratios for the seven predictors are summarized in ##FIG##3##\nFigure 4\n##.</p>", "<title>Clinical Application of the Model</title>", "<p>\n##FIG##4##\nFigure 5\n## presents the model in the form of a nomogram that provides the median survival time and long-term survival probabilities corresponding to a particular total score. The total score for a patient is obtained by adding up the scores for each of the seven predictors. We use the following hypothetical but typical patients to illustrate how the nomogram is used and how the short-term prognosis of a patient can be quite different from the long-term prognosis.</p>", "<p>\n<bold>Patient A:</bold>\n</p>", "<p>A 40-year old male, without pre-existing co-morbidities (ie Charlson co-morbidity index = 0), was admitted to the ICU because of septic shock with an APACHE II predicted mortality of 80%. He required vasopressor or inotropic therapy, mechanical ventilation, and hemofiltration therapy during the first 5 days in the ICU.</p>", "<p>The gender of this patient scores 5 points, age scores 28 points, Charlson co-morbidity scores zero points, the APACHE II predicted mortality or risk scores 30 points, 5 days of vasopressor or inotropic therapy scores 7 points, 5 days of mechanically ventilation scores 15 points, and 5 days of hemofiltration scores 20 points. The total score of this patient is therefore 105 which gives an estimated median survival time of about 4 years, &gt;70% 1-year survival probability, &gt;50% 3-year survival probability, &gt;45% 5-year survival probability, and &gt;20% 10-year survival probability.</p>", "<p>\n<bold>Patient B:</bold>\n</p>", "<p>A 70-year old female, with chronic obstructive airway disease and non-insulin dependent diabetes mellitus with no end-organ damage (ie Charlson co-morbidity index = 2), was admitted to the ICU because of severe community acquired pneumonia with an APACHE II predicted mortality of 30%. She required vasopressor or inotropic therapy and mechanical ventilation but not hemofiltration during the first 5 days in the ICU.</p>", "<p>The gender of this patient scores zero points, age scores 70 points, Charlson co-morbidity index scores 12 points, the APACHE II predicted mortality scores 16 points, 5 days of mechanical ventilation scores 15 points, and 5 days of vasopressor or inotropic therapy scores 7 points. The total score of this patient is therefore 120 which gives an estimated median survival time of about 2 years, 60% 1-year survival probability, 40% 3-year survival probability, 30% 5-year survival probability, and 10% 10-year survival probability.</p>", "<p>\n<bold>Patient C:</bold>\n</p>", "<p>A 80-year old male, with a history of myocardial infarction, congestive heart failure, peripheral vascular disease, cerebrovascular disease, and dementia (ie Charlson co-morbidity index = 5), was admitted to an ICU with bowel perforation and peritonitis with an APACHE II predicted mortality of 30%. He required vasopressor or inotropic therapy and mechanical ventilation but not hemofiltration during the first 5 days in the ICU.</p>", "<p>The gender of this patient scores 5 points, age scores 85 points, Charlson co-morbidity index scores 30 points, the APACHE II predicted mortality scores 16 points, 5 days of mechanical ventilation scores 15 points, and 5 days of vasopressor or inotropic therapy scores 7 points. The total score of this patient is therefore 158 which gives an estimated median survival time of &lt;0.5 years, 25% 1-year survival probability, and 10% 3-year survival probability.</p>", "<title>Discrimination and Calibration of the Prognostic Model</title>", "<p>The adjusted c-index for this prognostic model was 0.757 (95% confidence interval 0.745–0.769), Nagelkerke's R<sup>2</sup> was 0.255 and the bias-corrected calibration of the model over a 15-year period was reasonable (slope of the calibration = 0.98)(##FIG##5##\nFigure 6\n##). The Nagelkerke's R<sup>2</sup> remained unchanged and the adjusted c-index only increased marginally when the analysis was restricted to a maximum of 5 years follow up (c-index = 0.759, slope = 0.97) or data after 1997 (c-index = 0.762, slope of the calibration = 0.97).</p>" ]
[ "<title>Discussion</title>", "<p>This study showed that age, gender, co-morbidities (Charlson co-morbidity index), severity of acute illness (the APACHE II predicted mortality), and duration of intensive care therapy or organ support within the first 5 days of ICU admission are important prognostic factors for long-term survival of critically ill patients. To the best of our knowledge, this new prognostic model (<bold>P</bold>redicted <bold>R</bold>isk, <bold>E</bold>xisting <bold>D</bold>iseases, and <bold>I</bold>ntensive <bold>C</bold>are <bold>T</bold>herapy: the <bold>PREDICT</bold> model) is the first preliminary prognostic model that can be used to estimate the median survival time and long-term survival probabilities of critically ill patients up to 15 years after the onset of critical illness.</p>", "<p>The current prognostic model has confirmed that age, gender, co-morbidities, severity of acute illness, and duration of intensive care therapy or organ failure are important predictors of 6 months to 5 years survival of hospitalized or critically ill patients ##REF##7810938##[13]##, ##REF##12790812##[14]##, ##REF##3558716##[19]##, ##REF##16478903##[22]##, ##REF##1940999##[23]##. The current model is indeed built on the results of these previous studies but further extended the significance of these risk factors in predicting survival of critically ill patients beyond 6 months to 5 years. This current model also demonstrated that most of these predictors have a relatively linear relationship to the long-term survival probability. More importantly, our results also showed that age and co-morbidities are the most important determinants of long-term prognosis of critically ill patients. This latter finding has at least two significant clinical implications. First, the factors that determine long-term survival of a critically ill patient are different from those that affect short-term prognosis. Previous evidence suggested that diagnosis and acute physiological derangement of a patient are most important in determining hospital survival ##REF##3928249##[15]##, ##REF##17924888##[31]##. In our three hypothetical patients, Patient A has in fact the most severe form of acute critical illness and worst short-term prognosis. Nevertheless, because this patient is younger and has no co-morbidities, this patient has a very reasonable and better long-term prognosis than Patient B and C. If we use the prognostic model developed by Wright et al. ##REF##12790812##[14]## to estimate the long-term survival of our three hypothetical patients, Patient B will have the best 5-year prognosis (risk score is estimated to be 68) followed by Patient C (risk score 75) and then Patient A (risk score 87). The lack of detailed co-morbidity data and a heavy emphasis on severity of acute illness in the model developed by Wright et al. is the most likely explanation why our results are different from theirs.</p>", "<p>Many clinicians may intuitively consider the intensity of organ failure as very important in affecting a patient's prognosis ##REF##15856171##[32]##, ##REF##18188103##[33]##. Our findings suggest that the effect of acute organ failure on long-term survival is not strong and mostly captured by age, co-morbidities, and the APACHE II predicted mortality on admission to ICU. Our previous studies have also showed that the intensity of organ failure alone is not as important as the APACHE II score in predicting hospital mortality ##REF##17448058##[34]##, ##REF##18020069##[35]##. Therefore, our findings suggest that clinicians should be very careful not to place undue emphasis on the severity of acute illness and intensity of organ failure when making long-term prognostications of critically ill patients.</p>", "<p>Second, because the contributions by intensive care therapy are relatively small when compared to age, Charlson co-morbidity index, and the APACHE II predicted mortality, using the data after the first 24 to 48 hours of ICU stay is unlikely to underestimate the final total prediction score significantly (&lt;20 points)(##FIG##4##\nFigure 5\n##). Therefore, early estimation of a slightly ‘optimistic’ long-term survival probability and median survival time is feasible after the first 24 to 48 hours of ICU stay; and in patients with either extremes of prognosis, this early estimation is unlikely to be significantly different from the final prediction by collecting all data after five days of intensive care therapy. Nevertheless, the current prognostic model utilizes the APACHE II predicted mortality after ICU admission as a predictor to estimate long-term survival, as such, the model cannot be used, in its current form, as a tool to triage ICU admission.</p>", "<p>This study has significant limitations. First, patients' wishes and the anticipated quality of life before and after their critical illness are important factors in making treatment decisions ##REF##15648988##[36]##, ##REF##15803296##[37]##. The median survival time and long-term survival probabilities is only one of the many factors that patients and clinicians may consider in making treatment decisions. Furthermore, the c-statistics of this model is only about 0.76 and this leaves considerable uncertainty in its applicability in predicting long-term survival of individual patients. As such, the predicted survival probabilities of this prognostic model should only be considered as an average estimate of patients with similar characteristics and should not be used for individual patients. Second, evidence suggests that combining an objective prognostic model with physicians' intuition may improve the accuracy of outcome prediction ##REF##7810938##[13]##. Whether combining this current prognostic model with physicians' intuition will improve its predictive performance further remains uncertain, but this merits further investigation. Third, although we studied a large cohort of critically ill patients, and also the case-mix, severity of illness, and in-hospital survival of this cohort is very similar to many other ICUs in Australia ##REF##14963646##[38]##, validation of this model by other ICUs that have access to data linkage is essential to assess its generalizability. Finally, although the APACHE II prognostic model is still widely used for risk adjustment purposes in many ICUs ##REF##16084255##[39]##, ##REF##15163774##[40]##, it is possible that using newer prognostic models instead of the APACHE II prognostic model may improve our current model ##REF##16540951##[41]##. Similarly, the performance of the current model may be improved if we consider more predictors in the model although this will also increase the complexity of the model. In this regard, we hope that the <bold>PREDICT</bold> model developed in this study will be of value to others who aim to develop a new prognostic model to enhance our understanding of long-term survival of critically ill patients.</p>", "<p>In summary, Age, gender, co-morbidities, severity of acute illness, and the intensity and duration of intensive care therapy can be used to estimate long-term survival of critically ill patients. Age and co-morbidity are the most important determinants of the long-term prognosis of critically ill patients. The current prognostic model, the <bold>PREDICT</bold> model, provides a framework for prognostications and risk adjustment when long-term survival of critically ill patients is considered.</p>" ]
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[ "<p>Conceived and designed the experiments: KMH MK JF SAW. Analyzed the data: KMH MK JF SAW. Contributed reagents/materials/analysis tools: KMH. Wrote the paper: KMH MK JF SAW.</p>", "<title>Background</title>", "<p>Long-term survival outcome of critically ill patients is important in assessing effectiveness of new treatments and making treatment decisions. We developed a prognostic model for estimation of long-term survival of critically ill patients.</p>", "<title>Methodology and Principal Findings</title>", "<p>This was a retrospective linked data cohort study involving 11,930 critically ill patients who survived more than 5 days in a university teaching hospital in Western Australia. Older age, male gender, co-morbidities, severe acute illness as measured by Acute Physiology and Chronic Health Evaluation II predicted mortality, and more days of vasopressor or inotropic support, mechanical ventilation, and hemofiltration within the first 5 days of intensive care unit admission were associated with a worse long-term survival up to 15 years after the onset of critical illness. Among these seven pre-selected predictors, age (explained 50% of the variability of the model, hazard ratio [HR] between 80 and 60 years old = 1.95) and co-morbidity (explained 27% of the variability, HR between Charlson co-morbidity index 5 and 0 = 2.15) were the most important determinants. A nomogram based on the pre-selected predictors is provided to allow estimation of the median survival time and also the 1-year, 3-year, 5-year, 10-year, and 15-year survival probabilities for a patient. The discrimination (adjusted c-index = 0.757, 95% confidence interval 0.745–0.769) and calibration of this prognostic model were acceptable.</p>", "<title>Significance</title>", "<p>Age, gender, co-morbidities, severity of acute illness, and the intensity and duration of intensive care therapy can be used to estimate long-term survival of critically ill patients. Age and co-morbidity are the most important determinants of long-term prognosis of critically ill patients.</p>" ]
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[ "<fig id=\"pone-0003226-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003226.g001</object-id><label>Figure 1</label><caption><title>The proportional hazards assumption of the predictors in the Cox model was assessed by plotting the logarithm of the negative logarithm of the Kaplan Meier survivor estimates and the assumption was found to be acceptable for the three pre-selected continuous predictors; APACHE II predicted mortality, Charlson co-morbidity index, and age.</title><p>(a) Graph assessing the proportionality of hazards associated with severity of acute illness measured by the APACHE II predicted mortality risk categories (0–20%, 20–40%, 40–60%, 60–80%, 80–100%). (b) Graph assessing the proportionality of hazards associated with co-morbidities measured by Charlson co-morbidity index categories (0, 1, 2, 3, 4–5, &gt;5). (c) Graph assessing the proportionality of hazards associated with age measured by age categories (16–30, 30–50, 50–60, 60–70, 70–80, &gt;80 years old)</p></caption></fig>", "<fig id=\"pone-0003226-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003226.g002</object-id><label>Figure 2</label><caption><title>Contribution of each predictor in predicting the survival time in the Cox proportional hazards model.</title></caption></fig>", "<fig id=\"pone-0003226-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003226.g003</object-id><label>Figure 3</label><caption><title>The relationship between relative hazard and each predictor after adjusting for other predictors in the model.</title></caption></fig>", "<fig id=\"pone-0003226-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003226.g004</object-id><label>Figure 4</label><caption><title>The estimated (adjusted) hazard ratios and multilevel confidence bars (0.70 as illustrated by the black bar to 0.99 as illustrated by the orange bar) for the effects of predictors in the model are summarized in the figure below.</title><p>An increase of 20 years of age and an increase in Charlson co-morbidity index from 0 to 5 approximately doubled the risk of death. Doubling the APACHE II predicted mortality from 20% to 40% increased the relative risk of death by about 30 to 40%. Similarly, increased the number of days of intensive care therapy from 1 to 5 increased the relative risk of death by between 10% and 50%.</p></caption></fig>", "<fig id=\"pone-0003226-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003226.g005</object-id><label>Figure 5</label><caption><title>Nomogram for predicting long-term survival probabilities and median survival time.</title><p>Note: gender: 2 = female, 1 = male. Predicted.mortality = APACHE II predicted mortality in %.</p></caption></fig>", "<fig id=\"pone-0003226-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003226.g006</object-id><label>Figure 6</label><caption><title>Bootstrap estimate of calibration accuracy for 15-year estimates from the Cox proportional hazards model.</title><p>Dots correspond to apparent predictive accuracy and x marks the bootstrap-corrected estimates.</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003226-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003226.t001</object-id><label>Table 1</label><caption><title>Charlson co-morbidity index component and its weighting.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Co-morbidity</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Weighting</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Myocardial infarction</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Congestive heart failure</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Peripheral vascular disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cerebrovascular disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Dementia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Chronic pulmonary disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Connective tissue disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Peptic ulcer disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mild liver disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Diabetes mellitus</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hemiplegia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Moderate or severe renal disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Diabetes with end-organ damage</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Any tumour</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Leukemia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Lymphoma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Moderate to severe liver disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Metastatic solid tumour</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">AIDS</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003226-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003226.t002</object-id><label>Table 2</label><caption><title>Characteristics of the cohort (n = 11,930).</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Variables</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean (median, standard deviation), unless stated otherwise</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age, yrs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">53.8 (57.0, 19.0)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gender (male/female), no. (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7489 (62.8)/4441 (37.2)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Elective surgery admission, no. (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4318 (36.2)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">APACHE II score</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.7 (13.0, 6.8)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">APACHE II predicted mortality, %</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.5 (7.0, 17.8)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">No. of APACHE co-morbidities</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1 (0, 0.3)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(a) Cardiovascular, no. (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">592 (5.0)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(b) Respiratory, no. (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">210 (1.8)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(c) Renal, no. (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">109 (0.9)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(d) Immunosuppressed, no. (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">197 (1.7)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(e) Liver, no. (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">76 (0.6)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">No. of Charlson co-morbidities</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.8 (0, 1.2)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Charlson co-morbidity index</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.0 (0, 1.7)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Length of ICU stay, days</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.6 (3.0, 8.3)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Length of hospital stay, days</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.3 (13.0, 25.9)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">No. of patients mechanically ventilated (%) <xref ref-type=\"table-fn\" rid=\"nt101\">#</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8034 (67.3)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">No. of patients on inotrope (%) <xref ref-type=\"table-fn\" rid=\"nt101\">#</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3921 (32.9)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">No. of patients on dialysis (%) <xref ref-type=\"table-fn\" rid=\"nt101\">#</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">608 (5.1)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">No. of ICU survivor (%)<xref ref-type=\"table-fn\" rid=\"nt102\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11557 (96.9)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">No. of hospital survivor (%)<xref ref-type=\"table-fn\" rid=\"nt102\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11101 (93.1)</td></tr><tr><td colspan=\"2\" align=\"left\" rowspan=\"1\">No. of survivor/total no. of patients followed up (%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(a) at 1-year</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10334/11101 (93.1)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(b) at 3-year</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8031/10019 (80.2)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(c) at 5-year</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6109/8212 (74.4)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(d) at 10-year</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2609/4238 (61.6)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(e) at 15-year</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">441/887 (49.7)</td></tr></tbody></table></alternatives></table-wrap>" ]
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[ "<table-wrap-foot><fn id=\"nt101\"><label>#</label><p>During the first 5 days in ICU.</p></fn><fn id=\"nt102\"><label>*</label><p>Excluding patients died within 5 days of ICU admission.</p></fn><fn id=\"nt103\"><label/><p>ICU, intensive care unit.</p></fn><fn id=\"nt104\"><label/><p>APACHE, Acute Physiology and Chronic Health Evaluation.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>A grant was received from BUPA to provide support for the cost of linking the databases used in this study. The funding agency has no involvement in the study design, collection of data, analysis and interpretation of data, writing of the report, or in the decision to submit the article for publication.</p></fn></fn-group>" ]
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[{"label": ["1"], "element-citation": ["\n"], "collab": ["Acute Health Division DoHS"], "year": ["1997"], "article-title": ["Review of intensive care in Victoria [Phase 1 report]."], "publisher-loc": ["Melbourne"], "publisher-name": ["Department of Human Services"]}, {"label": ["5"], "element-citation": ["\n"], "collab": ["The Audit Commission"], "year": ["1999"], "article-title": ["Critical to Success. The place of efficient and effective critical care services within the acute hospital."], "publisher-loc": ["London"], "publisher-name": ["Audit Commission for Local Authorities and the National Health Service in England and Wales"]}, {"label": ["20"], "element-citation": ["\n"], "surname": ["Holman", "Bass", "Rouse", "Hobbs"], "given-names": ["CD", "AJ", "IL", "MS"], "year": ["1999"], "article-title": ["Population-based linkage of health records in Western Australia: development of a health services research linked database."], "source": ["ANZ J Public Health"], "volume": ["23"], "fpage": ["453"], "lpage": ["459"]}, {"label": ["21"], "element-citation": ["\n"], "surname": ["Cox"], "given-names": ["DR"], "year": ["1972"], "article-title": ["Regression models and life tables (with discussion)."], "source": ["Journal of the Royal Statistical Society"], "volume": ["34"], "fpage": ["187"], "lpage": ["220"]}, {"label": ["24"], "element-citation": ["\n"], "surname": ["Wyatt", "Altman"], "given-names": ["JC", "DG"], "year": ["1995"], "article-title": ["Prognostic models: clinically useful or quickly forgotten?"], "source": ["BMJ"], "volume": ["311"], "fpage": ["1539"], "lpage": ["1541"]}, {"label": ["25"], "element-citation": ["\n"], "surname": ["Harrell"], "given-names": ["FE"], "suffix": ["Jr"], "year": ["2001"], "article-title": ["Regression Modeling Strategies."], "publisher-loc": ["New York"], "publisher-name": ["Springer"]}, {"label": ["28"], "element-citation": ["\n"], "surname": ["Efron", "Tibshirani"], "given-names": ["B", "R"], "year": ["1993"], "article-title": ["An Introduction to the Bootstrap."], "publisher-loc": ["New York"], "publisher-name": ["Chapman & Hall"]}, {"label": ["30"], "element-citation": ["\n"], "surname": ["Nagelkerke"], "given-names": ["NJ"], "year": ["1991"], "article-title": ["A note on a general definition of the coefficient of determination."], "source": ["Biometrika"], "volume": ["78"], "fpage": ["691"], "lpage": ["692"]}]
{ "acronym": [], "definition": [] }
41
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 17; 3(9):e3226
oa_package/c4/13/PMC2528946.tar.gz
PMC2528947
18802459
[ "<title>Introduction</title>", "<p>The <italic>Mycobacterium tuberculosis</italic> complex (MTBC) is composed of closely related bacterial sub-species that have plagued human and animal populations for thousands of years. The most famous member of the MTBC is <italic>M. tuberculosis</italic>, the etiological agent of tuberculosis in humans that killed 1.7 million people in 2004 according to the World Health Organization ##UREF##0##[1]##. A new threat is the worldwide emergence of multi-drug resistant (MDR) and extremely drug-resistant (XDR) strains. Recent data suggest that the propensity to gain drug resistance as well as the pathogen's transmissibility profile may be influenced by the genetic and evolutionary background of <italic>M. tuberculosis</italic> strains ##REF##16789833##[2]##. Thus, understanding the relationships and dynamics of the MTBC lineages will undoubtedly help to unravel the basis for the considerable success and spread of tuberculosis, in both humans and animals. The MTBC is essentially clonal with little evidence of horizontal gene exchange ##REF##15041743##[3]##,##UREF##1##[4]##,##REF##12519202##[5]##, and probably derived from a pool of ancestral tubercle bacilli, collectively called “<italic>Mycobacterium prototuberculosis</italic>” ##REF##16201017##[6]##. However, despite the highly successful worldwide spread of the MTBC, the evolutionary timing of this spread remains largely unknown.</p>", "<p>This lack of knowledge is largely due to the limitations of the genetic markers used so far. All efforts to time MTBC evolution with single nucleotide polymorphisms (SNPs) have been based on a non-warranted hypothesis of universal bacterial mutation rates, itself extrapolated from a very hypothetical time of divergence between <italic>Escherichia coli</italic> and <italic>Salmonella enterica</italic>\n##REF##3125340##[7]##.</p>", "<p>In this study, we used a completely new approach by employing genetic markers based on mycobacterial interspersed repetitive units (MIRUs) to determine the timing of divergence, population diversity and spread of the MTBC. MIRU loci comprise variable numbers of tandem repeat (VNTR) sequences, which allow them to be used as powerful genotyping markers ##UREF##2##[8]##,##REF##10844663##[9]##. In terms of genetic diversity and mutation rates, they resemble human microsatellites, which are widely used in human population genetics studies ##REF##12493913##[10]##. Similar to microsatellites, MIRUs behave as selectively neutral phylogenetic markers if sufficient numbers of loci are used to buffer against potential biases.</p>", "<p>Here we used experimental data on the variability and evolution of these markers in clinical isolates of infected patients, which allowed us to calculate the MIRU molecular clock and model their evolution in coalescence approaches. Based on this information and extensive analysis of a large collection of representative MTBC strains, we obtained new insights into the origin and demography of the MTBC and its dynamic association with the human host.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Sampling and data collection</title>", "<p>The 355 <italic>M. tuberculosis</italic> and <italic>M. prototuberculosis</italic> isolates were genotyped by multiplex PCR amplification as described previously ##UREF##2##[8]##,##REF##11574573##[47]##. The samples were subjected to electrophoresis using ABI 3100 and 3730 automated sequencers. Sizing of the PCR fragments and assignment of the VNTR alleles of the 24 loci was done using the GeneScan and customized Genotyper, as well as the GeneMapper software packages (PE Applied Biosystems).</p>", "<title>Genetic diversity estimation</title>", "<p>The number of alleles (allelic richness) in each <italic>M. tuberculosis</italic> complex population was estimated and sample sizes were corrected by the rarefaction procedure using HP-RARE ##UREF##3##[23]##. Comparison tests as well as <italic>P</italic>-values were estimated using the STATISTICA v.6.1 package.</p>", "<title>Phylogenetic inferences</title>", "<p>Nei et al.'s <italic>D<sub>A</sub></italic> distance ##REF##6571220##[48]## was used to construct both isolate and population trees using a neighbour-joining algorithm as implemented in the software Populations version1.2.28. Support for the tree nodes was assessed by bootstrapping over loci (1, 000 iterations).</p>", "<title>Inferring population structure and recombination in the <italic>M. tuberculosis</italic> complex</title>", "<p>Using the no-admixture model ##REF##10835412##[18]## (STRUCTURE version 2), three to ten parallel Markov chains were run for all models of K with a burn-in of 100,000 iterations and a run length of 10<sup>6</sup> iterations following the burn-in. For each run, the ln likelihood of each model was calculated. The full data set was analysed for all models from K = 1 through to 3 without specifying prior information concerning the geographical sources or former designations. For K = 3, a clear splitting solution was found in which the sampled populations clustered into two main tuberculosis groups plus the outgroup (<italic>M. prototuberculosis</italic>); a result fully consistent with the neighbour-joining population tree (##FIG##0##Figure 1B##). For further analysis the data set was sub-divided into clades 1 and 2, and these were subsequently tested for K = 1 through to 6. Using the linkage model ##REF##12930761##[49]## of STRUCTURE version 2, ten parallel Markov chains were run for each model with a burn-in of 100,000 iterations and a run length of 10<sup>6</sup> iterations following the burn-in. For each run, <italic>M. tuberculosis</italic> strains were specified as belonging to pre-determined source clusters. We estimated the ancestry in each source cluster and the proportion of each strain genome having ancestry in each cluster.</p>", "<title>Stepwise mutation model (SMM) and mutation rate estimates</title>", "<p>To estimate the validity of SMM model, we built a minimal spanning tree of all MTBC strains based on the degree of allele sharing, by using BIONUMERICS (Applied Maths, Belgium). We then evaluated the proportion of single-locus variants (i.e. strains that differed from their closest relative) that differed by one or by multiple repeat-changes. To further evaluate the validity of the SMM model and to detect a potential bias towards increase or decrease in repeat numbers, eBURST analysis was performed on a larger dataset from two population-based studies. The first one included 807 isolates from different TB cases notified in the Brussels-Capital Region (Belgium) from September 1<sup>st</sup>, 2002 to December 31<sup>st</sup>, 2005 ##UREF##4##[50]##, while the second one is an ongoing study including 1907 isolates from different TB cases notified in the Netherlands over 2004 and 2005 (Van Soolingen et al., unpublished). In total, the dataset included 1,733 MIRU-VNTR profiles, with no missing data or incomplete repeats. On this dataset, the differences in the number of repeats were calculated for each pair of ancestor/descendant genotypes along the evolutionary path inferred by eBURST analysis ##REF##14973027##[51]##. The occurrence of each value of repeat difference was recorded for each group (defined as groups of strains with at most one allelic mismatch with at least one other member of the group), and values were pooled over all eBURST groups. This analysis was performed using software Multilocus Analyzer (S. Brisse, unpublished), which is an independent implementation (coded in Python) of the eBURST algorithm, to which the SMM test function was added.</p>", "<p>MIRU mutation rates were estimated by using data on VNTR changes among large sets of serial or epidemiologically-linked isolates ##REF##17009866##[11]##. Single-locus mutation rates of 5 most variable loci were estimated from corresponding frequencies of observed repeat changes. Repeat changes among serial or epidemiologically-linked isolates were not detected among the remaining, less variable loci. Therefore, the relative frequencies of single-locus variations among closely related isolates in a global MTBC isolate dataset ##REF##17009866##[11]## and in the population based dataset (see above) were then used as a surrogate for estimating mutation rates of less variable markers relatively to these most variable loci.</p>", "<title>Coalescence, TMRCA and demography</title>", "<p>In a first step, we used a Bayesian approach ##REF##10581303##[25]## that assumes a stepwise mutation model and estimates the posterior probability distributions of the genealogical and demographic parameters of a sample using Markov chain Monte Carlo simulations based on MIRU data. This method permits to extrapolate important biological parameters like the TMRCA of a given sample in years, the past and present effective population size and the latest demographic changes (decline, constant population size or expansion). In order to assess the age of the main <italic>M. tuberculosis</italic> lineages, an alternative algorithm, YTime ##REF##13680527##[24]## was used to calculate the TMRCAs and their confidence intervals. For the MSVAR procedure ##REF##10581303##[25]##,##UREF##5##[52]##, we focused on lineages of which at least 30 isolates were available, in order to obtain a reliable coverage of the TMRCA and to avoid small sample size artefacts. The estimated parameters were scaled in terms of current population size, and two main demographic parameters were quantified: <italic>t<sub>f</sub></italic>, which is a measure of time in generations, was defined as <italic>t<sub>a</sub></italic>/<italic>N<sub>0</sub></italic>, where <italic>t<sub>a</sub></italic> denotes the number of generations that have elapsed since the decline or expansion began, and <italic>r</italic>, which was defined as <italic>N<sub>0</sub></italic>/<italic>N<sub>1</sub></italic>, where <italic>N<sub>0</sub></italic> is the current effective number of chromosomes, and <italic>N<sub>1</sub></italic> is the number of chromosomes at some previous point in time <italic>t<sub>f</sub></italic>. For a declining population <italic>r</italic>&lt;1, for a stable population <italic>r</italic> = 1 and for expanding populations <italic>r</italic>&gt;1. The procedure also estimates <italic>θ</italic>, which is defined as <italic>N<sub>0</sub>μ</italic>, where <italic>μ</italic> is the mutation rate (mutation locus<sup>−1</sup> generation<sup>−1</sup>). The analyses were performed assuming exponential demographic change. Three different chains were run for each analysis to confirm the convergence of the results. In the analyses, rectangular priors of the log parameter values have been used. The method was found to converge appropriately for both single and multilocus data sets and supported a model of population expansion for all MTBC populations. We present only the multilocus data in the present report. Expansion signatures were robust and were confirmed in runs where decline was assumed as a prior (10<sup>−2</sup>–10<sup>−3</sup>).</p>", "<p>YTime ##REF##13680527##[24]##: YTime is a Matlab function which calculates the TMRCA for haplotype linked loci under the assumption of an S-SSM, which allows for unbiased +/−1 steps. YTime calculates confidence intervals using a simulation approach and is independent of the shape of the genealogy. We used all available loci (N = 24) as an input. The strains were grouped according to their lineages (obtained by phylogenetic analyses). The ancestral genotype for every subgroup was calculated as the mean of every single locus in the particular subgroup. The mutation rate was 10<sup>−4</sup> per year per locus. For the growth rate parameters we assumed a mean effective population size of 10<sup>8</sup> for every sub-population and a growth of 10<sup>3</sup> (the mean of the results is not affected by the growth rate, just the confidence intervals).</p>" ]
[ "<title>Results</title>", "<title>\n<italic>M. tuberculosis</italic> phylogeny</title>", "<p>To infer the MTBC evolutionary history, we used a sample collection of 355 isolates, representative of well-identified primary branches of the MTBC world distribution (##SUPPL##1##Table S1##). A recently standardized combination of 24 MIRU loci (##SUPPL##2##Figure S1##), which does not comprise saturated loci ##REF##17009866##[11]##, was utilized. To illustrate the power of MIRUs to reconstruct geographical patterns of genetic differentiation and their level of resolution, a distance-based tree was constructed using individual genotypes and a neighbour-joining algorithm (##FIG##0##Figure 1A##). The tree grouped all <italic>M. tuberculosis sensu stricto</italic> isolates (all from human patients) in a distinct lineage with the notable exception of the East African-Indian (EAI) population whose affiliation is unclear based on this approach. Another major lineage encompassed all MTBC strains from animals (<italic>M. microti</italic>. <italic>M. bovis</italic>, <italic>M. caprae</italic> and <italic>M. pinipedii</italic>) and the human isolates from West-Africa (<italic>M. africanum</italic> West African 1 and 2). From the resulting tree, it appears that the groupings of isolates within the primary MTBC branches based on SNPs, spoligotyping and large sequence polymorphisms (LSPs) ##REF##11891304##[12]##,##REF##16477032##[13]##,##REF##12524330##[14]##,##REF##16519816##[15]##,##REF##17448936##[16]##,##REF##16323140##[17]## (##SUPPL##3##Figure S2##) are highly congruent with those based on the MIRU typing, albeit the branch resolution was higher in the latter. In order to more robustly define the relationships between the lineages (by reducing the number of individuals vs the number of markers), we then grouped individual isolates into the populations defined by the above groupings and built a tree based on MIRU allelic frequencies in these populations (##FIG##0##Figure 1B##). The tree was rooted with samples of <italic>M. prototuberculosis</italic> (including <italic>M. canettii</italic>), which was recently reported to represent the progenitor of the MTBC ##REF##16201017##[6]##. This approach clearly revealed the distinctiveness of the two major lineages with strong bootstrap support, called hereafter clades 1 and 2. A further geographic sub-structuring within clade 1 became apparent, with distinct branches for the African (Uganda, Cameroon and S), Asian (Beijing and CAS), Latin American-Mediterranean and African-European populations (X, Ghana and Haarlem). Clade 2 is composed of both animal and human pathogenic isolates. A basal position of EAI (human tuberculosis) in clade 2 has strong statistical support, indicating a human origin for this predominantly animal-associated MTBC lineage. However, low bootstrap values within clade 2 prevent us from drawing further inferences on the branching order.</p>", "<title>A population genetics perspective</title>", "<p>To confirm the groupings and the deep dichotomy obtained with the MIRUs, we used an independent approach, based on the ‘no-admixture’ model of the STRUCTURE program ##REF##10835412##[18]##. In this Bayesian approach, multilocus genotypic data are used to define a set of populations with distinct allele frequencies and assign individuals probabilistically to them, with or without prior knowledge of geographic sampling information. We applied STRUCTURE to the global data set (including the outgroup) and in ten independent runs, at K = 3 populations (##FIG##0##Fig. 1C##) STRUCTURE detected the same two deeply divergent clades 1 and 2 that were identified with the neighbour joining analysis (see ##FIG##0##Figure 1B##). Notably, this separation is independently supported by the fact that TbD1 (<italic>M. tuberculosis</italic> deletion 1) is lacking in all clade 1 strains but present in all clade 2 strains, including those from EAI (##FIG##0##Figure 1B## and ##SUPPL##3##S2##) ##REF##11891304##[12]##. The robustness of these clades was further evidenced by STRUCTURE analysis, because each isolate derived all of its MIRU's from only one of the three ancestral sources of clade 1, clade 2 or <italic>M. prototuberculosis</italic> (see ##SUPPL##0##Protocol S1##). We further modelled the Bayesian assignments of the two main clades by sub-dividing them into additional clusters (##SUPPL##4##Figure S3A##). The bacterial isolates were consistently split into the same major clusters as those defined by the distance-based approach (see above). The highest likelihoods were obtained for <italic>K</italic> = 6 populations in each of the two main clades. Only three isolates (0.85%) were assigned to unexpected clusters by the Bayesian approach (##SUPPL##4##Figure S3A##), further illustrating the consistency of MIRU-VNTR cluster designations. To detect possible horizontal genetic transfer events, we used the STRUCTURE ‘linkage model’ as was done to detect ongoing genetic exchange in <italic>Helicobacter pylori</italic>\n##REF##15051885##[19]##,##REF##12624269##[20]##, <italic>Escherichia coli</italic>\n##REF##16689791##[21]## and <italic>Moraxella catarrhalis</italic>\n##REF##17895425##[22]##. Runs without prior knowledge of population source (##SUPPL##4##Figure S3B##) suggested that the vast majority of the MTBC strains are clonal, while some <italic>M. prototuberculosis</italic> strains might be hybrids with MTBC genotypes, in accordance with previous results ##REF##15041743##[3]##,##REF##12519202##[5]##,##REF##16201017##[6]##.</p>", "<title>MTBC ancestral lineages and genetic diversity</title>", "<p>To further assess the deep dichotomy, we calculated the allelic richness (the number of alleles) of the populations within the two main clades after correcting for sample size effects ##UREF##3##[23]## (##FIG##1##Fig. 2##). High levels of genetic diversity are a surrogate indication of ancestral origins as illustrated in the highly divergent African human populations. The mean allelic richness per locus was close to five for both clades, and the difference was not significant (##FIG##1##Fig. 2C##), arguing for a simultaneous split of the two clades. As expected, LAM and EAI, the most basal populations in clades 1 and 2 respectively, contained the highest number of alleles (##FIG##1##Fig. 2A, 2B##). However, some uncertainty remains on a basal position for LAM because it conflicts with groupings based on internal deletions of the pks15/1 gene and on SNPs ##REF##16477032##[13]##.</p>", "<title>Dating the disease and the evolutionary radiation steps</title>", "<p>In order to estimate the time to the most recent common ancestor (TMRCA) in the MTBC, we made use of recent analytical tools ##REF##13680527##[24]##,##REF##10581303##[25]##, which make these estimations possible. They rely on Bayesian statistics and apply a stepwise mutation model (SMM) for genetic markers. This model is a reasonable assumption for MIRU mutations, as initially shown for MIRU locus 4 in the BCG evolutionary framework ##REF##10844663##[9]##. To test the validity of this model for the total set of the MIRU loci used, we built a minimal spanning tree of all MTBC strains based on the degree of allele sharing. We then evaluated the proportion of strains that differed from their closest relative by one step (single-locus variants- SLVs) or by multiple steps, which would violate the SMM model. This simple method will certainly overestimate any violations of the SMM model because our sampling scheme is not exhaustive, resulting in some spurious missing links (intermediate strains) that falsely invalidate the SMM model. However, the data showed that at least 64% of the allelic changes fit the stepwise mutation model, a result that is close to the 75% and 81% observed in <italic>E. coli</italic> and yeast VNTRs, respectively ##REF##16740932##[26]##,##REF##9215886##[27]##.</p>", "<p>To further evaluate the validity of the SMM model, eBURST analysis was performed on a much larger dataset comprising 1,733 MIRU-VNTR profiles from two population-based studies performed at regional and national levels (see <xref ref-type=\"sec\" rid=\"s5\">Materials and Methods</xref>). This analysis identified 142 groups and 1061 singletons. In order to determine whether tandem repeats evolve following a SMM model and to detect a potential bias towards increase or decrease in repeat numbers, we computed within each eBURST group all differences in number of repeats along the evolutionary path, starting from the putative founder of the group to its surrounding SLVs (##SUPPL##5##Figure S4##). For all but two of the 24 loci, the most frequent change was either −1 or +1 repeat unit, with the symmetric change generally being the next most frequent. The only minor exceptions were loci MIRU-VNTR 3007 and 2347, which contain little information, because the only changes were one occurrence each of −2 and +1 repeat units, and four occurrences of −2 and two of +1 respectively. Both for the individual loci and for data cumulated over the 24 loci (##SUPPL##6##Figure S5##), the distribution of occurrences was unimodal and centered on 0 (average of −0.07±0.23, CI = 0.95, for cumulated data). At least sixty-five percent of the allelic changes matched the stepwise mutation model. It is noteworthy that missing links falsely invalidating the SMM model probably occur even in this population-based dataset, because many patients from the population studied (from the Brussels region and the entire Netherlands) were foreign-born and have probably acquired their infection abroad. Therefore, tandem repeats in <italic>M. tuberculosis</italic> most frequently evolve by progressive gain or loss of single repeat units without significant general bias towards increase or decrease.</p>", "<p>To estimate MIRU mutation rates, we used data from large sets of serial or epidemiologically-linked isolates. The probability of showing a repeat change over periods of up to 7 years was estimated to be about 1% for five of the most variable loci ##REF##17009866##[11]##. This corresponds to a single-locus mutation rate of 1.4×10<sup>−3</sup> per year. Consistently, 4 of these 5 loci composed the top 4 in the hierarchy of single-locus variation frequencies measured among the MIRU loci, both in a global MTBC isolate dataset ##REF##17009866##[11]## and in the above population-based dataset (data not shown). This supports the use of these frequencies as a surrogate for estimating relative mutation rates of the different markers, and especially those of the less variable loci, for which repeat changes among serial or epidemiologically-linked isolates were not observed ##REF##17009866##[11]##. We therefore somewhat arbitrarily chose a lower mean mutation rate per year of 10<sup>−4</sup> as a prior for the Bayesian inferences ##REF##10581303##[25]## over all loci, in order to accommodate the less variable loci which were associated with up to 38fold lower frequencies of single-locus variation. It is noteworthy that this initial value was well supported by posterior Bayesian analysis, as the calculated posterior mean for the mutation rate was 10<sup>−3.91</sup> (##FIG##2##Figure 3##). By applying this mutation rate and a generation time of one day for the tuberculosis bacilli, we estimated a mean TMRCA of ≈40,000 years before present for the complex (##TAB##0##Table 1##). The TMRCAs for clades 1 and 2 were estimated as 21,000 and 33,000 years, respectively, and two of the oldest lineages, EAI and LAM coalesced at 13,700 and 7,000 years, respectively (##TAB##0##Table 1##).</p>", "<p>In a second step, we used the MSVAR software ##REF##10581303##[25]## that infers past demographic changes and calculates additional parameters, including TMRCA of monophyletic populations using slightly different algorithms. For this procedure, we focused on lineages for which at least 30 isolates were included in the study, in order to avoid small sample size artefacts. The use of this method confirmed the TMRCA of the EAI population at ≈7,000 years (##FIG##3##Figure 4B## and ##TAB##1##Table 2##), albeit with very wide confidence intervals (150–190,000 years).</p>", "<title>\n<italic>M. tuberculosis</italic> demographic expansion</title>", "<p>Finally, genetic data can also unravel recent demographic change signatures in bacterial populations. By using Bayesian statistics, we tested whether a recent decline or expansion occurred in the MTBC population, and calculated <italic>t<sub>a</sub></italic>, which reflects the time that has elapsed since the decline or expansion began. All MTBC populations from human sources that we considered displayed markedly consistent expansion rates and EAI is typical in that respect (see ##FIG##2##Figure 3B##). The detected growth rates (on a log scale) ranged from a modest 0.6 value, as seen in Africa, to 2.7 for Beijing, which is probably the most successful present day lineage. This latter value translates into a recent 500-fold population size increase. The mean modal value of log10 <italic>t<sub>a</sub></italic> was 2.25 (range 2.00–2.5) for the different populations, with the exception of the LAM lineage. This corresponds to a tuberculosis expansion that began 180 years ago (see ##TAB##1##Table 2##).</p>" ]
[ "<title>Discussion</title>", "<p>Taken together, the findings presented in this study indicate that the MTBC is composed of two major lineages and has emerged approximately 40,000 years ago. This estimate is strikingly close to the proposed time of dispersal of founder modern human populations from the Horn of Africa ##REF##16902130##[28]##. However this dating must be considered with caution in the light of the large confidence intervals. Our results support the emergence of the MTBC clone from the <italic>M. prototuberculosis</italic> progenitor pool and its co-migration with modern humans out of Africa ##REF##16201017##[6]##. A similar trend was recently proposed for <italic>H. pylori</italic> and <italic>M. leprae</italic>\n##REF##17287725##[29]##,##REF##15894530##[30]##. We suggest that two main lineages arose later some 20,000 to 30,000 years ago from the common MTBC ancestor, one of which spread exclusively among humans, with subsequent waves of migration to Asia, Europe and continental Africa (##FIG##4##Figure 5##). This spreading scenario fits well with the current worldwide distribution of the main MTBC lineages, as reflected by the SpolDB4 database ##REF##11891304##[12]##,##REF##16477032##[13]##,##REF##12524330##[14]##,##REF##16519816##[15]##,##REF##17448936##[16]##,##REF##16323140##[17]## and LSP analysis ##REF##12524330##[14]##,##REF##16323140##[17]##. The second lineage (clade 2) arose from a human EAI-like population some 30,000 years ago and is the probable source of animal tuberculosis ##REF##11891304##[12]##,##REF##12788972##[31]##, a derivation that is strongly and convergently supported by both distance-based and probabilistic methods (i.e. NJ and STRUCTURE). This conclusion is consistent with the finding that extant representatives of <italic>M. tuberculosis</italic>, which derived from the proposed progenitor of MTBC, are human pathogens ##REF##16201017##[6]##. Thus it is likely that humans infected their livestock and not the other way around. Clade 2 secondary branches include <italic>M. bovis</italic> and <italic>M. caprae</italic>, the infectious agents of tuberculosis in a wide variety of animals including cattle and goat, which were first domesticated in the Near East ##REF##11323670##[32]##,##REF##10731145##[33]##. The transition from human to animal hosts may thus be linked to plant and animal domestication that took place in the Fertile Crescent some 13,000 years ago. This period corresponds to the estimated time of diversification of the oldest EAI and LAM populations (##TAB##1##Table 2##). In the Fertile Crescent, and during that era of human history, small nomadic hunter-gatherer groups were replaced by farming societies based on domesticated livestock and crops ##REF##12167878##[34]##. This paramount event in human history was probably not without consequence for an epidemic, infectious disease such as tuberculosis, where crowded farming populations may have promoted high infection rates, bacterial spread and transition to new niches and animal hosts ##REF##16156803##[35]##. Clade 2 also includes <italic>M. africanum</italic> strains that primarily infect humans. However, it has recently been speculated that <italic>M. africanum</italic> may not be primarily adapted to the human host but might have originated from an unknown animal reservoir ##REF##15297503##[36]##.</p>", "<p>All MTBC populations from human sources displayed markedly constant expansion rates, corresponding to an expansion that dates back to only about 180 years. Furthermore, the largest population size increase (500-fold) was detected for Beijing, which is thought to be the most successful present day lineage. These results suggest that the expansion of the most recent form of human tuberculosis was coupled to Western urbanization and industrialization. This expansion was synchronous with the modern demographic explosion of <italic>Homo sapiens</italic> and modern intercontinental movements. Evidence for strong phylogeographical structuring of the pathogen population and preferential sympatric combinations of pathogen populations with particular ethnic groups has indicated a close association between <italic>M. tuberculosis</italic> and its human host ##REF##15041743##[3]##,##REF##16477032##[13]##,##REF##16519816##[37]##. Our results indicate recent parallel demographic changes between the pathogen and its host and reveal the tell-tale dynamic dimension of this association. The coalescence approach may also be useful in the future to monitor demographic changes in emerging MDR <italic>M. tuberculosis</italic> strains.</p>", "<p>Some of the conclusions presented here on the basis of MIRU data have also been reached previously, e.g. data from comparative genomics ##REF##11891304##[12]## after the completion of <italic>M. bovis</italic> genome ##REF##12788972##[31]## indicated that the MTBC did not arise as a zoonosis ##REF##7697265##[38]##. In contrast, the validity of efforts to date the origins of the common ancestor of MTBC by using SNP-based methods ##REF##9275218##[39]##,##REF##11832515##[40]##,##REF##12453367##[41]##, has remained questionable ##REF##10535975##[42]##. Furthermore, preliminary SNP-based phylogenetic reconstructions may have been affected by hitch-hiking, and ascertainment bias ##REF##15347815##[43]##, because those SNPs were associated with genes involved in drug-resistance ##REF##15498158##[44]## or were selected from a non-representative set of available genomes ##REF##12524330##[14]##,##REF##16323140##[17]##,##REF##16385065##[45]##. Such markers evolve too slowly for recent pathogens, as is also the case for LSPs and their use often results in uninformative phylogenies that consist of multifurcated unresolved trees ##REF##16477032##[13]##,##REF##15498158##[44]##. Unlike previous studies, the novel analyses presented here rely on globally neutral markers with mutation rates that have been estimated from human <italic>M. tuberculosis</italic> infection cases, a descent-sampling scheme and multiple, convergent population genetic estimators. As they are based on intrinsically rare and stochastic VNTR changes in clonal populations, our mutation rate estimates do involve some special assumptions. The accuracy of the demographic and temporal estimates could be improved with long-term analyses, and we are aware that the use of a mean mutation rate for all loci is suboptimal, leading to an increase of the variance of parameters. However, our estimates were consistently corroborated by posterior Bayesian calculations in independent runs over different strain populations (ranging from 10<sup>−4.19</sup> for LAM to 10<sup>−3.82</sup> for EAI), ruling out the risk of some local maxima. To gain further insights into the host-pathogen interactions, it would certainly be important to account for the biogeographic history and distribution of the different <italic>M. tuberculosis</italic> lineages, because recent adaptations to local host populations might play a major role ##REF##16477032##[13]##. Furthermore, it is known, that genetic diversity can influence the transmission dynamics of drug-resistant bacteria ##REF##16789833##[2]##,##REF##16809538##[46]##, and, in terms of vaccination, it would be advisable to scrutinize independently the highly polymorphic clade 2 EAI strains that markedly differ in their genetic structure from the other human tuberculosis strains.</p>" ]
[]
[ "<p>Conceived and designed the experiments: KK DvS PS SN. Performed the experiments: CAB FW TK KK. Analyzed the data: TW FH SB PS SN. Contributed reagents/materials/analysis tools: DvS PS SN. Wrote the paper: TW DvS SRG CL AM PS SN.</p>", "<p>The evolutionary timing and spread of the <italic>Mycobacterium tuberculosis</italic> complex (MTBC), one of the most successful groups of bacterial pathogens, remains largely unknown. Here, using mycobacterial tandem repeat sequences as genetic markers, we show that the MTBC consists of two independent clades, one composed exclusively of <italic>M. tuberculosis</italic> lineages from humans and the other composed of both animal and human isolates. The latter also likely derived from a human pathogenic lineage, supporting the hypothesis of an original human host. Using Bayesian statistics and experimental data on the variability of the mycobacterial markers in infected patients, we estimated the age of the MTBC at 40,000 years, coinciding with the expansion of “modern” human populations out of Africa. Furthermore, coalescence analysis revealed a strong and recent demographic expansion in almost all <italic>M. tuberculosis</italic> lineages, which coincides with the human population explosion over the last two centuries. These findings thus unveil the dynamic dimension of the association between human host and pathogen populations.</p>", "<title>Author Summary</title>", "<p>The causative agents of tuberculosis, grouped in the <italic>Mycobacterium tuberculosis</italic> complex, have infected one-third of the present human population and a wide range of other mammals. However, paradigmatic questions, such as why, where and when the disease began and expanded, have largely remained unanswered. In this study, we provide genetic evidence indicating that the most common ancestor of the bacterial complex emerged some 40,000 years ago from its progenitor in East Africa, the region from where modern human populations disseminated around the same period. This initial step was followed 10,000 to 20,000 years later by the radiation of two major lineages, one of which spread from human to animals. In more recent years (approximately 180 years ago), coinciding with the human population explosion and the industrial revolution, the human-associated pathogen lineages have strongly expanded. These results thus reveal the strikingly parallel demographic evolution between humans and one of their primary pathogens.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>We are extremely grateful to all colleagues that have participated in previous studies allowing us to establish the large collection investigated here. We would also like to thank Géraldine Bollmann, Pascale Chesselet, Dave Gerrard, and Chiara Reggio for helpful comments on an earlier version of the manuscript and I. Radzio, T. Ubben, and P. Vock for excellent technical assistance.</p>" ]
[ "<fig id=\"ppat-1000160-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000160.g001</object-id><label>Figure 1</label><caption><title>Evolutionary relationships of the <italic>Mycobacterium tuberculosis</italic> complex.</title><p>(A) Unrooted MIRU Neighbour-joining phenogram depicting genetic distance relationships among tubercle bacilli isolates based on Nei et al.'s <italic>D<sub>A</sub></italic> distances. (B) Rooted MIRU population Neighbour-joining tree based on genetic distance. <italic>M. prototuberculosis</italic> was used as an outgroup. Values on the nodes represent the percentage of bootstrap replicates over individuals (N = 1000) showing the particular nodes. Branch lengths are proportional to the genetic distance between the tubercle lineages. It is noteworthy that low bootstrap values within clade 2 prevent us from drawing further inferences on the branching order in this clade (see also main text). Wa, West-Africa. (C) Population structure of 20 MTBC clonal lineages using the no-admixture model, where <italic>K</italic> = 3. Each colour represents one cluster, and the length of the color segment shows the strains' estimated proportion of membership in that cluster. Results shown are averages over 10 STRUCTURE runs. For clarity, strains codes are also given according to Gagneux et al. (2006).</p></caption></fig>", "<fig id=\"ppat-1000160-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000160.g002</object-id><label>Figure 2</label><caption><title>Genetic variability in the different MTBC lineages.</title><p>(A and B) MIRU allelic richness in each population within clade 1 and 2 respectively. Rarefaction included eight isolates per population (smaller populations were not considered in this analysis. (C) Clades mean allelic richness. Notice that the difference between clade 1 and 2 is not significant (t-test, <italic>P</italic> = 0.08).</p></caption></fig>", "<fig id=\"ppat-1000160-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000160.g003</object-id><label>Figure 3</label><caption><title>Calculated posterior mean for MIRU-VNTR mutation rate among loci using the MSVAR algorithm.</title><p>This graph corresponds to the output obtained for the Haarlem population sample and the 95% interval confidence is given (red dotted lines).</p></caption></fig>", "<fig id=\"ppat-1000160-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000160.g004</object-id><label>Figure 4</label><caption><title>Detection of recent expansion in different MTBC lineages.</title><p>(A) Posterior distribution of <italic>M. bovis</italic> TMRCA, including the 95% confidence interval and density plots of the marginal posterior distribution of log (<italic>N<sub>0</sub></italic>), where <italic>N<sub>0</sub></italic> is the current effective number of chromosomes and log (<italic>N<sub>d</sub></italic>), where <italic>N<sub>d</sub></italic> is the number of chromosomes before expansion. (B) Same plots for EAI. <italic>t<sub>a</sub></italic> is expressed in years (±SD) and denotes the time that has elapsed since the population growth began.</p></caption></fig>", "<fig id=\"ppat-1000160-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000160.g005</object-id><label>Figure 5</label><caption><title>\n<italic>M. tuberculosis</italic> evolutionary scenario (out of Mesopotamia).</title><p>The main migrations events are numbered and correspond to: 1, <italic>M. prototuberculosis</italic>, the ancestor of the MTBC, this bacterium reached the Fertile Crescent some 40,000 years ago by sea or land; 2 and 3, two distinct basal lineages arose, EAI and LAM and spread out of Mesopotamia some 10, 000 years ago; 4, 5 and 6, later on (8–5000 years ago) derived populations from clade 1 followed main human migration patterns to Africa, Asia and Europe, giving rise to locally adapted tubercle strains and further diversifications. Note that the depicted borders are “artificial” and are used for the demonstration. Global movements and intercontinental exchanges tend to blur this phylogenetic signal though strong enough to be detected nowadays.</p></caption></fig>" ]
[ "<table-wrap id=\"ppat-1000160-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000160.t001</object-id><label>Table 1</label><caption><title>Estimated Times (in years) since the most recent common ancestor (TMRCA).</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">TMRCA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age in years</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CIs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hierarchic level</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">LAM-Beijing</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21,300</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(14,300–31,600)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clade 1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Beijing-CAS</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17,100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(11,600–25,400)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Asian TB</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">LAM-LAM</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7,060</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(4,370–11,100)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">LAM</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CAS-CAS</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9,450</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(6,100–14,700)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CAS</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">EAI-WA2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32,800</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(27,900–38,300)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clade2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">EAI-EAI</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13,700</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(9,100–21,000)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">EAI</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>M. bovis</italic>-<italic>M. bovis</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5,750</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(4,560–7230)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>M. bovis</italic>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">EAI-LAM</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41,500</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(29,100–60,000)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">MTBC</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">EAI-Beijing</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37,500</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(25,800–55,100)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">MTBC</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"ppat-1000160-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.ppat.1000160.t002</object-id><label>Table 2</label><caption><title>Time to the most recent common ancestor (TMRCA), time elapsed since the last expansion began (<italic>t<sub>a</sub></italic>) and growth rate estimates based on the MSVAR software.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td colspan=\"3\" align=\"left\" rowspan=\"1\">TMRCA</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">\n<italic>t<sub>a</sub></italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Growth</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">lower</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">modal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">upper</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">lower</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">modal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">upper</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">modal</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Africa</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.024</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.510</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.085</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.418</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.193</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.604</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.60</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Asia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.126</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.620</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.022</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.833</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.006</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.774</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.16</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Europe</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.257</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.701</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.029</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.710</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.321</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.527</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.12</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Beijing</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.939</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.040</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.396</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.566</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.514</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.421</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.71</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CAS</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.865</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.540</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.156</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.389</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.345</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.624</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.67</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">LAM</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.378</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.007</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.758</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.341</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.989</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.381</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.80</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">EAI</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.208</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.854</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.282</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.134</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.145</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.274</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.81</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>M. bovis</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.379</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.687</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.859</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.316</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.184</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.222</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.83</td></tr></tbody></table></alternatives></table-wrap>" ]
[]
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[ "<supplementary-material content-type=\"local-data\" id=\"ppat.1000160.s001\"><label>Protocol S1</label><caption><p>(0.06 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"ppat.1000160.s002\"><label>Table S1</label><caption><p>List of the MTBC isolates used in this study.</p><p>(0.08 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"ppat.1000160.s003\"><label>Figure S1</label><caption><p>Bubble-graph representation of allele frequencies for the different MIRU loci. Allele size (number of repeats) on the y-axis, and source populations on the x-axis.</p><p>(2.07 MB PDF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"ppat.1000160.s004\"><label>Figure S2</label><caption><p>MIRU and region of deletion (RD) patterns of 176 random selected <italic>M. tuberculosis</italic> and <italic>M. prototuberculosis</italic> strains. A visualisation of MIRU and RD data was added to the rooted population neighbour-joining tree based on genetic distances (see ##FIG##0##Figure 1B##). Representative results are shown for 89 isolates. The copy numbers of the 24 MIRU loci are displayed in blue shades ranging from 0 (white) to 13 (dark blue). For RD-analysis, black and white boxes correspond respectively to presence and absence of the considered region. The deletions distribution and the spolygotype patterns (data not shown) were in good congruence with the MIRU typing. Several clusters defined by MIRU typing also showed specific deletions such as RD726 for the Cameroon lineage or RD711 for West-African 1 strains. The presence or absence of the deletions also supported the dichotomy of the tree as all clade 1 strains are TBD1 negative and all clade 2 strains are TBD1 positive. However, it must be noted, that MIRU typing allowed a fin grain resolution, for example, several lineages e.g. West African 1a and West African 1b belong to two different lineages but remain undistinguishable by RD-typing. The presence or absence of the 16 Rds was determined by PCR as described previously ##REF##16519816##[15]##,##REF##17448936##[16]##,##REF##16323140##[17]##,##REF##10835412##[18]##.</p><p>(1.21 MB EPS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"ppat.1000160.s005\"><label>Figure S3</label><caption><p>MTBC population structure. (A) Population structure of 355 <italic>M. tuberculosis</italic> and <italic>M. prototuberculosis</italic> isolates. Each strain is represented by a single vertical line divided into <italic>K</italic> colours, where <italic>K</italic> is the number of clusters assumed. Each colour represents one cluster, and the length of the coloured segment shows the strain's estimated proportion of membership in that cluster. Black lines separate the main lineages. (B) Population structure for <italic>K</italic> = 3, as in a, but with the implementation of the linkage model.</p><p>(7.91 MB EPS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"ppat.1000160.s006\"><label>Figure S4</label><caption><p>Evolution of repeat copy number among MIRU-VNTR single-locus variants. To evaluate the validity of the stepwise mutation model and to detect a potential bias towards increase or decrease in repeat numbers, EBURST analysis was performed on a large dataset comprising a total of 2714 isolates from two population-based studies. Genotypes from a selected clonal complex are represented as circles. Stepwise and non-stepwise allelic changes between genotypes, along with corresponding marker number, are highlighted in green and gray, respectively. Insets show examples of allelic identification by analysis of marker amplicons using GENEMAPPER. Gray ladders and axis in insets define amplicon size bins expected for MIRU-VNTR alleles and measured amplicon sizes in base pairs, respectively. Code numbers in the upper left of insets define sample and marker identity, respectively. M, marker (from 1 to 24).</p><p>(2.07 MB EPS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"ppat.1000160.s007\"><label>Figure S5</label><caption><p>Distribution of repeat copy number changes among MIRU-VNTR single-locus variants. The difference in the number of repeats was calculated for each pair of ancestor/descendant genotypes along the evolutionary path inferred by EBURST analysis, on a large dataset comprising a total of 2714 isolates from two population-based studies. The occurrence and nature of each repeat difference was recorded for each strain group (defined as groups of strains with at most one allelic mismatch with at least one other member of the group), and values were pooled over all EBURST groups.</p><p>(0.96 MB EPS)</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"nt101\"><label/><p>Estimates and 95% confidence intervals were calculated with the software YTime.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt102\"><label/><p>Modal values and 95% confidence intervals are presented. The results are on a log scale.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p>Parts of this work were supported by the Germany Ministry of Health and the German Federal Ministry for Education and Research (BMBF) within the PathoGenomikPlus Network (S.N). P.S. is a researcher of the Centre National de la Recherche Scientifique (CNRS).</p></fn></fn-group>" ]
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[ "<media xlink:href=\"ppat.1000160.s001.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"ppat.1000160.s002.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"ppat.1000160.s003.pdf\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"ppat.1000160.s004.eps\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"ppat.1000160.s005.eps\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"ppat.1000160.s006.eps\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"ppat.1000160.s007.eps\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["1"], "element-citation": ["\n"], "surname": ["Organization"], "given-names": ["WH"], "year": ["2006"], "source": ["Global Tuberculosis Control, WHO Report"], "publisher-loc": ["Geneva"], "publisher-name": ["W.H.O"]}, {"label": ["4"], "element-citation": ["\n"], "surname": ["Liu", "Gutacker", "Musser", "Fu"], "given-names": ["X", "MM", "JM", "YX"], "year": ["2006"], "article-title": ["Evidence for Recombination in Mycobacterium tuberculosis."], "source": ["J Bacteriol"]}, {"label": ["8"], "element-citation": ["\n"], "surname": ["Supply", "Allix", "Lesjean", "Cardoso-Oelemann", "Rusch-Gerdes"], "given-names": ["P", "C", "S", "M", "S"], "year": ["2006"], "article-title": ["Proposal for standardization of optimized Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat typing of Mycobacterium tuberculosis."], "source": ["J Clin Microbiol"]}, {"label": ["23"], "element-citation": ["\n"], "surname": ["Kalinowski"], "given-names": ["ST"], "year": ["2005"], "article-title": ["HP-rare: a computer program for performing rarefaction on measures of allelic diversity."], "source": ["Molecular Ecology Notes"], "volume": ["5"], "fpage": ["187"], "lpage": ["189"]}, {"label": ["50"], "element-citation": ["\n"], "surname": ["Allix-Beguec", "Fauville-Dufaux", "Supply"], "given-names": ["C", "M", "P"], "year": ["2008"], "article-title": ["Three-year population-based evaluation of standardized Mycobacterial Interspersed Repetitive Unit-Variable Number of Tandem Repeat typing of Mycobacterium tuberculosis."], "source": ["J Clin Microbiol"]}, {"label": ["52"], "element-citation": ["\n"], "surname": ["Storz", "Beaumont"], "given-names": ["JF", "MA"], "year": ["2002"], "article-title": ["Testing for genetic evidence of population expansion and contraction: an empirical analysis of microsatellite DNA variation using a hierarchical Bayesian model."], "source": ["Evolution Int J Org Evolution"], "volume": ["56"], "fpage": ["154"], "lpage": ["166"]}]
{ "acronym": [], "definition": [] }
52
CC BY
no
2022-01-13 03:40:34
PLoS Pathog. 2008 Sep 26; 4(9):e1000160
oa_package/3c/2f/PMC2528947.tar.gz
PMC2528948
18800166
[ "<title>Introduction</title>", "<p>Over the past few decades, researchers as well as clinicians have made great strides in understanding the pathophysiological mechanisms of heart failure. Whereas heart failure was once thought of as a series of symptoms simply due to a poorly functioning heart, it is now understood to be a syndrome whose causes are both multifactorial and complex ##REF##12748317##[1]##. Several diverse mechanisms contribute to this syndrome including structural and functional abnormalities of the heart, vascular disease, biological and neurohormonal factors, oxidative stress, genetics, environment and coexisting conditions ##REF##12748317##[1]##. Yet, while these advancements in understanding have indeed led to better treatment of heart failure, it remains a major cause of morbidity and mortality worldwide.</p>", "<p>More recently, considerable evidence has shown that heart failure is associated with tissue ischemia and endothelial dysfunction, as assessed by impaired flow-mediated dilatation, as well as increases in specific plasma markers such as von Willebrand factor and soluble thrombomodulin ##REF##11751734##[2]##–##REF##12860861##[5]##. A newer method to identify endothelial damage and dysfunction is the quantification of circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in the peripheral circulation. CECs are mature endothelial cells that have detached from the intimal monolayer of blood vessels in response to endothelial injury ##REF##17045885##[6]##, whereas EPCs are immature, bone-marrow derived cells with the capacity to transform into mature endothelial cells and promote postnatal angiogenesis and vasculogenesis ##REF##12778169##[7]##–##REF##9020076##[9]##. EPCs can be characterized by the expression of surface markers, such as CD34, CD133 and VEGFR-2 (KDR or Flk-1) in various combinations ##REF##15601578##[10]##. It has, in fact, recently been shown by us that patients with heart failure have elevated circulating EPCs, which may be an independent predictor of mortality in CHF ##REF##17277352##[11]##.</p>", "<p>There are small membrane particles, known as endothelial microparticles, which are associated with endothelial cell damage and apoptosis. These endothelial microparticles have been shown to be elevated in conditions such as acute coronary syndrome (ACS) and myocardial infarction ##REF##16239600##[12]##–##REF##11723013##[14]##. Recently, we identified, for the first time, a new population of apoptotic progenitor cells (APCs) which were elevated in patients with ACS ##REF##17332488##[15]##. The apoptotic progenitors can be divided into early, reversible apoptotic cells and late, irreversible apoptotic cells. In this study, we sought to quantify the number of apoptotic progenitor cells in patients with heart failure. In so doing, we learned that while CHF patients did not exhibit higher levels of total or early apoptotic progenitors than controls, the more severe CHF patients exhibited elevated numbers of late apoptotic progenitors compared to those with less severe CHF.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Study Subjects</title>", "<p>We studied a total of 58 patients (median age 76.5) arbitrarily with various classes of heart failure according to the New York Heart Association (NYHA) classification. The control group comprised of 23 subjects with a similar profile of age (a median of 74 years, range 42–81), gender, a normal ejection fraction by echocardiography and no evidence of heart failure. The incidence of risk factors for atherosclerosis including diabetes, hypertension, smoking and treated hyperlipidemia did not differ between the study and control groups. With regard to medication use, study group patients had significantly increased use of warfarin and renal failure was more common as compared with controls. In addition, as seen in ##TAB##0##Table 1##, we subdivided the 58 CHF patients into two subgroups: 33 patients with mild/moderate CHF (NYHA class I–II) and 25 patients with advanced CHF (NYHA class III–IV). There were no significant differences between the demographic characteristics of these two subgroups.</p>", "<p>Of the CHF patients, 43 were male and 15 were female. Of the controls, 15 were male and 8 were female. ##TAB##0##Table 1## summarizes demographic and clinical characteristics of the patient population. Institutional ethics committee approved the study and informed consent was obtained from all patients.</p>", "<title>Preparation of Blood Samples</title>", "<p>Peripheral blood mononuclear cells (PBMNCs) were isolated from 20 ml of freshly drawn heparinized blood using Isopaque-Ficoll (Amersham Biosciences, Buckinghamshire, United Kingdom) gradient centrifugation.</p>", "<title>Flow Cytometry evaluation of early and late apoptotic progenitor CD34+ cells</title>", "<p>After Ficoll gradient separation, PBMNCs were washed with phosphate-buffered saline (PBS), and 10<sup>6</sup> cells were stained with (FITC)-anti-CD34 MAb for 30 minutes at 4°C in 100 microliters of FACS staining buffer (PBS and 2% fetal calf serum (FCS). Apoptosis in progenitor CD34+ progenitor cells was assessed using Southern Biotech ApoScreen Annexin V Apoptosis detection kit (Annexin V-PE, Propidium Iodide (PI) solution and Annexin V binding buffer). This assay involves staining peripheral blood mononuclear cells with Annexin V-PE (a phospholipid-binding protein binding to disrupted cell membranes) in combination with propidium iodide (PI, a vital dye binding to DNA penetrating into apoptotic cells). FACS analysis of CD34+ progenitor cells that are in early (annexin V+/PI−) or late (annexin V+/PI+) apoptotic phase was performed.</p>", "<p>The percentage of apoptotic CD34+ progenitor cells (out of total circulating CD34+ progenitor cells) was assessed by staining peripheral blood mononuclear cells for 3 color FACS analysis employing (FITC)-anti-CD34 MAb (IQ products), Annexin V-PE and Propidium Iodide (SouthernBiotech). The cells were then washed again with PBS and resuspended in 100 microliters of Annexin V-PE binding buffer and incubated with 5 microliters of Annexin V-PE for 15 minutes at room temperature. Without washing, another 200 microliters of binding buffer and 5 microliters of PI solution were added, and 800,000 cells were acquired by flow cytometry (FACSCalibur, Becton Dickinson) and analyzed by CellQuest software (BD Bioscience). All analyses and readings were made by technicians who were blinded to the study questions.</p>", "<title>Determination of Erythropoietin, Thrombomodulin/CD141 and Antibodies to Oxidized LDL levels</title>", "<p>ELISA kits were used to detect levels of Erythropoietin (Stem Cell Technologies) and Thrombomodulin/CD141 (Diaclone). Antibody to oxidative LDL levels were detected by ELISA method with anti-human antibody and solutions created in the laboratory ##REF##16061420##[19]##.</p>", "<title>Statistical Analysis</title>", "<p>All data was summarized and displayed as mean (SD) for the continuous variables and as number of patients plus the percentage in each group for categorical variables. The one-way Kolmogorov-Smirnov test was used to assess the distributions. Levels of late apoptotic cells could not be converted to normal distribution, thus we categorized the variables into tertiles.</p>", "<p>For all categorical variables the Chi-Square statistics was used for assessing the statistical significance between the groups of CHF severity, while for all continuous variables, the independent samples one way ANOVA test was used. Regression analysis and analysis of variance for APCs by independent variables was performed in all cases.</p>", "<p>All above analyses were considered significant at p&lt;0.05 (two tailed). The SPSS 15 statistical package was used to perform all statistical evaluations (SSPS Inc., Chicago, IL, USA).</p>" ]
[ "<title>Results</title>", "<p>We employed a novel assay for determining apoptotic CD34 cells. Progenitor CD34+ cells were initially gated from the side scatter/CD34 dot plot according to the Milan protocol ##REF##10504123##[20]##–##REF##1702334##[21]##. As can be seen in ##FIG##0##Figure 1a##, the progenitor CD34+ cells can be outlined and gated using this FACS dot plot. The percentage of apoptotic CD34+ progenitor cells was then determined by FACS analysis of Annexin V/PI staining. In apoptotic cells, the membrane phospholipids phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the extracellular environment. Annexin V is a 36 kDa Ca<sup>2+</sup> dependent phospholipid-binding protein that has a high affinity for PS and binds to cells when PS is exposed to the external cellular environment, which occurs when membrane integrity is affected in early phase of apoptosis ##REF##8155692##[16]##–##REF##8068938##[17]##. Propidium Iodide (PI) is a vital dye binding to DNA, a process implying disrupted cellular membrane and exposed DNA, compatible with late, irreversible cell necrosis. Cells that are viable are Annexin V-PE and PI negative; cells that are in early apoptosis are Annexin V-PE positive and PI negative (lower right quadrant–##FIG##0##figure 1b##); and cells that are in late apoptosis or necrosis are both Annexin V-PE and PI positive (upper right quadrant–##FIG##0##figure 1b##) ##REF##8068938##[17]##–##REF##7595224##[18]##.</p>", "<p>As can be appreciated in ##TAB##0##Table 1##, there were no significant statistical differences between the demographic characteristics of the two study groups (mild-moderate CHF and severe CHF) and the control group which comprised ACS patients not having CHF. With regard to the number of early apoptotic cells, no statistically significant difference was found between the age matched controls vs. CHF groups (once adjusted for age, gender and risk factors) or between the two CHF groups (mild/moderate vs. severe) (##FIG##1##figure 2##). Within the CHF groups, however, there was a positive correlation between the number of early apoptotic progenitor cells and levels of hemoglobin (r = 0.279, p = 0.016) and total CD34+ cells (r = 0.261, p = 0.023). With regard to the late apoptotic cells, there was an elevated number of cells in the severe CHF group compared to the mild/moderate CHF group (p =  0.013), as shown in ##FIG##2##figure 3##. Interestingly, there was a positive association between late apoptotic cells and NYHA class (r = 0.223, p = 0.046) as well as a negative correlation between late apoptotic cells and ejection fraction (r = −0.252, p = 0.028). There was also a negative association between hyperlipidemia and total CD34+ progenitors in the CHF groups. ##TAB##1##Tables 2## and ##TAB##2##3## demonstrate the parameters tested for correlations and associations. As shown in ##TAB##1##table 2##, neither erythropoietin (r = 0.072; p = 0.3), thrombomodulin/CD141 (r = 0.09; p = 0.25), nor antibodies to oxidized LDL levels (r = −0.13; p = 0.19) correlated with early apoptotic progenitor cells.</p>" ]
[ "<title>Discussion</title>", "<p>Over the past decade, since the isolation of a circulating angioblast (later referred to as EPC) from adults with the capacity to differentiate into mature endothelial cells in response to ischemia ##REF##9020076##[9]##, ##REF##10436164##[22]##, much work has been done to further classify and characterize the role of endothelial progenitor cells. EPCs are released from the bone marrow in response to endothelial damage in order to facilitate in angiogenesis and vasculogenesis. Previous studies have shown elevated numbers of EPCs in ACS ##REF##15734770##[23]##–##REF##15345590##[25]## as well as in heart failure ##REF##17277352##[11]##, presumably due to the release of angiogenic factors and activation of multiple neurohormonal axes triggered by tissue ischemia ##REF##12778169##[7]##. It is also known that all major cardiovascular risk factors negatively influence these factors ##REF##16322535##[26]##.</p>", "<p>In CHF there is increased oxidative stress due to an imbalance between reactive oxygen species (including the superoxide anion, hydrogen peroxide, and the hydroxyl radical) and endogenous antioxidant defense mechanisms ##REF##18480207##[32]##. Oxidative stress may damage cellular proteins and cause myocyte apoptosis and necrosis. Markers of oxidative stress that are increased in CHF include plasma-oxidized low-density lipoproteins, malondialdehyde and myeloperoxidase (an index of leukocyte activation), urinary levels of biopyrrins (oxidative metabolites of bilirubin), and isoprostane levels in plasma and urine ##REF##18480207##[32]##.</p>", "<p>Endothelial cell (EC) apoptosis is an additional biomarker of endothelial damage and hemostasis that has more recently been explored ##REF##14977533##[27]##. The number of circulating endothelial microparticles positively correlates with the severity of coronary endothelial dysfunction, suggesting a close relationship between coronary endothelial-dependent vasodilation and EC apoptosis ##REF##16239600##[12]##. In addition, their functional properties such as their procoagulant activity, involvement in inflammation and direct effect on endothelial dysfunction play a large role ##REF##15200490##[28]##. We have recently described a novel assay to detect and quantify circulating apoptotic CD34+ progenitor cells showing that they were elevated in ACS patients compared with healthy controls ##REF##17332488##[15]##.</p>", "<p>Similar to ACS, CHF is associated with myocardial and peripheral tissue ischemia. Thus, CHF patients were found to exhibit endothelial dysfunction that is correlated with disease severity ##UREF##0##[29]##. Previous studies have demonstrated elevated levels of EPCs in heart failure patients ##REF##17277352##[11]##, yet levels of apoptotic EPCs in these patients have not been determined so far. In our study, we sought to quantify these apoptotic progenitor cells in patients with heart failure. In so doing, we divided these apoptotic cells into two groups: early, reversible apoptotic CD34+ cells and late, irreversible apoptotic progenitors. The late apoptotic progenitor cells represent cells whose plasma membrane is no longer intact. Our results show that, while there was no significant difference in numbers of early or late apoptotic cells between CHF patients and healthy controls, there was an elevated number of late apoptotic progenitors in the more severe CHF patients compared to the less severe heart failure patients. Furthermore, there was a negative correlation between late apoptotic progenitors and ejection fraction as well as a positive association between late apoptotic CD34 cells and NYHA class.</p>", "<p>Several possible mechanisms could be postulated to explain these findings. Oxidative stress is known to cause endothelial dysfunction, and, as mentioned previously, CHF is associated with increased oxidative stress ##REF##18480207##[32]##. It has been demonstrated by Dernbach et al that EPCs are equipped with antioxidative enzyme systems, allowing for improved survival of cells undergoing severe oxidative stress ##REF##15161665##[30]##. Oxidized LDL has been shown to increase the rate of EPC senescence/apoptosis ##REF##15236625##[31]##. Antibodies to oxidized LDL are thought to mirror oxidative stress and have been shown to be increased in patients with CHF ##REF##16061420##[19]##. Although it would be plausible to explain the increase in late apoptotic cells in the more severe CHF (low EF and high NYHA class) patients as being due to increased oxidative stress, we did not find a statistically significant correlation between antibodies to oxidized LDL and apoptotic progenitor cells. Therefore, the relationship of additional indirect markers of oxidative stress which are known to be increased in CHF ##REF##18480207##[32]## should be further explored in future studies.</p>", "<p>Erythropoietin and thrombomodulin have also been shown to play a role in endothelial function and heart failure. Both these markers, erythropoietin being a mobilizer of progenitor cells and thrombomodulin being associated with endothelial dysfunction, were not found to correlate with the number of apoptotic CD34 cells.</p>", "<p>In view of the interplay between cytokines enhancing progenitor cell mobilization and those precipitating their apoptosis, our results support the hypothesis that increasing severity of heart failure shifts the balance towards enhanced progenitor cell apoptosis. The lower the ejection fraction, the poorer the forward flow, which may increase tissue ischemia and therefore endothelial damage.</p>", "<p>It should be mentioned, however, that the relatively small number of circulating CD34+ cells poses a question as to the true functional importance of these cells and imposes a difficulty in determining their accurate number. In addition, our study is limited by the relatively small sample size.</p>", "<p>In conclusion, we found that patients with advanced CHF have higher levels of late apoptotic progenitors than those with mild/moderate CHF and that levels of late apoptotic progenitors were positively associated with NYHA class and had a negative correlation with ejection fraction. These findings support the hypothesis that increasing severity of heart failure shifts the balance towards enhanced progenitor cell apoptosis. Therefore, apoptotic progenitor cells could be evaluated in future studies as a potential predictive biomarker in CHF.</p>" ]
[]
[ "<p>Conceived and designed the experiments: DG KG. Performed the experiments: DG SMA. Analyzed the data: DG SS AF JA AR SMA DW GK KG. Wrote the paper: DG SS.</p>", "<title>Background</title>", "<p>Circulating CD34+ endothelial progenitor cells (EPCs) are capable of differentiating into mature endothelial cells to assist in angiogenesis and vasculogenesis. We sought to quantify the numbers of apoptotic progenitors in patients with congestive heart failure.</p>", "<title>Methods and Results</title>", "<p>Peripheral blood mononuclear cells were isolated by Ficoll density-gradient from 58 patients with various degrees of heart failure and 23 matched controls. Apoptosis in progenitor CD34+ cells was assessed using the Annexin V-PE/PI detection kit, and FACS analysis was performed with triple staining for CD34, annexin-V and propidium iodide. The percentage of early and late apoptotic progenitor cells was determined in the subject groups and was correlated with clinical characteristics. While there was no significant difference in total CD34 positive cells or early apoptotic progenitors between control subjects and CHF patients (p = 0.42) or between severe and mild/moderate CHF groups (p = 0.544), there was an elevated number of late apoptotic progenitors in the severe CHF group compared with the mild/moderate CHF group (p =  0.03). Late apoptotic progenitors were significantly increased in CHF patients as compared to matched controls. There was also an inverse correlation between late apoptotic progenitors and ejection fraction (r = −0.252, p = 0.028) as well as a positive association with NYHA class (r = 0.223, p = 0.046).</p>", "<title>Conclusion</title>", "<p>Severe heart failure patients exhibited higher numbers of late apoptotic progenitors, and this was positively associated with NYHA class and negatively correlated with ejection fraction. This finding may shed light on the numerous factors governing the pathophysiology of CHF.</p>" ]
[]
[]
[ "<fig id=\"pone-0003238-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003238.g001</object-id><label>Figure 1</label><caption><title>Representative flow cytometric dot plots: SSC/CD34 scatter (1a) and flow cytometric evaluation of progenitor CD34+ apoptotic cell percentage (1b).</title><p>Dots in the lower right quadrant represent early apoptotic cells (Annexin-V positive and PI negative) while dots in the upper right quadrant represent late apoptotic cells (Annexin-V and PI positive).</p></caption></fig>", "<fig id=\"pone-0003238-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003238.g002</object-id><label>Figure 2</label><caption><title>Early apoptotic progenitor CD34+ cell percentage (out of total circulating progenitor CD34+ cells) in mild/moderate and severe CHF groups.</title><p>P-value is &gt;0.05 after adjustment for variables.</p></caption></fig>", "<fig id=\"pone-0003238-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003238.g003</object-id><label>Figure 3</label><caption><title>Late apoptotic progenitor CD34+ cell percentage (out of total circulating progenitor CD34+ cells) in mild/moderate (NYHA class 1–3) and severe (NYHA class 4) CHF groups.</title><p>Chi-square P value of the distribution of tertiles between NYHA 1–3 &amp; NYHA = 4 is 0.038.</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003238-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003238.t001</object-id><label>Table 1</label><caption><title>Baseline characteristics CHF and control patients.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Group</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Controls</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NYHA I–II</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NYHA III–IV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">n = 23</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">n = 33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">n = 25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">Demographic data</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male/Female</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15/8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25/8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18/7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median age (range)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">74 (42–81)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">73 (48–89)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">74 (47–79)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Current Smoker</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (9%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (18%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (16%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">Comorbidities</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hypertension</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12 (52%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21 (63%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (56%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Diabetes Mellitus</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12 (52%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20 (61%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (56%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hyperlipidemia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (65%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26 (79%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15 (60%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">Drug Treatment</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Statin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (65%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27 (82%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13 (52%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Beta Blocker</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (26%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25 (76%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19 (76%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ACEI/ARB</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (65%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27 (82%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19 (76%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Spironolactone</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23 (70%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17 (68%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Furosemide</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33 (100%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25 (100%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;.001 &lt;.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Coumadin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (42%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10 (40%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;.01</td></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">Measurements</td></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">Ejection Fraction<xref ref-type=\"table-fn\" rid=\"nt101\">*</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> &gt;40%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">13 (39%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10 (40%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> ≤40%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">20 (61%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15 (60%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">Creatinine</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> &gt;1.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">20 (61%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (64%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> ≤1.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">13 (39%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (36%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td colspan=\"5\" align=\"left\" rowspan=\"1\">Ischemic vs. Non-Ischemic<xref ref-type=\"table-fn\" rid=\"nt102\">Δ</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Ischemic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (64%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Non-ischemic</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">20 (36%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003238-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003238.t002</object-id><label>Table 2</label><caption><title>Correlations between early apoptotic/late apoptotic/total CD34+ EPCs and various parameters.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Parameter</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Early Apoptotic Cells</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Late Apoptotic Cells</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Total CD34+ cells</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">r</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">p-value</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">r</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">p-value</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">r</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">p-value</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.013</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.460</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.052</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.350</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.123</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.180</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Heart failure duration</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.063</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.318</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.018</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.447</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.058</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.332</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">LVEF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.107</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.211</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.252</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.028<xref ref-type=\"table-fn\" rid=\"nt103\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.152</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.127</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Creatinine</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.033</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.403</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.027</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.420</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.049</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.357</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Erythropoietin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.072</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.298</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.157</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.123</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.031</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.409</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Thrombomodulin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.093</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.247</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.091</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.251</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.031</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.411</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">OxLDL antibodies</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.117</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.194</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.037</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.391</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.041</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.380</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hemoglobin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.279</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.016<xref ref-type=\"table-fn\" rid=\"nt103\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.027</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.420</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.176</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.092</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CD34% of total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.261</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.023<xref ref-type=\"table-fn\" rid=\"nt103\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.213</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.053</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003238-t003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003238.t003</object-id><label>Table 3</label><caption><title>Associations between early apoptotic/late apoptotic/total CD34+ EPCs and various parameters.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Parameters</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Early apoptotic cells</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Late apoptotic cells</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Total CD34+ cells</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">r</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">p-value</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">r</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">p-value</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">r</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">p-value</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gender</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.027</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.420</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.124</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.176</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.214</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.053</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hyperlipidemia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.130</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.165</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.186</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.082</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.236</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.038<xref ref-type=\"table-fn\" rid=\"nt104\">*</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Smoking</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.094</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.241</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.052</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.351</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.170</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.101</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hypertension</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.017</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.450</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.074</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.291</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.157</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.120</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Diabetes Mellitus</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.108</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.210</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.023</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.433</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.113</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.200</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ischemic heart disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.040</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.384</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.081</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.274</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.106</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.215</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">TIA/CVA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.065</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.313</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.174</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.096</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.103</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.221</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PTCA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.109</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.209</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.243</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.033<xref ref-type=\"table-fn\" rid=\"nt104\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.124</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.178</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CABG</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.071</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.300</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.028</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.417</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.113</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.199</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NYHA Class</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.174</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.096</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.223</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.046<xref ref-type=\"table-fn\" rid=\"nt104\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.098</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.232</td></tr></tbody></table></alternatives></table-wrap>" ]
[]
[]
[]
[]
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[]
[ "<table-wrap-foot><fn id=\"nt101\"><label>*</label><p>LV ejection fraction was estimated by 2D echocardiography.</p></fn><fn id=\"nt102\"><label>Δ</label><p>Ischemic cardiomyopathy refers to patients with ischemic heart disease (prior MI, PTCA or CABG) and ejection fraction &lt;40%.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt103\"><label>*</label><p>Statistically Significant.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt104\"><label>*</label><p>Statistically Significant.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>The authors have no support or funding to report.</p></fn></fn-group>" ]
[ "<graphic id=\"pone-0003238-t001-1\" xlink:href=\"pone.0003238.t001\"/>", "<graphic xlink:href=\"pone.0003238.g001\"/>", "<graphic xlink:href=\"pone.0003238.g002\"/>", "<graphic xlink:href=\"pone.0003238.g003\"/>", "<graphic id=\"pone-0003238-t002-2\" xlink:href=\"pone.0003238.t002\"/>", "<graphic id=\"pone-0003238-t003-3\" xlink:href=\"pone.0003238.t003\"/>" ]
[]
[{"label": ["29"], "element-citation": ["\n"], "surname": ["Fischer", "Rossa", "Landmesser", "Spiekermann", "Engberding"], "given-names": ["D", "S", "U", "S", "N"], "year": ["2005"], "article-title": ["Endothelial dysfunction in patients with chronic heart failure is independently associated with increased incidence of hospitalization, cardiac transplantation, or death."], "source": ["Eur Heart J"], "volume": ["25"], "fpage": ["65"], "lpage": ["69"]}]
{ "acronym": [], "definition": [] }
32
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 18; 3(9):e3238
oa_package/60/c8/PMC2528948.tar.gz
PMC2528949
18795100
[ "<title>Introduction</title>", "<p>Some of the most complex cognitive abilities of humans, such as planning, are commonly attributed to a disproportionate enlargement of the human frontal lobe during evolution. However, recent comparative studies of the relative size of the frontal cortex taken as a whole indicate that the human frontal cortex is not larger in comparison to those of the great apes ##REF##11850633##[1]##, ##REF##11241188##[2]##. Rather, the specific cognitive capacities of humans may be due to differences in specific individual cortical areas (such as the frontopolar cortex), as well as to richer interconnectivity between the frontal lobe and other higher-order association areas, none of which require an increase in the overall relative size of the frontal lobe during hominid evolution.</p>", "<p>Of particular importance in the cognitive capacities that are uniquely human seems to be the most anterior part of the prefrontal cortex, namely the fronto-polar cortex (Brodmann's area 10), which is larger in humans relative to the rest of the brain than it is in the ape's brain ##REF##11850633##[1]##, ##REF##11241188##[2]##. The specific function of this brain region remains poorly understood, but one recent hypothesis states that its role is to hold goals in mind while exploring and processing secondary goals ##REF##10335843##[3]##, ##REF##11848695##[4]##, ##REF##11257280##[5]##, ##REF##12667527##[6]## – a process that we refer to as multitasking in the remainder of this paper-. Neither keeping in mind a goal over time (working memory) nor successively allocating attentional resources between alternative goals (dual-task performance) could by themselves selectively activate the fronto-polar cortex while a highly specific super-additive effect was demonstrated in the frontopolar cortex when subjects held in mind goals while processing secondary goals at the same time ##REF##10335843##[3]##.</p>", "<p>This functional hypothesis about a key role of Brodmann's area (BA) 10 in multitasking is based on the results of functional neuroimaging studies that can only support inferences about the association of brain regions with a specific cognitive process. In contrast, neuropsychological studies are crucial for inferring whether a brain region is necessary to mediate a cognitive process.</p>", "<p>Here we examined patients with focal prefrontal cortex lesions to test whether the fronto-polar cortex is necessary for multitasking. Our hypothesis was that the extent of damage to the fronto-polar cortex should correlate with impairment in this process. The results confirmed this hypothesis by demonstrating that the extent of damage to Brodmann's area 10 correlated with impaired multitasking.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Participants</title>", "<p>We tested 13 patients with focal frontal lobe lesions (see ##TAB##0##\nTable 1\n## and ##FIG##0##\nFigs 1\n##\n<bold>–</bold>\n\n##FIG##2##\n3\n##\n<bold>for</bold> patient's demographic and lesion's sites). Patients were divided into two groups on the basis of the location of their lesion: one group had lesions that included the fronto-polar cortex (n = 7, 4 males, mean age = 49±6) and one group had lesions that excluded the fronto-polar cortex (n = 5, 3 males, mean age = 49±3.5). The two groups of patients did not differ in age (t = 0.03, P = 0.98), level of education (t = 0.29, P = 0.8) and Full Scale WAISIII IQ scores (t = 0.74, P = 0.48). In addition, 7 control subjects (5 males, mean age = 50.8±6) were matched in age (t = 0.2, P = 0.82, n.s) and level of education (t = 0.72, P = 0.48) with the patients with fronto-polar lesions. All subjects were screened for a prior history of neurological disease, substance abuse, and psychiatric disorder.</p>", "<p>The etiology of fronto-polar patients was as followed: 3 were Vietnam veterans who suffered a penetrating brain injury, one patient had a resection of a malignant tumor, one had an aneurysm of the right anterior communicating artery and two patients had closed head injuries. Reconstruction of lesions for each patient with or without frontopolar cortex damage is provided in ##FIG##1##figures 2## and ##FIG##2##3##. All subjects were screened for a prior history of neurological disease, substance abuse, and psychiatric disorder. The control subjects were paid for their participation. Subjects provided written informed consent approved by the NINDS Institutional Review Board.</p>", "<title>Lesion analysis</title>", "<p>The penetrating brain injury patients were scanned using a standard CT sequence since they had retained metal in their brain. Other patients were scanned by MRI using a three-dimensional set acquisition in the axial plane with a SPGR T1-weighted sequence and a T2-weighted axial sequence. All lesions were traced using the Analysis of Brain Lesions software package ##REF##12060992##[7]## and normalized in Talairach space to the Damasio's template ##UREF##0##[8]##. Then, the percentage of the approximate Brodmann areas contained within the boundaries of the lesions was computed for each subject using this standardized, semi-automated software that can determine the extent of brain lesions in terms of cytoarchitectonic regions in Talairach space ##REF##12060992##[7]## (see ##FIG##0##\nFig. 1\n##).</p>", "<title>Behavioral paradigm</title>", "<p>The tasks were identical to those used in a previous fMRI study ##REF##10335843##[3]## and were designed to systematically vary keeping in mind a main goal over time (working memory) and allocation of attentional resources between alternative subgoals (dual-task) (##FIG##3##\nFig. 4\n##). The experiment consisted of 6 runs in which 3 tasks (delay, dual and multi-tasking, described hereafter) were administered in pseudo-random order (28 trials by task, inter-stimuli interval = 3 s). This pseudo-random order was built in such a way that each condition appears at all serial positions within a run and two conditions appeared once or twice in immediate succession to prevent confounding order effects. The first run was used for training and is not included in the present analysis.</p>", "<p>Subjects responded to visually presented letters (500 ms duration, 3000 ms stimulus-onset-asynchrony) by pressing response buttons with their right (match) or left (no match) hand, respectively. Subjects were given standard instructions to respond quickly and accurately. Single-letters (upper or lower-case) from the word “tablet” (i.,e A, B, E, L, T, a, b, e, l, t) were successively presented and subject's decisions were recorded using the two single response-buttons. Matching proportions were maintained between 40 and 43% of trials in each condition. In all conditions lower-case letters were pseudorandomly presented in 64% of trials and the mean SOA between two successive upper-case letters was strictly maintained at 6.3 s. The tasks were administered using the Expe6 software package ##UREF##1##[9]##.</p>", "<p>\n<underline>In the delay condition</underline>, subjects decided whether two successively presented upper-case letters were also in immediate succession in the word “TABLET” by pressing the right button for yes and the left button if they were not in succession, and they had to ignore lower case letters that were presented in order to delay the response required for upper-case letters.</p>", "<p>\n<underline>In the dual-task condition</underline>, subjects decided whether two successively presented letters were also in immediate succession in the word “tablet” or “TABLET” (this time both for upper and lower case letters), by pressing the right button for yes and the left button if they were not in succession, except that they had to decide whether every first letter indicating a case change was the letter T (or t).</p>", "<p>\n<underline>In the multitasking condition</underline>, subjects responded to upper case letters exactly as in the delay condition and to lower case letters exactly as in the dual task condition. Thus, the multitasking condition requires maintenance of the primary task information in memory (primary goal) so that it can be returned to after completing a secondary task (subgoal). In other words, for successive upper case letters, or for successive lower case letters, subjects decided whether the current letter followed immediately the previously presented letter in the word “TABLET” or ‘tablet” by pressing the right button for yes and the left button if they were not in succession, and they had to decide whether every first letter indicating a case change was the letter T (or t).</p>" ]
[ "<title>Results</title>", "<title>Behavioral performance</title>", "<p>First, a 2*3 repeated measures ANOVA was conducted on correct reaction time (RT) (&lt;3000 ms) and on error rates with group (patients with fronto-polar cortex lesions and age-matched controls) as the between-subject factor and with conditions (delay, dual task, multi-tasking) as the within-subject factor (##FIG##4##\nFig. 5\n##). In this group analysis of variance, for response times, there was a main effect of task [F(2, 36) = 10.6, P&lt;0.0005], indicating that additional processes are engaged successively in the delay, dual-task and multi-tasking conditions. No main effect of group [F(1,36) = 2.0, P = 0.16] and no group*task interaction [F(2,36) = .25, P = .8] were observed.</p>", "<p>For error rates, patients with lesions affecting the fronto-polar cortex made more errors than controls (main effect of group [F(1, 36) = 5.01, P&lt;0.05]). There was also a main effect of task [F(2, 36) = 9.8, P&lt;0.0005]. Overall, the multi-tasking condition led to more errors than the delay condition (F(1,26) = 21.5, P&lt;0.0001) but did not differ from the dual-task condition [F(1,26) = 1.2, P = 0.3]. We did not observe any group*task interaction [F(2,36) = 0.5, P = 0.6] on performance accuracy. However, when performing a new 2*2 analysis of variance grouping the dual task and multi-tasking conditions together (both involving putting information into sequences) compared to the delay condition, we found a significant difference between patients with frontopolar lesions and controls [F(1,26) = 5.04, P&lt;0.05]. No between group difference was observed when performing another 2*2 analysis of variance grouping the delay and multi-tasking conditions together (both involving working memory) compared to the dual task condition [F(1,26) = 1.24, P = 0.27].</p>", "<p>We also directly compared the performance of the two groups of patients. For response times, there was a main effect of task [F(2,30) = 11.6, P&lt;0.0005], confirming additional engagement of processes from the delay to the dual-task and to the multi-tasking conditions. No main effect of group [F(1,30) = .74, P = 0.39] and no group*task interaction [F(2,30) = .63, P = .53] were observed for response times. For error rates, there was a trend towards significance in patients with lesions affecting the fronto-polar cortex compared to patients without lesion of the frontopolar cortex (main effect of group [F(1,30) = 3.1, P = 0.08]). There was also a main effect of task [F(2,30) = 16.9, P&lt;0.00005] due to the lower error rate in the delay condition. No group*task interaction [F(2,30) = 0.59, P = 0.55] was observed on performance accuracy. When we performed another 2*2 analysis of variance grouping the dual task and multi-tasking conditions together (both involving switching between tasks) compared to the delay condition, we found a trend towards significance between patients with <italic>versus</italic> without frontopolar lesions [F(1,32) = 3.68, P = 0.06]. These data suggest that frontopolar lesions impair switching processes, both compared to controls and compared to patients without frontopolar lesions. No difference between patient group was observed when performing another 2*2 analysis of variance grouping the delay and multi-tasking conditions together (both involving working memory) compared to the dual task [F(1,32) = 0.96, P = 0.33].</p>", "<title>Correlations between performance and damage to the fronto-polar cortex</title>", "<p>In order to test our specific hypothesis that the fronto-polar cortex (Brodmann's area 10) is necessary for performing a subgoal while maintaining primary goal related information in memory, we correlated the proportion of damage to each approximate Brodmann's area contained within the boundaries of the lesion with the error rates in the multi-tasking condition. Only the left BA 10 (Spearman rank correlation coefficient R<sub>1</sub> = 0.94, P&lt;0.005) showed a significant positive correlation with performance (6 patients had left frontopolar damage) (##FIG##5##\nFig. 6.A\n##). No other Brodmann's area was significantly correlated with performance in the multi-tasking condition. Moreover, lesion size of the left BA 10 did not show a significant correlation with error rates in the dual-task condition (Spearman rank correlation R<sub>2</sub> = 0.65, P = 0.16, n.s) (##FIG##5##\nFig. 6.B\n##). This demonstrates that variability of frontopolar cortex patients in multi-tasking performance is primarily explained by the size of the lesion in BA 10.</p>", "<p>Note that this analysis did not include the only patient with lesion restricted to the right fronto-polar cortex because there may be a functional lateralization of this brain region. However, when including the data of this subject, the correlation between error rates in the dual-task condition and the size of fronto-polar lesions was still non-significant (R = 0.1, P = 0.5) and the correlation between error rates in the branching condition and size of fronto-polar lesions remained significant (Spearman rank correlation coefficient R = 0.82; P&lt;0.05).</p>", "<p>Further analyses of the total volume of brain lesions revealed no relationship between the total volume of damaged tissue (which included extra fronto-polar damage) and error rates in the multi-tasking condition (R = 0.36, P = 0.43), ruling out the possibility of a confound between total lesion size and lesions of the fronto-polar cortex <bold>(</bold>\n##FIG##5##\nFig. 6.C\n##\n<bold>)</bold>. Moreover, task difficulty or mental effort alone can not explain our findings since error rates in the multi-tasking and the dual-task condition did not differ significantly in patients with fronto-polar cortex lesions ((F1,19) = 0.4, P = 0.5).</p>" ]
[ "<title>Discussion</title>", "<p>Based on recent neuroimaging findings ##REF##10335843##[3]##, ##REF##11848695##[4]##, we could have expected that patients with frontopolar lesions would exhibit a specific increase of errors only in the multi-tasking condition. The fact that we did not observe such a group by task interaction may be due to the extent of the lesions of the frontopolar group, which included, but were not restricted to, the frontopolar region. This may also explain why performance is altered in the dual task condition, thereby masking the behavioral effect of more restricted frontopolar lesions. Note that it is also possible that the frontopolar cortex contains multiple subregions, each contributing to different processes, and/or that brain regions outside the frontopolar cortex are part of a functionally distributed network that is necessary to perform multitasking computations.</p>", "<p>Although we did not find a group*task interaction on performance accuracy, likely due to group size and low statistical power, our results suggest that what is crucially impaired in patients with frontopolar lesions is the ability to put information into sequences as required by the dual-task and the multi-tasking conditions rather than simply holding a goal in working memory (such as in the delay condition). Indeed, when performing an analysis of variance grouping the dual task and multi-tasking conditions together (both involving putting information into sequences) compared to the delay condition, we did find a significant difference between patients with frontopolar lesions and controls and a trend towards significance between patients with frontopolar lesions <italic>versus</italic> without frontopolar lesions.</p>", "<p>Our main finding is that only the extent of the lesion of the left fronto-polar cortex showed a positive correlation with performance in the multitasking condition. This demonstrates that damage to the frontopolar cortex is necessary to impair multitasking, i.e. a process dependent upon the ability to put tasks in pending sequences. These results provide new evidence that specific executive functions are subserved by distinct prefrontal regions ##REF##12482070##[10]##, ##REF##12391312##[11]##, ##REF##12631562##[12]##, ##REF##12183390##[13]##, ##REF##15917482##[14]##, ##REF##12563285##[15]##, contradicting the view that the functions of distinct prefrontal regions cannot be distinguished ##REF##11006464##[16]##. Thus, variability of frontopolar cortex patients in multi-tasking performance is primarily explained by the size of the lesion in BA 10. This correlation between left BA 10 lesion volume and performance is consistent with the linguistic nature of the task. Nevertheless, since there was only one subject with strictly unilateral right frontopolar damage among our subjects, we cannot be certain about whether the effect is truly lateralized.</p>", "<p>Although the lesion size of the left BA 10 did not show a significant correlation with error rates in the dual-task condition (Spearman rank correlation R = 0.65, P = 0.16, non significant), it could be argued that the significant correlation observed between lesion size in the fronto-polar cortex and multi-tasking (R = 0.94, p&lt;0.005) does not prove that the effect is specific. However, the inference we tested was that the correlation coefficient was significantly different from zero in the multitasking condition and/or in the dual task condition, not that the correlation in the multitasking condition was significantly higher than the correlation in the dual task. Thus, it would only be statistically justified to compare correlation coefficients (between overlapping pair of variables) if both of these correlations coefficients were significantly different from zero (which is not the case). To further ensure the specificity of our findings, we tested the robustness of our results by recomputing the correlation coefficient in the multitasking and dual-task conditions for each possible n-1 subset of data sample (see <bold>supplementary ##SUPPL##0##tables S1## and ##SUPPL##1##S2##</bold>). These tables show that the significance of the correlation coefficient remains P&lt;0.05 in the multitasking condition and is non-significant in the dual-task condition. This demonstrates that variability of frontopolar cortex patients in multitasking is primarily explained by the size of the lesion in BA 10 and that the size of the left BA 10 lesion is a good anatomical predictor of multitasking but not of dual-task related errors.</p>", "<p>Although there may be many functional subregions within the frontopolar cortex, we believe that BA 10 is particularly important for multitasking. This process may be involved in a number of functions previously associated with the frontopolar cortex besides multitasking ##REF##10689059##[17]##, including integrating the outcomes of two or more separate cognitive operations in the pursuit of a higher behavioural goal ##REF##14976518##[18]##, processing of internally generated information ##UREF##2##[19]##, ##REF##14674837##[20]##, memory retrieval ##REF##8134341##[21]##, ##REF##9990104##[22]##, carrying out delayed intentions (prospective memory) ##REF##11257280##[5]##, ##REF##12667527##[6]##, relational integration ##REF##11697945##[23]##, ##REF##9212721##[24]##, ##REF##16750818##[25]##, integration of diverse information content ##REF##10607400##[26]## and exploratory decisions ##REF##16778890##[27]##.</p>", "<p>It should be noted that there is a fundamental qualitative difference between multitasking and the dual task. Multitasking combines not only a dual-task component but also a working memory component. It successively allocates processing resources between concurrent tasks, as in dual-task performance and it keeps relevant information in working memory to allow a return to the main task after completing a secondary task. In contrast to multitasking, which specifically involves the fronto-polar cortex, dual task performance induces higher inferior and middle frontal sulcus activity as compared to single task performance ##REF##12631562##[12]##, ##REF##11532885##[28]##, ##REF##16175414##[29]##, ##REF##12495525##[30]##. However, our results are not conclusive about other regions than the frontopolar cortex because we did not test patients with specific dorsolateral prefrontal cortex lesions.</p>", "<p>A general model, integrating the recent cascade model by Koechlin et al. ##REF##14615530##[31]## and the multi-tasking view of the fronto-polar cortex has recently been proposed in a review paper ##REF##17475536##[32]##. This general model explains at an information processing level, using information theory, what is called branching (renamed here as multitasking). The overview of this general model is that cognitive control operates according to three nested levels of control processes (contextual, episodic and multi-tasking) implemented from posterior to polar prefrontal regions. In this model, H(a) measures the total amount of control information required for selecting action “a” and is processed in the premotor cortex. H(a) is the sum of two control terms: bottom-up information conveyed by a stimulus S (I(s,a), sensorimotor control) and the remaining top-down information Q(a|s) processed in the posterior lateral PFC and measuring cognitive control. Cognitive control, in turn, is the sum of two control terms: bottom-up information conveyed by the context c in which stimulus s occurs (I(c,a|s), contextual control); and the top-down remaining information Q(a|s,c) processed in the anterior lateral PFC. Finally, this latter control term is the sum of bottom-up information conveyed by a past event u (I(u,a)|s,c), episodic control) and the remaining top-down information processed in the polar lateral PFC (multi-tasking control). Multi-tasking control is related to the information conveyed by events preceding <italic>u</italic> and maintained in a pending state until completion of the ongoing episode. Thus, according to this model, during execution of the current episode <italic>u</italic>, the most anterior portions of the PFC maintain (in a distractor-resistant fashion) pending information from a yet more temporally distant episode, enabling this information to be flexibly retrieved when this episode is re-instantiated. This model explains the pattern of prefrontal activations observed in several experimental paradigms, including learning ##REF##12160754##[33]##, episodic memory ##REF##9448256##[34]##, working memory ##REF##10834847##[35]## and task switching paradigms ##REF##11848695##[4]##, ##REF##12482070##[10]##, ##REF##12183390##[13]##, ##REF##15917482##[14]##. In these experiments, caudal and rostral LPFC activations were observed, depending on whether the executive control of behavior was based on contextual or episodic signals.</p>", "<p>A recent review, consistent with our interpretation of frontopolar function, is that a common process across these studies may be that the frontopolar cortex is recruited to integrate the results of two or more cognitive operations, fulfilling a higher behavioural goal ##REF##14976518##[18]##. This view predicts that the process of integration should be reflected in frontopolar activity beyond the activity observed for processing the component elements to be integrated. Confirming this prediction, a highly specific super-additive effect was previously demonstrated in the frontopolar cortex using fMRI when subjects held in mind goals while processing secondary goals at the same time ##REF##10335843##[3]##.</p>", "<p>To conclude, we have demonstrated that managing subgoals while maintaining information about primary goals is a process that is critically and selectively disrupted with increasing size of fronto-polar cortex damage. From an evolutionary point of view, it is interesting to note that during hominoid evolution, the frontopolar cortex (area 10) may have undergone not only a shift in its extent but also of its topographic location and a specific increase in connectivity with other higher-order association areas (the supragranular layers having more space available for connections with other higher-order association area) ##REF##11850633##[1]##, ##REF##11241188##[2]##. It has recently been proposed that the frontopolar cortex may be the only prefrontal region that is predominantly (and possibly exclusively) interconnected with supramodal areas in the prefrontal cortex and anterior temporal cortex ##REF##14976518##[18]##, allowing the frontopolar cortex to dynamically monitor and assign positional priority to information received from more caudal areas of supramodal cortex. Since the frontopolar cortex in the human brain appears to have evolved in size and organization, this suggests that complex functions requiring the temporary interruption of a current plan to achieve subgoals (such as planning of future actions and reasoning) associated with this part of the cortex have become particularly important during hominid evolution.</p>" ]
[]
[ "<p>Conceived and designed the experiments: JCD EK JG. Performed the experiments: JCD MT. Analyzed the data: JCD. Contributed reagents/materials/analysis tools: JCD. Wrote the paper: JCD JG.</p>", "<p>Current address: Cognitive Neuroscience Center, ‘Reward and decision making’ team, CNRS UMR 5229, Lyon, France</p>", "<p>Current address: INSERM U742, Paris VI University, Paris, France</p>", "<title>Background</title>", "<p>A major question in understanding the functional organization of the brain is to delineate the functional divisions of the prefrontal cortex. Of particular importance to the cognitive capacities that are uniquely human is the fronto-polar cortex (Brodmann's area 10), which is disproportionally larger in humans relative to the rest of the brain than it is in the ape's brain. The specific function of this brain region remains poorly understood, but recent neuroimaging studies have proposed that it may hold goals in mind while exploring and processing secondary goals.</p>", "<title>Principal Findings</title>", "<p>Here we show that the extent of damage to the fronto-polar cortex predicts impairment in the management of multiple goals. This result reveals that the integrity of the fronto-polar cortex is necessary to perform tasks that require subjects to maintain a primary goal in mind while processing secondary goals, an ability which is crucial for complex human cognitive abilities.</p>", "<title>Conclusion/Significance</title>", "<p>These results provide important new insights concerning the cerebral basis of complex human cognition such as planning and multitasking.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>We thank MK Colvin and K Detucci for assistance with patient recruitment.</p>" ]
[ "<fig id=\"pone-0003227-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003227.g001</object-id><label>Figure 1</label><caption><title>(A). Location and degree of lesion overlap in patients with fronto-polar cortex lesions.</title><p>(B). Location and degree of lesion overlap in control patients without fronto-polar cortex lesions. Slices are oriented in radiological convention (i.e. the left side of the image is the right hemisphere). Lighter colors denote the degree to which lesions involve the same structure in multiple subjects. The darker color at the bottom of the color scale indicates no overlap between brain region.</p></caption></fig>", "<fig id=\"pone-0003227-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003227.g002</object-id><label>Figure 2</label><caption><title>Reconstruction of lesions for each patient with frontopolar cortex damage (P1–P7) based on computerized tomography and MRI scans.</title><p>The shaded area represents the lesion. Axial slices from ventral (left) to dorsal (right). According to radiological convention right is left.</p></caption></fig>", "<fig id=\"pone-0003227-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003227.g003</object-id><label>Figure 3</label><caption><title>Reconstruction of lesions for each patient without frontopolar cortex damage (NFP1–NFP5) based on computerized tomography and MRI scans.</title><p>The shaded area represents the lesion. Axial slices are displayed in radiological convention from ventral (left) to dorsal (right).</p></caption></fig>", "<fig id=\"pone-0003227-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003227.g004</object-id><label>Figure 4</label><caption><title>Behavioural tasks.</title><p>Single-letters (upper or lower-case) from the word “tablet” were successively presented and subject's decisions were recorded using two single response-buttons, one for each hand. Delay condition: subjects decided whether two successively presented upper-case letters were also in immediate succession in the word “tablet” by pressing the right button for yes and the left button if they were not in succession, and they had to ignore lower case letters that were presented in order to delay the response required for upper-case letters. Dual-task condition: subjects decided whether two successively presented letters were also in immediate succession in the word “tablet” by pressing the right button for yes and the left button if they were not in succession, this time both for upper and lower case letters, except that they had to decide whether every first letter indicating a case change was the letter T (or t). Multi-tasking condition: subjects responded to upper case letters exactly as in the delay condition and to lower case letters exactly as in the dual task condition. Thus, the multitasking condition requires maintenance of the primary task information in memory (primary goal) so that it can be returned to after completing a secondary task (subgoal).</p></caption></fig>", "<fig id=\"pone-0003227-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003227.g005</object-id><label>Figure 5</label><caption><title>Behavioral performance.</title><p>(A). Graph representing responses times (for correct responses) in patients with fronto-polar cortex lesions (blue), in patients without fronto-polar cortex lesions (green) and in controls (red). (B). Percentage of errors in the three conditions (delay, dual and multi-tasking) in patients with fronto-polar lesions (blue), in patients without fronto-polar cortex lesions (green) and in normal controls (red). Error bars represent the 95% confidence intervals.</p></caption></fig>", "<fig id=\"pone-0003227-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003227.g006</object-id><label>Figure 6</label><caption><title>Relationships between lesion size and behavior.</title><p>(A) A positive significant correlation was observed between the percentage of errors in the multi-tasking condition and the percentage of damage to the left Brodmann's area 10 in patients with fronto-polar cortex lesions (Spearman rank correlation coefficient R = 0.94, P&lt;0.005). (B) No significant correlation was observed between the lesion sizes of the left BA 10 with error rate in the dual-task condition. (C) No significant correlation was observed between total volume of brain lesions and error rate in the multi-tasking condition.</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003227-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003227.t001</object-id><label>Table 1</label><caption><title>Demographic of patients and healthy control subjects.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Group</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Level of Educa-tion</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Sexr</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hand</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Time since lesion (years)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">WAIS-III Full Scale IQ</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">WMS - III Working Memory Index Score</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">WMS - III Auditory Immediate Index Score</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">WMS - III Auditory Recognition Delayed Index Score</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fluency Total Raw Score</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">BNT Total Raw Score</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">BDI-2 Total Raw Score</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NART Full IQ</td></tr></thead><tbody><tr><td colspan=\"14\" align=\"left\" rowspan=\"1\">\n<bold>FP Lesions</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FP1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">47</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">108</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">89</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">89</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FP2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">121</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">88</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">120</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">105</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">74</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">59</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FP3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">56</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">102</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">96</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">57</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FP4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">104</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">111</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">114</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">95</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">56</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FP5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">132</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">124</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">117</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">77</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FP6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">137</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">108</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">114</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">110</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">57</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FP7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">49</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">97</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">96</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">65</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">65</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td colspan=\"14\" align=\"left\" rowspan=\"1\">\n<bold>No FP lesion</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NFP1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">L</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">99</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">91</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">102</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">90</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">57</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NFP2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">101</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">77</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">95</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NFP3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">98</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">108</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">92</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">105</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">51</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NFP4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">118</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">115</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">74</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">85</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NFP5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">38</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">93</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">108</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">110</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">38</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">58</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td colspan=\"14\" align=\"left\" rowspan=\"1\">\n<bold>Controls</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">110</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">63</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">112</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">L</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">102</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">91</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">121</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">112</td></tr></tbody></table></alternatives></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003227.s001\"><label>Table S1</label><caption><p>Correlation coefficient in the branching condition for each possible n-1 subset of data sample.</p><p>(0.03 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003227.s002\"><label>Table S2</label><caption><p>Correlation coefficient in the dual-task condition for each possible n-1 subset of data sample.</p><p>(0.02 MB DOC)</p></caption></supplementary-material>" ]
[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>JG was funded by the National Institute of Neurological Disorders and Stroke and JCD was funded by the Fyssen Foundation. This research was in part supported by the Intramural Research Program of the NIH, NINDS.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"pone.0003227.g001\"/>", "<graphic xlink:href=\"pone.0003227.g002\"/>", "<graphic xlink:href=\"pone.0003227.g003\"/>", "<graphic id=\"pone-0003227-t001-1\" xlink:href=\"pone.0003227.t001\"/>", "<graphic xlink:href=\"pone.0003227.g004\"/>", "<graphic xlink:href=\"pone.0003227.g005\"/>", "<graphic xlink:href=\"pone.0003227.g006\"/>" ]
[ "<media xlink:href=\"pone.0003227.s001.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003227.s002.doc\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["8"], "element-citation": ["\n"], "surname": ["Damasio", "Press"], "given-names": ["AR", "OU"], "year": ["1995"], "article-title": ["Brain anatomy in computerized images."], "publisher-loc": ["New York"]}, {"label": ["9"], "element-citation": ["\n"], "surname": ["Pallier", "Dupoux", "Liddle", "Frackowiak"], "given-names": ["C", "E", "PF", "R"], "year": ["1997"], "article-title": ["EXPE: an expandable programming language for on-line psychological experiments."], "source": ["Behav Res Methods Instrum Comput"], "volume": ["29"], "fpage": ["322"], "lpage": ["327"]}, {"label": ["19"], "element-citation": ["\n"], "surname": ["Christoff", "Gabrieli"], "given-names": ["K", "JDE"], "year": ["2000"], "article-title": ["The frontopolar cortex and human cognition: evidence for a rostrocaudal hierarchical organization within the human prefrontal cortex."], "source": ["Psychobiology"], "volume": ["28"], "fpage": ["168"], "lpage": ["186"]}]
{ "acronym": [], "definition": [] }
35
CC0
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 16; 3(9):e3227
oa_package/fb/91/PMC2528949.tar.gz
PMC2528950
18800167
[ "<title>Introduction</title>", "<p>Hepatitis C virus (HCV) infection affects about 170 million people worldwide, about 3% of the world's population ##REF##16122679##[1]##, and is the major cause of liver disease and a potential cause of substantial morbidity and mortality in the future ##REF##15483230##[2]##. Hepatitis C virus is the only species of the genus <italic>Hepacivirus</italic> within the family <italic>Flaviviridae</italic>. It has a single stranded, positive-sense, nonsegmented RNA genome of about 9600 nucleotides (nt) with a single, long open reading frame encoding a polyprotein of about 3000 amino acids with the gene order C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The structural proteins are C (core) and E1 and E2 (envelope glycoproteins). The function of the p7 product is presently unknown. The NS2 through NS5 regions encode the non-structural proteins ##REF##12827459##[3]##.</p>", "<p>Six major HCV genotypes and about 50 subtypes have been described ##REF##8245854##[4]##, ##REF##16149085##[5]## based on levels of sequence divergence. HCV genotypes have been shown to be distributed over distinct geographical areas and although they share most basic biological features, there seems to be some differences in their susceptibility to interferon (IFN)-based therapies ##REF##14996676##[6]##, ##REF##15751759##[7]##. Genotype 1 is the predominant variant in developed countries and shows the poorest response to therapy. Patients with genotypes 2 and 3 are also common in Europe, although with a lower frequency than genotype 1, and show the best response to IFN therapy.</p>", "<p>HCV is mainly transmitted by parenteral routes and differences in their transmission rates can be an important factor to explain the differences in prevalence of a genotype/subtype in different geographic regions ##REF##15639745##[8]##–##REF##17170444##[10]##. Needle sharing among intravenous drug users (IDUs) currently represents the most common route of acquisition of HCV in the developed world ##REF##11439948##[11]##. Because HCV and human immunodeficiency virus (HIV) share blood-borne transmission routes, HIV/HCV co-infection is relatively frequent, especially in regions such as Spain, where the major proportion of newly diagnosed AIDS patients belong to the IDU category (44.2%) ##UREF##0##[12]##.</p>", "<p>Like HIV-1, HCV is characterized by high levels of genetic heterogeneity ##UREF##1##[13]##, ##REF##9018053##[14]## which impact heavily on different aspects such as HCV persistence, susceptibility to treatment, progression of infection, among others ##REF##10755999##[15]##–##REF##10895435##[17]##. Although it is well known that recombinant forms of HIV-1 have a relatively high prevalence all over the world ##UREF##2##[18]##, there has been limited evidence of HCV recombination between different genotypes/subtypes ##REF##8818959##[19]##, suggesting that these events are rare <italic>in vivo</italic> and that the resulting recombinants are usually not viable ##REF##9018053##[14]##, ##REF##8175159##[20]##, ##REF##10697786##[21]##. In the last few years, a few natural intergenotypic recombinants of HCV have been identified (RF1_2k/1b, RF2_1a/1b and RF3_2b/1b) and the crossover points have been mapped to the NS2, NS5B and NS3 regions, respectively ##REF##11907242##[22]##–##REF##16998890##[25]##. Very recently, a new natural intergenotypic (2/5) recombinant of HCV has been found whose crossover point is located between genes NS2 and NS3 ##REF##17267503##[26]##. All these reports have described HCV recombination between different genotypes/subtypes but, to date, there is only a single case of an HCV intra-subtype recombinant strain, detected by analysis of NS5A sequences from intra-patient populations belonging to six patients undergoing anti-viral therapy ##REF##17062150##[27]##.</p>", "<p>Given the important role that recombination seems to play in the evolution of RNA viruses ##REF##10573145##[28]##, ##UREF##3##[29]##, by creating genetic variation, and the important implications that the production of new pathogenic recombinant strains could have, for example, on the development of vaccines to control RNA viruses, our aim in this study has been to assess the extent and, eventually, the frequency of intragenic recombination on HCV. For this, we have retrospectively analyzed a large data set (over 17700) of HCV sequences from intra-patient viral populations. These sequences were obtained from two separate studies of our group none of which was specifically designed for this objective ##REF##17170444##[10]##, ##REF##17622623##[78]##, ##UREF##8##[79, and unpublished results]##. One included only HCV-monoinfected patients and the other HCV/HIV coinfected patients, with a common genome region for both studies (E1-E2 region) and another, the NS5A region, analyzed only in the former study. Both studies included treatment-naïve and patients non-responding to antiviral treatment. We found evidence of intrapatient recombination in HCV sequences from over 10% of the patients thus revealing that recombination in HCV can be a much more common phenomenon than previously recognized. The possibility of this result arising from artifacts during the experimental procedure has been considered by performing an “ad hoc” experiment in which serum samples from two closely related, but clearly differentiated patients were mixed in equal proportions and the resulting mixture was subjected to the same experimental procedure used in the previous analyses. No evidence of artifactual recombination was found.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Patients and Samples</title>", "<p>136 serum samples from 111 HCV-infected patients were analyzed in this study. Patients belonged to two different groups: (i) infected only with HCV, either treated with IFN-α plus ribavirin (denoted HCV T) or treatment naïve (HCV 0), and (ii) HCV-HIV co-infected patients with (HCV 0-T) and without (HCV 0-0) highly active antiretroviral treatment (HAART) against HIV. Samples from the former group were included in a molecular epidemiology study of HCV in the Comunidad Valenciana and have been described in detail elsewhere ##REF##17170444##[10]##, ##REF##17622623##[78]##, ##UREF##8##[79]##. Samples from the second group were obtained from the Hospital General de Valencia (Valencia, Spain) and informed, written consent was obtained from all the patients. Both studies were approved by the corresponding ethics committees of the institutions involved. Treatment response for all HCV treated patients is shown in ##SUPPL##0##Table S1##.1 (Supplementary Material). For non-responder patients from the HCV T group a second serum sample taken after interruption of treatment (6 or 12 months after its start) was available and included in the study.</p>", "<p>HCV genotyping was initially performed by a commercial reverse hybridization genotyping assay (Inno-LIPA HCV II; Innogenetics) and later confirmed by nucleotide sequence comparison in the analyzed genome regions. Genotype 1b represented 61.5% of the total HCV-monoinfected patients analyzed, and genotype 1a the remainder 38.5% whereas for HCV/HIV-coinfected patients, the frequency of the different HCV genotypes were 39.4%, 30.3%, 3.0%, 18.1% and 9.1% for genotypes 1a, 1b, 2b, 3a and 4, respectively.</p>", "<p>Two HCV genome regions were considered in this study. The first one corresponds to a fragment encompassing the genes encoding envelope glycoproteins E1 and E2. This fragment spanned from positions 1322 to 1793 in the HCV reference genome sequence [GenBank accession no. AF009606, 80] for HCV mono-infected samples (472 nt) and up to position 1855 in HCV-HIV co-infected samples (534 nt). This region will be referred to as “E1–E2 region”. The second region corresponded to a 743 nt fragment from gene NS5A (positions 6742–7484 in the HCV reference genome), referred to as “NS5A region”.</p>", "<p>These two genome fragments were chosen because of the biological relevance of the regions included therein. On the one hand, three hypervariable regions are included in the E1-E2 region: HVR1, which seems to be involved in target cell recognition and virus attachment ##REF##11356980##[81]##; HVR2, which could be involved in cell surface receptor binding ##REF##10861927##[82]##; and HVR3, which could play a role in the process of binding with host cell receptors and virus entry into host cells ##REF##16781757##[83]##. On the other hand, two remarkable domains are included in the NS5A region: the V3 domain, seemingly involved in responsiveness to interferon ##REF##9634077##[70]##, ##REF##12629645##[84]##, and PKR-BD, which contains the putative interferon sensitivity determining region (ISDR) and seems to be involved in blocking the cellular antiviral response induced by interferon ##REF##7542279##[61]##, ##REF##8531962##[85]##–##REF##9593328##[87]##.</p>", "<title>RNA Extraction, cDNA Synthesis and Amplification</title>", "<p>Virus RNA was extracted from 200 µl serum by using a High Pure Viral RNA kit (Roche). Reverse transcriptions were performed in a 20 µl volume containing 5 µl eluted RNA, 4 µl 5× RT buffer, 0.5 mM each deoxynucleotide, 0.5 µl random hexamers, 100 U Moloney murine leukaemia virus reverse transcriptase (Promega) and 20 U RNasin ribonuclease inhibitor (Promega). The reactions were incubated at 42°C for 45 min, followed by 3 min at 95°C.</p>", "<p>A first PCR round was then carried out in a 100 µl volume containing 10 µl of the reverse transcription product, 0.2 mM each dNTP, 400 nM genomic primer, 400 nM antigenomic primer and 1.25 U <italic>Pfu</italic> DNA polymerase (Promega). For the first set of samples, i.e. those obtained from HCV-monoinfected patients, we used the primers detailed in ##TAB##0##Table 1## of ##UREF##9##[88]## unless specified otherwise. These primers yielded a 472 nt fragment for the E1–E2 region, while a 543 nt fragment was obtained in this region from HCV/HIV coinfected samples using primers 1-Em1 (<named-content content-type=\"gene\">5′-CGCATGGCHTGGRAYATGAT</named-content>), 1-Em2 (5′-GGRATATGATRAATGAAYTGGTC) and 1-Em1 (<named-content content-type=\"gene\">5′-GGRGTGAARCARTAYACYGG</named-content>) for genotypes 1a, 1b, 2b and 4, and primers 3-Eg1 (<named-content content-type=\"gene\">5′-CGWATGGCTTGGGAYATGAT</named-content>), 3-Eg2 (<named-content content-type=\"gene\">5′-GGGAYATCATGATGAAYTGGT</named-content>), 3-Ea1 (<named-content content-type=\"gene\">5′- GGRGTRAAGCAGTABACRGG</named-content>) for genotype 3. For region NS5A, subtype 1a: 1-Ng1, 2-Ng1, 1-Ng2, 2-Ng2, 1-Na and 2-Na. For the NS5A region we used primers Ng1 (<named-content content-type=\"gene\">59-TGGAYGGRGTRCGGYTGCACAGGTA</named-content>), Ng2 (<named-content content-type=\"gene\">59-CAGGTACGCTCCRGYRTGCA</named-content>) and Na (<named-content content-type=\"gene\">59-CCYTCRAGGGGGGGCAT</named-content>), which yielded a 743 nt fragment. This region was analyzed only in HCV-monoinfected samples. In all cases, PCRs were performed in an Applied Biosystems 2400 thermal cycler as described ##UREF##9##[88]##.</p>", "<title>Cloning and Sequencing of Viral Populations</title>", "<p>Amplified DNA products for each region were purified and cloned directly into <italic>Eco</italic>RV-digested pBluescript II SK(+) phagemid (Stratagene). Cloned products for the E1–E2 region or NS5A region were sequenced by using vector-based primers KS and SK (Stratagene).</p>", "<p>Sequencing was carried out by using the ABI PRISM BigDye Terminator v3.0 system (Applied Biosystems) on an ABI 3700 automated sequencer. Sequences were verified and both strands were assembled using the STADEN package ##UREF##10##[89]##. HCV sequences obtained in this study have been deposited in GenBank and the corresponding accession numbers are shown on ##SUPPL##0##Table S1## in the supplementary material along with the numbers of previously determined sequences ##REF##17170444##[10]##, ##REF##17622623##[78]##, ##UREF##8##[79]##.</p>", "<title>Sequence and Phylogenetic Analyses</title>", "<p>Sequence alignments were obtained with CLUSTALX v1.81 ##REF##9396791##[90]##. Optimal models of nucleotide substitution were assessed using the maximum likelihood approach implemented in Modeltest v3.7 ##UREF##11##[91]##. Likelihood scores for each model were estimated in PAUP*4.0b10 ##UREF##12##[92]## and the best model was determined using the Akaike Information Criterion (AIC) ##UREF##13##[93]##. Maximum likelihood phylogenetic trees were obtained with PHYML 2.4.4 ##REF##14530136##[94]## using the previously determined models of nucleotide substitution for each genome region and sample, and support for the nodes were evaluated by bootstrapping with 1000 pseudorreplicates.</p>", "<title>Intrapatient Recombination</title>", "<p>Putative recombination events in intrapatient sequence alignments of the two genome regions were detected using RDP 3.0b03 ##REF##15377507##[95]##. This program implements several methods for the identification of recombinant sequences and recombination breakpoints. We choose six of them: two phylogenetic methods, which infer recombination when different parts of the genome result in discordant topologies, RDP ##REF##10980155##[96]##; and Bootscanning ##REF##8573403##[97]##; and four nucleotide substitution methods, which examine the sequences either for a significant clustering of substitutions or for a fit to an expected statistical distribution: Maxchi ##REF##1556748##[98]##, Chimaera ##REF##1556748##[98]##, GeneConv ##REF##10600594##[99]## and Sis-scan ##REF##11038328##[100]##.</p>", "<p>We only considered recombination events that were identified by at least three methods. Common settings for all methods were to consider sequences as linear, to require phylogenetic evidence, to polish breakpoints and to check alignment consistency. Statistical significance was set at the P&lt;0.05 level, after considering Bonferroni correction for multiple comparisons as implemented in RDP. Consensus daughter sequences and breakpoints were determined whenever possible.</p>", "<p>In order to test the phylogenetic congruence of the two ML trees derived from each of the segments identified by the recombination breakpoints reported, we used TreePuzzle v.5.2 ##REF##11934758##[101]## to compare both phylogenetic trees using the SH ##UREF##14##[102]## and the ELW tests ##UREF##15##[103]##.</p>" ]
[ "<title>Results</title>", "<p>Sequences used in this work were derived from two previous studies on HCV genetic variation before and after antiviral treatment. One study included HCV-monoinfected patients whereas the other analyzed HCV/HIV-coinfected individuals. None of these studies was designed with the goal of detecting recombination in HCV. In both cases the E1-E2 region of the HCV genome was analyzed by sequencing viral clones and the former study also included clones from the NS5A region. We obtained sequences from the E1-E2 region from 110 patients and a total of 136 samples (##TAB##0##Table 1##), since two samples (before and after antiviral treatment) were available for 26 patients, all from the HCV-monoinfection study. For the NS5A region we obtained cloned sequences from 78 patients and a total of 98 samples, since amplification failed for 4 and 1 samples from the pre- and post-treatment groups, respectively.</p>", "<p>The average number of clones sequenced for each region, group and patient is described in ##TAB##0##Table 1##. For the E1-E2 region, 11746 cloned sequences were obtained and average number of sequenced clones per sample for the treatment-naïve mono-infected group (HCV 0) was 105.74±21.43 whereas the average for HCV treated mono-infected patients who did not respond to a combined antiviral treatment with IFN-α plus ribavirin (HCV T) was 106.15±5.24. In the case of HCV/HIV coinfected patients, an average of 28.69±3.86 and 28.88±7.32 E1-E2 viral sequences were obtained for treatment naïve (HCV 0-0) and HIV treated patients (HCV 0-T), respectively. The data set for the NS5A region comprised 5966 clonal sequences. The average number of NS5A clones sequenced per sample for the non-treated group (HCV 0) was 63.33±18.51 and 53.72±18.00 for the antiviral treated group (HCV T).</p>", "<p>Putative recombination events between the viral strains infecting the same patient were found in 20 of the 111 patients studied (18.01%). These intragenic recombination events were detected in 25 of the 234 independent samples analyzed (10.7%). The detected recombination events belonged to HCV samples from all the infection groups described and the two genomic regions analyzed (E1-E2 and NS5A), as well as to different HCV subtypes (1a, 1b and 3a). Five events were detected among the E1-E2 sequences derived from HCV/HIV coinfected patients, 11 corresponded to events in the E1-E2 region from HCV-monoinfected patients, and the remaining 9 were detected in the NS5A region. No differences between the recombination frequencies from the two genomic regions analyzed were found neither for treatment-naïve (Mann-Whitney test: z = −0.104, p-value = 0.917) nor for interferon plus ribavirin treated groups (Mann-Whitney test: z = −0.810, p-value = 0.418). ##FIG##0##Figures 1##–\n\n##FIG##3##4## show in detail the results of recombination analyses only for samples in which the presence of putatively significant recombination events was detected. Intragenic recombination analyses were performed for each locus independently using 6 different methods for the detection of recombination implemented in the program RDP 3.0. We considered as significant recombination events only those for which the corrected probability for simultaneous inference of the event was lower than 0.05 and were significantly detected by at least 3 of the 6 methods used.</p>", "<p>A large proportion (84%, 21/25) of the samples in which recombination was detected included more than one recombination event (##FIG##0##Figures 1##–\n\n##FIG##3##4##). Each recombination event detected in a sample was described according to the following features: i) average p-value for the event and analysis method, ii) most likely parental and daughter sequences as well as similarity among them, and iii) most likely limits for the location of breakpoint(s) in the sequenced fragment.</p>", "<p>We found 46 single recombination events (17 in the NS5A and 29 in the E1-E2 region) and 12 double recombination events (3 in the NS5A and 9 in the E1-E2 region) in the 25 alignments where recombination was detected. A single recombination event was defined by a single, significant breakpoint detected in the daughter sequence following the described methodology, while a double event was defined as two significant breakpoints identified in the same daughter sequence, delimiting the extension of the recombinant fragment. When one of the parental sequences implicated in an event could not be inferred within the sequence alignment, it was denoted as “unknown parental”.</p>", "<p>Among the methods used, those detecting a larger number of significant events were Siscan, Chimera, and Maxchi, followed by Genconv and the phylogenetic methods, Bootscan and RDP, which detected very few events.</p>", "<p>Maximum likelihood phylogenetic trees were constructed for the two regions delimited for each single breakpoint event identified. For the double events detected, three maximum likelihood phylogenetic trees were constructed; one was derived from the alignment for the region involved in the recombinant segment (delimited by the two breakpoints detected) and the others from the two resulting flanking segments. Two different tests, Shimodaira–Hasegawa (SH) and Expected Likelihood Weight (ELW), were applied to the two or three resulting topologies for each single or double event, respectively, to further verify the results obtained with RDP3. Tests for double events were made between the recombinant segment topology and each of the two topologies derived from the flanking segments independently. These tests resulted in significant differences between both topologies in all single and double event cases (detailed results of the tests are available in the Supporting Information ##SUPPL##1##Table S2##).</p>", "<title>Recombination in the E1-E2 Region</title>", "<p>Statistically significant recombination events were detected by at least three methods in 11.8% (16/136) of the amplified samples in the E1-E2 region. The total number of events identified in these recombinant samples was 38, corresponding to 29 single and 9 double events, belonging to 15 patients from all the studied groups. The breakpoints detected were located mainly in the segments flanking the HVR1 (##FIG##4##Figure 5##). No differences in the distribution of breakpoints were found between the different studied groups, viral subtypes or treatment response. The frequency of double recombination events detected in this region was 23.7% (9/38) and the average length of the derived recombinant fragments was 147.5 nt (ranging from 66.5 to 212 nt), representing between 27.2% and 31.2% of the total region sequenced (543 nt for HCV/HIV co-infected and 472 nt for HCV single-infected patients). In 8 of the 9 double recombination event cases the derived recombinant regions comprised a large portion of the complete HVR1, while in the remaining case the recombinant fragment involved the entire HVR3 region (##FIG##4##Figure 5##).</p>", "<p>In the analysis of HCV-monoinfected samples (103 E1-E2 amplified samples from 78 patients), the frequency of significant recombination cases was 9.1% (7/77) for treatment-naïve patients (HCV 0) and 15.4% (4/26) for IFN-α plus ribavirin treated patients (HCV T, ##TAB##0##Table 1##). All samples in which recombination events were detected were derived from patients infected with genotype 1b of HCV, including both responders and non-responders to antiviral treatment, with the only exception of a sample from an untreated patient infected with genotype 1a. The frequencies of samples with detected recombination events among coinfected patients were 12.5% (2/16) and 17.64% (3/17) for treatment-naïve (HCV 0-0) and HAART-treated (HCV 0-T) patients, respectively (##TAB##0##Table 1##). These five samples were obtained from individuals infected with genotype 3a (3 patients) and genotype 1b (2 patients) of HCV. No significant differences were found in the frequencies of recombination-positive samples from naïve and treated groups for HCV mono-infected and HCV/HIV co-infected patients (Mann-Whitney test for single infected patients: z = −0.894 p-value = 0.371; and z = −0.406 and p-value = 0.685, for co-infected patients), although a larger proportion of recombination events was detected among samples from treated patients both in single-infected and in co-infected individuals. Similarly, no differences were found between HCV/HIV co-infected and HCV single infected, treatment-naïve groups (z = −0.417, p-value = 0.676). Complete Mann-Whitney tests results are available in ##SUPPL##2##Table S3## of the Supporting Information.</p>", "<p>\n##FIG##5##Figure 6## shows an example of a recombination event detected by phylogenetic analysis in the E1-E2 region reflected in the incongruence (reciprocal tests with SH and ELW were highly significant, see Supplementary material) between the maximum likelihood trees derived from the two regions (delimited by nucleotides 1–264 and 265–534, respectively) defined by the breakpoints assigned to the recombination event from sample EC5703. Variable positions in the alignment of the viral sequences involved in this recombination event are shown in ##FIG##6##Figure 7##, marking the recombinant parental sequences involved in the detected recombination event.</p>", "<title>E1-E2 Region. Analysis of Two Time-Point Samples</title>", "<p>Two samples were available for 24 HCV-monoinfected patients who did not respond to antiviral treatment. One sample was taken before the onset of treatment (T0 sample) and the second one was obtained when it was discontinued, 6 or 12 months later (T1 sample). For these cases an additional analysis was performed by simultaneously considering all the sequences obtained from both samples. We detected significant recombination events in 4 patients, C29, A21, G16 and G26.</p>", "<p>One recombination event was detected in the joint analysis of the two samples from patient C29 (C29T0-T1).The parental sequences from this event were derived one from the T0 sample (before treatment) and the other from the T1 sample (taken 6 months later), while the daughter sequences were detected only in the T0 sample. The same event, involving the same daughter sequences, was also detected in the analysis of sequences obtained only from the T0 sample, although in this case only one parental sequence was identified while the other was described as “unknown parental” in the analysis (##FIG##0##Figure 1##).</p>", "<p>Similarly, in the joint analysis of the two samples from patient A21, we detected one significant recombination event whose corresponding breakpoint was located between positions 210 and 214. This event was originated by two parental sequences from the T0 sample while the resulting daughter sequences from such event (A21_T1-47 and A21_T1-80) corresponded to sequences from the sample obtained after six months of unsuccessful HCV antiviral treatment (##FIG##3##Figure 4##). The same event was detected in the analysis of sequences derived only from the T1 sample but, obviously, the identified parental sequences belonged also to this sample. However, when the parental similarities for both events (the one detected only with T1 sequences and the other joining T0 and T1 sequences) were compared, the similarity between daughter and the corresponding parental sequences for both cases were lower in the T1 sample (97.4 and 99.4% for the two putative parental sequences) than in the joint T0-T1 analysis (98.4 and 100%). In consequence, T1 daughter sequences derived from this recombination event had been more likely generated by parental sequences from the T0 sample than from those in the T1 one. This would imply that the recombination event occurred at the earlier time point and the resulting daughter sequences were able to persist in the viral population six months later and under antiviral treatment. Finally, in the case of patients G16 and G26, all significant events detected in the joint analysis involved sequences from the same time-point and were also detected in the analysis of only this sample.</p>", "<title>Recombination in the NS5A Region</title>", "<p>Significant events of recombination were detected by at least three methods in 9.18% (9/98) of the amplified samples for the NS5A region. The total number of recombination events detected in these samples was 20, with 17 single and 3 double events, and they were identified in samples from 8 patients, all infected with HCV genotype 1b, including treatment-naïve and non-responder patients. The breakpoint(s) detected for each event were located mainly in a segment flanking the PKR-BD (protein-kinase binding domain) and within the ISDR (interferon sensitivity-determining region) regions (##FIG##7##Figure 8##). The frequency of double events detected in this region was 15% (3/20) and the average recombinant fragment length was 315 nt (ranging from 305 to 337 nt) corresponding to the 42.4% of the total sequenced region (743 nt). In 2 of the 3 double event cases, the recombinant region comprised the complete PKR-BD region, including also the ISDR, while in the other case the recombinant region involved only the non ISDR fragment of PKR-BD region (##FIG##7##Figure 8##). No differences were observed in the distribution of breakpoints between treated and non-treated samples or between treatment responses. Similar proportions of recombination events were detected in samples from treatment-naïve patients (9.6%, 7/73) than from IFN-α plus ribavirin treated patients (8.3%, 2/24) and no significant differences due to the HCV treatment were found (z = −0.236 p-value = 0.813). ##FIG##8##Figure 9## presents an example of recombination event detected in the NS5A region from the T0 sample of patient G08. In this case, the event corresponded to a single recombination breakpoint located between positions 345 and 420 of this fragment. Maximum likelihood trees obtained for each region using the GTR model of evolution showed a clear phylogenetic incongruence between the two derived region trees (##SUPPL##1##Table S2## and ##SUPPL##3##File S1## in Supplementary material). Variable positions in the alignment of the viral sequences involved in this recombination event are shown in ##FIG##9##Figure 10##.</p>", "<title>NS5A Region: Analysis of Two Time-Points Samples</title>", "<p>Two samples, one taken before (T0) and the other after unsuccessful antiviral treatment (6 or 12 months later, T1 or T2), were available for 24 HCV-monoinfected patients. The joint analysis of sequences from the two samples of each patient resulted in the detection of recombination events in only two cases (8.33%), for patients C29 and G07.</p>", "<p>The joint analysis of samples from patient C29 revealed several recombination events that involved parental sequences from samples T0 and T1 and daughter sequences only from the T0 sample. All these events were also detected in the analysis of the T0 sample but, again, with presumed parental sequences from this sample time point.</p>", "<p>For patient G07, different recombination events in the joint analysis of sequences from samples T0 and T2 were found. Two of these events were particularly interesting because they involved daughter sequences from the T2 sample with inferred parental sequences from the T0 sample which was obtained one year earlier. These parental sequences (T0-66 and T0-55) were involved in other recombination events detected in the T0 sample and the T2 resulting daughters (except T2-52, only detected in the joint analysis), T2-58 and T2-76 were detected in the T2 sample too, but parental inferred sequences for these events had lower similarity or were unknown (see ##FIG##2##Figures 3## and ##FIG##3##4##). Similarly to the previously commented case of E1-E2 sequences from two time-points samples of patient A21, the T2 daughter sequences obtained from this event were more likely generated by parental sequences from the T0 sample. Again, this indicates that the recombination event most likely took place at T0 and the resulting daughter sequences were able to persist for one year under HCV treatment.</p>", "<title>Joint Analysis of Recombination in the E1-E2 and NS5A Regions</title>", "<p>We obtained sequences for the E1-E2 and NS5A regions from 73 patients. These were all from the HCV monoinfected, non-treated group (HCV 0). Given the observed frequency of recombination events in the two analyzed regions, we calculated the expected probability of detecting recombination events simultaneously in both genome regions for the same patient. The frequency of samples with significant recombination events detected was 9.1% (7/77) and 9.6% (7/73), for the E1-E2 and NS5A regions, respectively (##TAB##0##Table 1##). Hence, the expected frequency of obtaining a patient with recombination events detected in the two genomic regions if these events were independent was 0.87%, whereas the observed frequency was 4.1% (3/73), 4.45 times higher than the expected value. Fisher's exact probability test resulted in a significant value with p = 0.03.</p>", "<p>We have also compared the frequencies of the recombinant sequences with those of the other sequence clones. A summary of these results is shown in ##TAB##1##Table 2##. While the frequencies of detected recombinant sequences varied widely, from a minimum of 0.003 to a maximum of 0.516, none of the recombinant sequences was present in more than 2 copies in the corresponding population, which corresponded to frequencies from 0.001 up to 0.074. Nevertheless, these figures must be compared with those corresponding to the frequency of the most common variant in the corresponding population, which varied between 0.030 and 0.20. In the joint analyses of two samples from the same patient in which recombinant sequences were detected, the highest frequency of a recombinant haplotype was 0.010. The most common sequence in this patient was only three times larger (0.031) but the largest number of identical sequences (111) and the highest haplotype frequency (0.547) were found in this group.</p>", "<title>Experimental Analysis of Artifactual Recombination</title>", "<p>In order to evaluate the level and extent of artifactual generation of recombinant sequences during the reverse transcription and PCR amplification, we performed an additional experiment using the same conditions previously described. The only difference was the starting sample used which, in this case, was a mixture of sera derived from two patients. These were chosen to maximize the possibility of detection of recombinant sequences, which requires clearly differentiated viral populations, while simultaneously maximizing the possibility of contiguous pairing enabling RNA polymerase shifting from one template to another while keeping the same position and reading-frame. This was achieved by selecting two patients HCV-1b infected from a common source who had within-patient nucleotide diversities of 0.0046 and 0.0007, respectively, with a net nucleotide differentiation of 0.0247, as estimated from a previous analysis of 10 clones from each sample (unpublished results).</p>", "<p>To ensure equimolar amounts of viral RNAs from both samples in the final mixture, we set up several mixtures with varying amounts of each sample that were subjected to the same RT and PCR-amplification procedures for the E1-E2 region described above. The resulting amplificates were directly sequenced and one mixture with equal peak heights in the automated sequencer electrophoregram in the polymorphic positions was chosen for further analysis. We cloned and sequenced PCR products from the selected mixture as described, obtaining 142 sequences. These were analyzed using the 6 methods implemented in RDP as described above and none of them detected any putative recombination event.</p>" ]
[ "<title>Discussion</title>", "<p>Several studies have reported recombination in different Flaviviruses ##REF##11413291##[30]##–##REF##12560576##[33]##, but until recently no evidence for recombination in natural populations of HCV had been found. Since the first identification of an intergenotypic (2k/1b) HCV recombinant in St. Petersburg ##REF##11907242##[22]##, several intergenotypic and intragenotypic HCV recombinant strains have been identified ##REF##15218169##[23]##–##REF##17267503##[26]##, ##REF##16840336##[34]##, ##REF##17107614##[35]## therefore incorporating recombination as a mechanism generating genetic variation in HCV. More importantly, the identification of these recombinant strains demonstrates that HCV is capable of successfully completing all the stages (simultaneous infection of the same cell, simultaneous replication of both viral genomes, template shift by the viral RNA polymerase while keeping the correct reading frame, encapsidation and release of the recombinant genomes) in the process. The resulting products will then be subjected to the same population processes governing the maintenance, expansion or disappearance of new variants in a heterogeneous viral population.</p>", "<p>All these reports have focused on HCV recombination between different genotypes/subtypes, but to date, there is only one single case of putative HCV intra-subtype recombinant strain, detected by the analysis of NS5A sequences from six intra-patient populations undergoing antiviral therapy ##REF##17062150##[27]##. In the present study we have identified a high frequency of intrasubtype recombination events (18.01% of the analyzed patients 20/111) analyzing a large data set of HCV sequences from intra-patient viral populations obtained from patients belonging to different groups: HCV-monoinfected patients, naïve and non-responding to antiviral treatment, and HCV/HIV-coinfected patients, treatment-naïve and under HAART. The relevance of recombination in HCV for its long-term evolution and its incidence in different aspects of HCV infection have not been explored yet, but these findings support a potentially significant role for recombination in the evolution of HCV by creating genetic variation through the reshuffling of independently arisen variants.</p>", "<p>Although these studies have firmly established the possibility of recombination in HCV, as in other Flaviviruses, no general mechanism has been proposed yet (but see below), and despite extensive analysis of genetic variation in HCV there has been only one report of recombination between strains from the same subtype. Hence, it seems adequate to start discussing our findings in terms of them being real or an artefact and, since we naturally accept the first option, why it has been so difficult to detect.</p>", "<p>The first question is how to discard the possibility that the detected recombination events were false positives, resulting from PCR-mediated recombination, especially since some experiments have failed to experimentally induce and detect recombination in HCV ##UREF##5##[36]##. We have performed one further such experiment, also with negative results. This is no proof of absence of artifacts in our experimental results, but they clearly indicate that, if present, recombinant sequences arising from the experimental procedures cannot account for the reported results. This conclusion is based on the following arguments, derived from considering different possible estimates of the artifactual recombination rate when no such event has been observed. If no false recombination event is observed among 143 clones then the probability of any such event must be lower than 1/143 (0.0075). With this upper limit, which would be our worst case scenario, under a Poisson distribution we would have expected to observe no false recombination events in 138.5 of the 234 independent samples analyzed with an average size of 75 sequences each. The actual number of samples with no recombinant sequences detected was 210; hence the presumed rate of artifactual recombination must be lower than 0.0075. If we had based our estimate of artifactual recombination rate on the number of negative cases (210), the inferred rate using again a Poisson distribution would be 0.0014. But with this rate it is not possible to account for the observation of 21 cases with two or more recombination events (##FIG##0##Figures 1##–\n##FIG##2##3##) nor of up to 9 events when sequences from the same patient taken at two different times were combined in a single analysis (##FIG##3##Figure 4##). Finally, should we consider the 58 events observed among 17712 sequences analyzed to be artifacts then we would expect 183 and 45 samples with none and one recombination event, respectively, while we obtained 210 and 3 cases. The discrepancy for cases with 2 or more events is even larger (5, 0.5 and 0, for 2, 3 and 4 events, while 13, 4 and 4 have been observed, respectively). In summary, the observed number and distribution of recombination events cannot be explained by artifacts during the experiments. Additional arguments for this conclusion are discussed next.</p>", "<p>There are two points in our experimental procedure that have been instrumental in obtaining the reported results and building our confidence in that they are not artifacts. First, instead of analyzing the full length master sequence of the viral distribution in each sample, we concentrated our efforts in sequencing a large number of clones in each of two genome regions. These were chosen on the basis of their biological relevance and not on the location of breakpoints identified in previous reports of recombination in HCV ##REF##15218169##[23]##–##REF##17267503##[26]##, ##REF##16840336##[34]##, ##REF##17107614##[35]##. Secondly, we minimized the chances of detecting false, artifactual recombination by using long extension times ##UREF##6##[37]## and a proofreading DNA polymerase (Pfu) ##REF##12153589##[38]## in our PCRs. Additional support for the actual occurrence of recombination events within HCV infected patients is provided by the following points: i) breakpoints or recombinant regions implicated in the recombinant events are not distributed at random along the analyzed genome regions (##FIG##4##Figures 5## and ##FIG##7##8##). On the contrary, the same breakpoints have been detected in HCV samples from different patients, indicating the presence of recombination hotspots, and entire biologically relevant regions are comprised within the recombinant fragments detected. ii) We have found evolution of the daughter sequences derived from a recombination event, with similarity to the parental sequences lower than the expected 100% if the recombination event was PCR-mediated. In addition, all recombination generated sequences preserve the polyprotein reading frame, which is not expected in PCR-mediated recombination where selection cannot purge deleterious mutants. iii) We have detected several recombination events in which the most likely parental sequences belonged to a previous time sample. Through the joint analysis of sequences from the two sample time points from a single patient, we have identified recombinant daughter sequences resulting from the cross-over between sequences sampled six or twelve months before under HCV treatment (see ##FIG##3##Figure 4##, results for A21_T0-T1 and G07_T0-T2). Parental and recombinant sequences were amplified and sequenced independently from the two different samples. Hence, these events probably occurred in the period between the two samples, and therefore the recombinant sequences were able to persist in the viral population under treatment and immunological selection pressures. iv) Finally, we have found patients with evidence for recombination events in the two genome regions analyzed 4.3 times more often than expected from the assumption of complete independence between both events. In PCR-mediated recombination we would expect a similar frequency of independent recombination events in both regions, but the joint expectation would be their product. A substantially larger value such as that observed might indicate the existence of some features, probably in the viral RNA-dependent RNA-polymerase (protein NS5B in HCV) that might either facilitate or prevent recombination, thus leading to the observed discrepancy.</p>", "<p>Intergenotype recombination in HCV has been related to homologous recombination during minus-strand synthesis via template switching ##REF##15218169##[23]##, although this proposal relies on the presence of two hairpin structures in the vicinity of the inferred breakpoint of recombination. Recombination breakpoints for the E1-E2 region were mainly located in the conserved region between hypervariable regions 1 (HVR1) and 3 (HVR3), and the recombinant fragments from the double recombinant sequences detected spanned the entire HVR1 or HVR3 regions. For the NS5A region, breakpoints were concentrated mainly at the end of the ISDR and the PKR-BD, and double recombinant fragments comprised the entire ISDR or PKR-BD regions. This distribution of breakpoints could be explained by the operation in HCV, like in other DNA or RNA viruses, of an intermolecular homologous replicative recombination system. This mechanism is associated with extensive nucleotide sequence identity between the two parental genomes around the cross-over site and copy choice or template switching during the replication process, that involves detachment from a template of the polymerase complex with a nascent product, and continuation of the copying process at the same position of another template molecule.</p>", "<p>This is the first study where recombination in HCV has been detected at the intrapatient intragenic level (the lowest possible level of diversity) by analyzing a large number of HCV sequences (17712) and samples (234) from 111 patients assigned to different clinical groups. The detection of recombinant strains in 18% of the HCV-infected patients studied implies that recombination events between the viral strains infecting the same patient may be relatively frequent, and still more if we consider that this might be an underestimate of the true frequency of HCV recombination because of the difficulty in detecting recombination events if they occur between genetically very similar variants of the same subtype or in conserved genome regions.</p>", "<p>The frequent detection of recombination events in all patient groups described makes the capability of HCV to produce recombinant forms not only relatively frequent but also effective and, depending on the recombinant strains produced, it might be selectively advantageous. However, we did not find any evidence for an increased frequency of recombinant sequences which might be explained by their presumed selective advantage. A more adequate analysis of positive selection on this same set of sequences does not show any indication of a selective advantage of these recombinant sequences (Sentandreu et al., in prep.).</p>", "<p>Given the previously reported results, a higher frequency of recombinant HCV strains than actually identified, with only 5 inter-genotypic and 1 intra-genotypic recombinants reported, might be expected. This might be explained by three different factors: Firstly, in recombination events between subtype viral strains, such as those reported here, there is a trade-off between the capability of homologous replicative recombination event to occur, which likely depend on the intrinsic recombination rate of HCV, and the intra-patient viral diversity, because homologous recombination requires a minimum length of sequence identity. Secondly, there is another trade-off between the intra-patient viral diversity and the power for the detection of recombination by the different methods used. Finally, recombination events between different genotypes/subtypes co-infecting the same patient are most probably easier to detect but, on the other hand, they are less likely to occur, given the higher differentiation between strains of different subtypes than those from the same subtype resulting in less likely template switching and, additionally, if a recombination event does happen it will likely generate recombinant sequences less viable than the parental ones. The action of some or even all these factors thus provides an explanation for the low frequency of recombinant HCV sequences reported up to date.</p>", "<p>We have detected recombination events in all the genotypes/subtypes analyzed. However, given the direct relationship between intrapatient genetic variability and our ability to detect recombination events, those viral populations with higher rates of intrapatient genetic variation are more likely to be involved in the detection of a recombination event if it ever occurs. This large genetic variation at the intrapatient level is usually associated to long persistent infections and/or to coinfection and superinfection with a strain from the same or a different subtype/genotype ##REF##12172410##[39]##–##REF##15378431##[41]##. Furthermore, there is increasing evidence for the presence of compartmentalization of HCV populations within infected patients ##REF##15113899##[42]##–##REF##17492592##[47]##, which would further facilitate lineage divergence within a patient. Despite some failed attempts to induce superinfection in cell cultures infected with HCV ##REF##17301154##[48]##, ##REF##17287280##[49]## this and previous reports of recombination in HCV clearly demonstrate that this process is not fully blocked, and more research is certainly necessary to establish under what circumstances and in which cellular types is superinfection with HCV more likely to occur.</p>", "<p>Our aim in this study has been to detect the presence of intragenic recombination and also to assess the extension and the frequency of these recombination events at the subtype level, analyzing two genome regions, E1-E2 and NS5A, from HCV-infected patients with different clinical and epidemiological backgrounds: mono-infected with HCV or coinfected with HIV, with or without antiviral treatment, and responder or non-responders to this treatment. No significant differences in the frequency of recombination events were detected between the two genomic regions studied (9.1% E1-E2 and 9.6 % NS5A, ##TAB##0##Table 1##) for the treatment-naïve, HCV-monoinfected patients group (HCV 0). On the contrary, large differences were found between these two regions for the group of HCV infected patients who did not respond to interferon plus ribavirin treatment therapy (HCV T). The observed recombination frequencies, 15.4% and 8.0% for the E1-E2 and NS5A regions, respectively, were not statistically significant due to the low sample size of the HCV T group but hinted to a larger genetic variation being generated in the E1-E2 region of non-responder patients. A positive relationship between genetic variability in this region ##REF##1380477##[50]##–##REF##15709027##[54]## and in the whole HCV genome ##REF##17522222##[55]## with lack of response to antiviral treatment and progression of the infection has been reported, although there are contradictory observations ##REF##10895435##[17]##, ##REF##1314389##[56]##–##REF##7686313##[58]##. In consequence, if this association is real, an increased rate of recombination in this region might contribute to viral resistance to treatment and, consequently, to a higher probability of detection of recombination in non-responder patients. To date, the sensitivity of recombinant forms of HCV to pegylated interferon-based therapy is still unknown, but recombinant forms for HIV do not have the same sensitivity to anti-retroviral therapy than wild type HIV-1 clade B isolates ##REF##16623640##[59]##. Furthermore, it has been recently suggested ##REF##17369105##[60]## that recombination plays an important role in the evolution of drug resistance in HIV-1 under various realistic scenarios. No significant differences were found in the frequency of recombination events in the E1-E2 region between the two treatment groups in HIV coinfected patients. Nevertheless, a higher proportion of cases of recombination were detected in the HAART treated group (12.5% for HCV 0-0 and 17.6% for HCV 0-T). This might be related to an increase in the selection pressure due to the decrease in HIV load and the restoration of the immune system in these patients.</p>", "<p>There are also reports correlating the degree of variability of the ISDR and responsiveness to interferon treatment ##REF##7542279##[61]##–##REF##9534954##[63]##, again without a complete consensus. However, the association in this case is an opposite one to that found in the E1-E2 region. Departure from a canonical sequence at ISDR has been associated to decreased response to interferon treatment, mainly in Japanese populations ##REF##9049229##[64]##–##REF##17133546##[66]##, but opposite results have been obtained for European and American ones ##REF##9334922##[67]##–##REF##10406179##[74]##. Nevertheless, recent meta-analyses of these reports have provided further support for this relationship ##REF##11130893##[75]##–##UREF##7##[77]##. Hence, the reversed relationship between genetic variability (departure from the canonical sequence) at ISDR and response to interferon treatment might be counterbalanced by recombination, which would allow the maintenance of the canonical sequence at ISDR while maintaining high levels of variation at other genome locations.</p>", "<p>Given the biological relevance described about the regions involved in the recombinant fragments, and the distribution of the recombinant cross-over points, it is clearly that the reported intragenic recombinant exploratory activity producing new genomic combinations could play an important role in the HCV evolution with significant consequences for treatment efficiency and the development of vaccines.</p>", "<p>Given the obtained results with a high frequency of HCV intragenic recombinant detected strains from patients belonging to the different described groups and the biological relevance related with the regions involved in this recombinant events, we conclude that, recombination must be considered as a potentially important mechanism generating genetic variation in HCV with serious implications in the vaccine and drug treatment optimal development and the response to antiviral therapy.</p>" ]
[]
[ "<p>Conceived and designed the experiments: VS AM FGC. Performed the experiments: VS NJH MTP MAB AV MJG. Analyzed the data: VS FGC. Contributed reagents/materials/analysis tools: EO. Wrote the paper: VS MAB AM FGC.</p>", "<p>Hepatitis C virus (HCV) is a major cause of liver disease worldwide and a potential cause of substantial morbidity and mortality in the future. HCV is characterized by a high level of genetic heterogeneity. Although homologous recombination has been demonstrated in many members of the family Flaviviridae, to which HCV belongs, there are only a few studies reporting recombination on natural populations of HCV, suggesting that these events are rare <italic>in vivo</italic>. Furthermore, these few studies have focused on recombination between different HCV genotypes/subtypes but there are no reports on the extent of intra-genotype or intra-subtype recombination between viral strains infecting the same patient. Given the important implications of recombination for RNA virus evolution, our aim in this study has been to assess the existence and eventually the frequency of intragenic recombination on HCV. For this, we retrospectively have analyzed two regions of the HCV genome (NS5A and E1-E2) in samples from two different groups: (i) patients infected only with HCV (either treated with interferon plus ribavirin or treatment naïve), and (ii) HCV-HIV co-infected patients (with and without treatment against HIV). The complete data set comprised 17712 sequences from 136 serum samples derived from 111 patients. Recombination analyses were performed using 6 different methods implemented in the program RDP3. Recombination events were considered when detected by at least 3 of the 6 methods used and were identified in 10.7% of the amplified samples, distributed throughout all the groups described and the two genomic regions studied. The resulting recombination events were further verified by detailed phylogenetic analyses. The complete experimental procedure was applied to an artificial mixture of relatively closely viral populations and the ensuing analyses failed to reveal artifactual recombination. From these results we conclude that recombination should be considered as a potentially relevant mechanism generating genetic variation in HCV and with important implications for the treatment of this infection.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>We thank D. Martin for his help with RDP, J. Abellán for comments on the statistical analyses and two anonymous reviewers for their suggestions.</p>" ]
[ "<fig id=\"pone-0003239-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g001</object-id><label>Figure 1</label><caption><title>Summary of positive recombination results in the E1-E2 region (HCV-monoinfected patients).</title><p>The columns represent: i) patient group before (HCV 0) or after (HCV T) antiviral treatment; ii) patient code; iii) amplified sequences for this patient and region; iv) HCV genotype; v) p-values for the different recombination detection methods implemented in RDP 3.0 (1 = RDP, 2 = Geneconv, 3 = Bootscan, 4 = Maxchi, 5 = Chimera, and 6 = Siscan) using the following color coding: non-significant p-values (white filling), p&lt;0.05 (grey) and p&lt;0.01 (black); vi) recombination event number; vii) Bkpt 1, location range for breakpoint 1; viii) Bkpt 2, location range for breakpoint 2; ix) parent1 sequence (% similarity to daughter sequence); x) parent2 sequences(% similarity to daughter sequence); and xi) daughter sequence(s). Only events detected as significant by at least three methods are shown.</p></caption></fig>", "<fig id=\"pone-0003239-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g002</object-id><label>Figure 2</label><caption><title>Summary of positive recombination results in the E1-E2 region (HCV-HIV coinfected patients).</title><p>See further details in legend to ##FIG##0##Figure 1##.</p></caption></fig>", "<fig id=\"pone-0003239-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g003</object-id><label>Figure 3</label><caption><title>Summary of positive recombination results in the NS5A region.</title><p>See further details in legend to ##FIG##0##Figure 1##.</p></caption></fig>", "<fig id=\"pone-0003239-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g004</object-id><label>Figure 4</label><caption><title>Summary of the joint analysis of two time-point samples from the same patients resulting in positive detection of recombination.</title><p>See further details in legend to ##FIG##0##Figure 1##.</p></caption></fig>", "<fig id=\"pone-0003239-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g005</object-id><label>Figure 5</label><caption><title>Location of recombination breakpoints detected in the E1E2region.</title></caption></fig>", "<fig id=\"pone-0003239-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g006</object-id><label>Figure 6</label><caption><title>Example of recombination detection in the E1E2 region (EC5703 sample).</title><p>The phylogenetic tree on the left corresponds to the analysis of the complete region whereas the other two are derived from the two regions defined by the detected recombination event.</p></caption></fig>", "<fig id=\"pone-0003239-g007\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g007</object-id><label>Figure 7</label><caption><title>Example of recombination detection in the E1E2 region (EC5703 sample).</title><p>Variable nucleotide positions in the daughter (EC5703-46) and two parental sequences (EC5703-34 and EC5703-36).</p></caption></fig>", "<fig id=\"pone-0003239-g008\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g008</object-id><label>Figure 8</label><caption><title>Location of recombination breakpoints detected in the NS5a region.</title></caption></fig>", "<fig id=\"pone-0003239-g009\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g009</object-id><label>Figure 9</label><caption><title>Example of recombination detection in the NS5a region (NG08T0 sample).</title><p>The phylogenetic tree on the left corresponds to the analysis of the complete region whereas the other two are derived from the two regions defined by the detected recombination event.</p></caption></fig>", "<fig id=\"pone-0003239-g010\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.g010</object-id><label>Figure 10</label><caption><title>Example of recombination detection in the NS5a region (NG08T0 sample).</title><p>Variable nucleotide positions in the daughter (NG08-47K) and two parental sequences (NG08-33K and NG08-63K).</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003239-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.t001</object-id><label>Table 1</label><caption><title>Summary of patients, sequence data sets and recombination analysis results.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Source</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Patients</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Group</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Amplified samples</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Average sequence/ sample</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SD</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total seqs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Positive cases</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Freq.</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">E1-E2 region</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">78</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">77</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">105.74</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21.43</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8142</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.091</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCVT</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">106.15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2680</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.154</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV-HIV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV0-0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28.69</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.86</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">433</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.125</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV0-T</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28.88</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.32</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">491</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.176</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">TOTAL</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">111</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">136</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">11746</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.118</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NS5A region</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">78</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">73</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">63.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.51</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4623</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.096</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCVT</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">53.72</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.00</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1343</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.080</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">TOTAL</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">78</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">98</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">5966</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.091</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003239-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003239.t002</object-id><label>Table 2</label><caption><title>Summary of frequency analysis of recombinant sequences in the different data sets where intrapatient recombination of HCV was detected.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Group</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Patient</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total Seqs.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Diff. Haplotypes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Numb. of recomb</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Freq. recomb.</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Recombinants</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Non-recombinants</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Seqs.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Freq.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Seqs.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Freq.</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NS5a region HCV 0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.036</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.018</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.036</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">C29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">87</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.057</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.011</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.184</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">C35</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.073</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.018</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.036</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">C36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">67</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.027</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.013</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.040</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G05</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.048</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.032</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.048</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G07</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">49</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.143</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.020</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.041</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.036</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.018</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.309</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV T</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">C05T1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.233</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.023</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.047</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G07T2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">49</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.265</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.020</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.041</td></tr><tr><td colspan=\"10\" align=\"left\" rowspan=\"1\">E1-E2 region</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV 0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">99</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">92</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.040</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.040</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">A21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">72</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.020</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.140</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">A28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">87</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.040</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.030</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">C28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">99</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">77</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.020</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.030</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">C29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.020</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.200</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">C32</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">99</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.040</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.051</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">104</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">93</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.058</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.038</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV T</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">108</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.046</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.019</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.056</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">81</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.030</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.060</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">79</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.030</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.060</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G26</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">102</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">58</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.196</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV 0-0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">C06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.400</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.080</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.120</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">V035</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.516</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.065</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.065</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HCV 0-T</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">C23</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.069</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.034</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.034</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">C30</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.222</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.074</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.111</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">C57</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.024</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.024</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.195</td></tr><tr><td colspan=\"10\" align=\"left\" rowspan=\"1\">Two joint samples</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NS5A</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">C29T0-T1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">147</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">59</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.027</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.007</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.231</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G07T0-T2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">98</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">89</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.133</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.031</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">E1-E2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A21T0-T1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">209</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">156</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.067</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">C29T0-T1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.015</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.005</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.100</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G16T0-T1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">203</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">82</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.005</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">111</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.547</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">G26T0-T1-T2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">302</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.003</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.003</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">85</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.281</td></tr></tbody></table></alternatives></table-wrap>" ]
[]
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[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003239.s001\"><label>Table S1</label><caption><p>Detailed information on patients, samples, sequences and accession numbers used in this research</p><p>(0.07 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003239.s002\"><label>Table S2</label><caption><p>Summary of SH and ELW tests for alternative topologies derived from the recombination events detected</p><p>(0.19 MB PDF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003239.s003\"><label>Table S3</label><caption><p>Summary of Mann-Whitney tests for differences in recombination frequency between clinical groups considered in this study</p><p>(0.09 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003239.s004\"><label>File S1</label><caption><p>Zip-compressed file with all the maximum likelihood trees used in the analyses</p><p>(0.13 MB ZIP)</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"nt101\"><label/><p>The “source” column represents the study (HCV-monoinfection, HCV/HIV-coinfection) in which samples were obtained originally. For the HCV-monoinfection study, samples were obtained from 78 patients and the numbers indicated in the “amplified samples” column yielded successful amplificates before (HCV0) and after (HCVT) antiviral treatment. For the HCV/HIV study, the two groups correspond to patients without any treatment (HCV0-0) or having been treated for HIV infection (HCV0-T). SD indicates standard deviation of the number of sequences obtained from each sample and “Freq.” denotes the frequency of samples in which at least one recombination event has been detected.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt102\"><label/><p>For each patient and sample(s) considered the table reports the total number of sequences analyzed, the number of different haplotypes, the total number of recombinant sequences and their frequency in the sample. Additional information is given on the absolute and relative frequencies of the most frequent haplotype among recombinant and non-recombinant sequences.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>This work was supported by the Conselleria de Sanitat i Consum Generalitat Valenciana (Spain) and project BFU2005 00503 from Ministerio de Educacion y Ciencia.</p></fn></fn-group>" ]
[ "<graphic id=\"pone-0003239-t001-1\" xlink:href=\"pone.0003239.t001\"/>", "<graphic xlink:href=\"pone.0003239.g001\"/>", "<graphic xlink:href=\"pone.0003239.g002\"/>", "<graphic xlink:href=\"pone.0003239.g003\"/>", "<graphic xlink:href=\"pone.0003239.g004\"/>", "<graphic xlink:href=\"pone.0003239.g005\"/>", "<graphic xlink:href=\"pone.0003239.g006\"/>", "<graphic xlink:href=\"pone.0003239.g007\"/>", "<graphic xlink:href=\"pone.0003239.g008\"/>", "<graphic xlink:href=\"pone.0003239.g009\"/>", "<graphic xlink:href=\"pone.0003239.g010\"/>", "<graphic id=\"pone-0003239-t002-2\" xlink:href=\"pone.0003239.t002\"/>" ]
[ "<media xlink:href=\"pone.0003239.s001.xls\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003239.s002.pdf\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003239.s003.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003239.s004.zip\"><caption><p>Click here for additional data file.</p></caption></media>" ]
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{ "acronym": [], "definition": [] }
103
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 18; 3(9):e3239
oa_package/98/46/PMC2528950.tar.gz
PMC2528951
18820724
[]
[]
[]
[ "<title>DISCUSSION</title>", "<p>The brown recluse spider, <italic>Loxosceles recluse</italic> is the most common of the <italic>Loxosceles</italic> species in the United States. Clinical sequelae of <italic>Loxosceles</italic> bites were described in the literature as early as 1879.<sup>##UREF##0##1##</sup> According to the Centers for Disease Control and Prevention, approximately 10,000 spider bites are annually reported to poison control centers, of these, 1835 bites were attributed to the brown recluse in 1994.<sup>##REF##8618542##2##</sup> The brown recluse spider mainly populates the southern central states, due to a preferred habitat of mild climates.<sup>##REF##3541795##3##</sup> The spiders prefer dark, quiet environments such as closets, basements, attics, and sheds. Brown recluse spiders do not usually bite unless provoked or threatened. The spider is generally 1 to 5 cm in length and has a tan to light brown color. Distinguishing features include fiddle-shaped brown markings on its dorsum and characteristic 3 dyads (6 eyes as opposed to the usual 8 for most spiders).<sup>##REF##3288080##4##</sup> Brown recluse spiders commonly bite exposed lower or upper extremities, but bites to the face have been reported.<sup>##REF##16297700##5##</sup></p>", "<p>Brown recluse spider bite injuries cause envenomation leading to major tissue destruction.<sup>##UREF##1##6##</sup> Pain and erythema are apparent within the first few hours after the bite. Progression to skin necrosis with purplish blue cyanotic bullae within 24 to 48 hours soon follows. The site of injury undergoes a cycle of erythema, ischemia, and thrombosis, referred to as the “red, white, and blue sign.”<sup>##REF##11260528##7##(p565)</sup> This is due to brown recluse venom components, including hyaluronidase, elastase, sphingomyelinase D, lipase, and serum amyloid protein.<sup>##REF##2403477##8##</sup> Each of these enzymes contribute to the extent of tissue necrosis.<sup>##REF##2403477##8##</sup> The envenomation attracts polymorphonuclear cells, which further propagate the necrosis. The polymorphonuclear cells degranulate within the vasculature leading to vessel thrombosis followed by tissue ischemia. The underlying area of necrosis is generally extensive in comparison to the minimal surface lesion as seen in our patient. The clinical picture may progress to signs of fever, chills, malaise, vomiting, and arthralgias. There have even been reports of brown recluse spider bite envenomation leading to multisystem organ failure and death.<sup>##REF##1573793##9##</sup></p>", "<p>The diagnosis and treatment of brown recluse bites is controversial: a classification system proposed by Anderson has been favored by expert entomologists to facilitate the diagnosis and, perhaps, to decrease overdiagnosis in nonendemic areas.<sup>##UREF##1##6##</sup><sup>,</sup><sup>##REF##11973562##10##</sup><sup>,</sup><sup>##REF##12145729##11##</sup> A number of entomologists have collected data from reported brown recluse spider bites, but they noted that positive identification of the spider has been absent.<sup>##REF##12145729##11##</sup><sup>,</sup><sup>##UREF##2##12##</sup> Laboratory tests using enzyme-linked immunosorbent assay techniques have been developed in attempt to accurately diagnose brown recluse envenomation but none have been widely accepted or approved for clinical use.<sup>##REF##11973553##13##</sup></p>", "<p>The differential diagnosis of brown recluse spider bite envenomation includes thromboembolic disease; focal vasculitis; drug reactions; pyoderma gangrenosum; Lyme disease, bacterial, viral, or fungal infections; neoplasms; chemical burns; and factitious injections.<sup>##REF##3288080##4##</sup><sup>,</sup><sup>##REF##16297700##5##</sup> Attempts to exclude other potential causes before attributing necrotic skin lesions to the probably overused diagnosis of brown recluse bite should be made. Nonendemic regions are especially unlikely to encounter the problem and other more probable diagnoses should be entertained.</p>", "<p>Treatment includes basic wound care measures, such as rest, ice compresses, and elevation of the involved extremity. Antibiotics are typically not indicated unless there is skin breakdown with secondary infection caused by typical skin flora. <italic>Dapsone</italic>, a leukocyte inhibitor, has shown some promise, if administered early. Dapsone's efficacy is based on its inhibitory effect on the polymorphonuclear cells that infiltrate and further propagate thrombosis, leading to further ischemia and tissue necrosis. Dapsone is usually reserved for severe cases because of its adverse effects, including dose-related hemolysis, agranulocytosis, aplastic anemia, cholestatic jaundice, and methemoglobinemia.<sup>##REF##12911188##14##</sup></p>", "<p>Operative intervention is indicated when frank abscess formation occurs, tissue necrosis is extensive or severe, or deep vital structures are involved or exposed.<sup>##REF##16297700##5##</sup><sup>,</sup><sup>##REF##11260528##7##</sup> Incision and drainage of a defined abscess is always indicated; however, as intense tissue inflammation and edema are more common findings than discrete abscesses, indiscriminate incision without the presence of an underlying abscess should be avoided. Debridement of necrotic tissue is necessary to avoid the negative metabolic effects of the breakdown products of cell death on wound healing. Deep exposed structures such as bone or vital tissues may require soft tissue coverage by an appropriate flap or graft after adequate wound bed preparation.</p>", "<p>Our patient presented with a prolonged course of a chronic, open wound with deep soft tissue and bone involvement. There was chronic infection manifested by osteomyelitis of the mandible. Appropriate soft tissue debridement and bone debridement were performed and primary wound closure was obtained. Long-term follow-up demonstrated resolution of the chronic draining wound with surgical management and antibiotic therapy. Although the spider is not considered endemic to Florida, the patient did identify environmental exposure and clinical presentation consistent with a brown recluse spider bite.</p>" ]
[ "<title>CONCLUSION</title>", "<p>Brown recluse spider bites and envenomations may be overreported. The course of injury may vary from mild erythema to frank necrosis of soft tissue and bone. The treatment regimen should include the basics of wound care and consideration of systemic antibiotics and topical antimicrobials as indicated. Dapsone may be of value if given early in the course of treatment. Surgical intervention is warranted in cases of abscess formation, extensive tissue necrosis, or deep tissue involvement. Injuries to the face are rare but follow the course of injury to other more common areas of the body.</p>" ]
[ "<p>This is an open-access article whereby the authors retain copyright of the work. The article is distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p><bold>Background:</bold> Brown recluse spider bites cause significant trauma via their tissue toxic venom. Diagnosis of these injuries and envenomation is difficult and many times presumptive. Treatment is varied and dependent upon presentation and course of injury. <bold>Materials and Methods:</bold> We present a case of a previously unreported incidence of osteomyelitis of the mandible as a result of a brown recluse spider bite. A review of the literature and discussion of diagnosis and treatment of brown recluse spider bites are presented. <bold>Results:</bold> Osteomyelitis of the mandible causing a chronic wound was the presenting finding of a patient with a history of spider bite and exposure to brown recluse spiders. Operative debridement and wound closure resulted in successful treatment. Brown recluse spider envenomation varies in its presentation and treatment is based on the presenting clinical picture. <bold>Conclusion:</bold> Treatment regimens for brown recluse spider bite envenomation should include the basics of wound care. Systemic antibiotics, topical antimicrobials, dapsone, and surgical debridement are valuable adjuncts of treatment, as indicated, based on the clinical course.</p>" ]
[ "<p>Brown recluse spider bite envenomation can be a significant traumatic injury. Probably overreported, brown recluse spider bites and their resultant tissue injury patterns are well documented; however, treatment regimens are somewhat controversial. We present an unusual case of a presumed brown recluse spider bite injury of the face, which developed into chronic osteomyelitis of the mandible, and discuss the evaluation and further management of this interesting clinical presentation.</p>", "<title>CASE REPORT</title>", "<p>A 52-year-old white male presented with a 6-month history of a tender, open wound in the submental area. The chronic wound had been draining serous and purulent material for 4 to 6 weeks prior to evaluation. The wound began as a “pimple” on his chin that progressed in a few days to erythema, skin breakdown, and then, eventually, purulent drainage. The patient related a history of having encountered multiple “fiddleback” spiders at his place of work. He also claimed to have suffered multiple spider bites on the extremities in the past, all of which healed without sequelae. Physical examination was significant for a 5-mm open wound of the mandible, slightly to the right of the midline of his chin (Fig ##FIG##0##1##). The area was tender and a small amount of murky fluid was expressed. The wound probed to bone, using a sterile hemostatic clamp. There was no palpable regional lymphadenopathy. Computed tomography revealed a large bony cavity eroding through the inferior cortex of the mandibular symphysis anteriorly and abutting the lingual cortex. The mandibular integrity was intact with no evidence, clinically or radiographically, of a pathological fracture (Fig ##FIG##1##2##). The bony destruction appeared chronic, as evidenced by the sclerotic margins of the cavity. <italic>Differential diagnosis</italic> included possible odontogenic infection, neoplastic process, and traumatic wound infection. An odontogenic infection was excluded because of the viability of associated dentition and lack of demonstrable dental pathology. As a soft tissue or bony neoplasm appeared unlikely because of the smooth, sclerotic margins seen on computed tomographic scan and the lack of a demonstrable soft tissue lesion, this was attributed most likely to a wound infection secondary to traumatic injury.</p>", "<p>Operative debridement was planned with a presumptive diagnosis of traumatic infection. The patient underwent excision of the wound and the chronic granulation/soft tissue inflammatory reaction within the bony cavity. The bone cavity was aggressively curetted and debrided to healthy bleeding bone (Fig ##FIG##2##3##). The mandibular symphysis was found to be extremely stable with adequate stock of bone to maintain its integrity without the need for mechanical reinforcement, either by plating or bone graft. The soft tissue was extensively mobilized and reapproximated in layers. The pathology report revealed chronic osteomyelitis. At 6 weeks follow-up, the wound had healed without complication or further sequelae (Fig ##FIG##3##4##).</p>", "<title>Acknowledgment</title>", "<p>The authors thank Chris R. Payne, Major, USAF (retired), for assistance in the preparation of photographs for this article. There was neither financial support nor do any authors have proprietary interest in any product, device, or method discussed in this article.</p>" ]
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[ "<fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Preoperative photograph of presenting wound.</p></caption></fig>", "<fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Computed tomographic scan demonstrating infection of mandibular symphyseal bone.</p></caption></fig>", "<fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Intraoperative photograph of debrided, stable mandibular symphysis.</p></caption></fig>", "<fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Postoperative photograph demonstrating healed, stable wound.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"eplasty08e45_fig1\"/>", "<graphic xlink:href=\"eplasty08e45_fig2\"/>", "<graphic xlink:href=\"eplasty08e45_fig3\"/>", "<graphic xlink:href=\"eplasty08e45_fig4\"/>" ]
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[{"label": ["1"], "surname": ["Caveness"], "given-names": ["WA"], "article-title": ["Insect bite, complicated with fever"], "source": ["Nashville J Med Surg."], "year": ["1872"], "volume": ["10"], "fpage": ["333"]}, {"label": ["6"], "surname": ["Anderson"], "given-names": ["PC"], "article-title": ["Spider bites in the United States"], "source": ["Dermatol Clinics"], "year": ["1997"], "volume": ["15"], "fpage": ["307"], "lpage": ["11"]}, {"label": ["12"], "surname": ["Edwards"], "given-names": ["GB"], "article-title": ["The present status and a reviewof brown recluse and related spiders, "], "italic": ["Loxosceles spp."], "source": ["Entomol Circ"], "year": ["2001"], "volume": ["406"], "fpage": ["1"], "lpage": ["6"]}]
{ "acronym": [], "definition": [] }
14
CC BY
no
2022-01-12 14:47:34
Eplasty. 2008 Aug 28; 8:e45
oa_package/b9/23/PMC2528951.tar.gz
PMC2528962
18797506
[ "<title>Introduction</title>", "<p>Dispersal exerts an important influence on population genetics and demography, as well as on our ability to predict population-level responses to environmental disturbance ##REF##15921049##[1]##, ##UREF##0##[2]##. Many vertebrates exhibit sex-biased dispersal, but the pattern differs among taxa: female-biased dispersal (FBD) is typical among birds, whereas males disperse and females are philopatric in most mammals ##UREF##1##[3]##–##UREF##3##[5]##. Evolutionary models of sex-biased dispersal have drawn comparative support from the prevalence of different mating systems in mammals and birds. Over 90% of bird species live in male-female pairs ##UREF##4##[behavioral monogamy, 6]##, whereas 95% or more of mammal species exhibit polygynous mating systems ##UREF##5##[7]##. Theoretical approaches suggest that the same sexual asymmetries driving the evolution of mating systems should also influence the evolution of dispersal patterns ##UREF##6##[8]##.</p>", "<p>Three non-mutually exclusive factors have been proposed to explain the association between mating systems and sex-biased dispersal: inbreeding avoidance, local resource competition (LRC), and local mate competition ##REF##9061966##[reviewed in 9]##. All three hypotheses predict male-biased dispersal in polygynous species, because male offspring may be more likely to mate with the care-giving parent (i.e. females often have longer tenure), face more intense local competition for mates, or compete for resources to attract females, respectively. Sexual asymmetries in mate competition and risk of inbreeding are not predicted under monogamy, because individuals of both sexes may have only one mate and the same number of offspring. However, intense local resource competition may lead to FBD in monogamous species when dispersing females gain critical resources for reproduction ##UREF##1##[3]##.</p>", "<p>Monogamy in mammals is associated with female use of exclusive territories ##UREF##7##[10]##, primarily as a strategy to minimize feeding competition when predation and other factors do not favor group-living ##REF##327542##[11]##–##UREF##8##[13]##. Reproduction in males is unlikely to be as severely limited by food resources as it is in females, and thus an asymmetry in the costs of philopatry may arise in monogamous species if females compete for access to feeding territories. LRC may also increase the rate of female aggression in multi-female groups, resulting in the expulsion of juvenile females by their mothers ##UREF##9##[e.g. primates 14]##, ##UREF##10##[15]##. However, comparative data suggest that most juvenile dispersal is “voluntary” ##UREF##11##[16]##, because the costs of dispersal may be low when unoccupied areas are available to immigrants ##UREF##12##[17]##.</p>", "<p>The predicted association between FBD and monogamy has rarely been examined in mammals, largely because most mammals are polygynous ##UREF##5##[7]##. Dobson's ##UREF##2##[4]## comparative study did not find an association between FBD and monogamy in mammals, but few data were (and still are) available for monogamous species. Lawson Handley ##REF##17402974##[18]## found evidence for FBD in only four monogamous mammals, although other cases may exist ##UREF##13##[e.g. 19]##. Unbiased measures of dispersal are difficult to obtain using traditional techniques, especially for pair-living species that are widely dispersed in space and time. Sex biases in dispersal may also be obscured by the geographic scale at which a given study is conducted ##REF##11412373##[20]##, ##REF##15154563##[21]##. However, genetic methods to detect both sex-biased dispersal and gene flow at varying spatial scales have recently become available that ameliorate these logistical problems ##REF##12030985##[22]##, ##REF##11920116##[23]##.</p>", "<p>Several polygynous mammals have been studied using these genetic techniques, and as predicted either no sex bias ##REF##11928704##[e.g. river otters, Lontra canadensis, 24]## or male-biased dispersal ##REF##15548278##[e.g. brush]##-##REF##15969731##[tailed rock wallabies, Petrogale penicillata, 25, talar tuco–tucos, Ctenomys talarum, 26]## has been detected in most cases. However, genetic analyses have revealed FBD multiple times in polygynous species ##UREF##14##[common wombats]##, ##REF##11555272##[ Vombatus ursinus]##, ##REF##10849290##[ 27]##, ##UREF##15##[bush hyraxes, Heterohyrax brucei, 28, kinkajous, Potos flavus, 29, greater white–lined bats, Saccopteryx bilineata, 30]##, especially among catarrhine primates ##REF##17912352##[chimpanzees]##, ##REF##11528385##[Pan troglodytes]##, ##REF##15043817##[31]##, ##REF##17561888##[humans]##, ##REF##16599958##[Homo sapiens]##, ##REF##16615216##[32, western gorillas, Gorilla beringei, 33,34, bonobos, Pan paniscus, 35, hamadryas baboons, Papio hamadryas, 36]##. Genetic studies conducted on behaviorally monogamous mammals have found no evidence of sex-biased dispersal ##UREF##16##[fat–tailed dwarf lemurs, Cheirogaleus medius, 37]##, or contrasting patterns. Male alpine marmots (<italic>Marmota marmota</italic>) disperse more often than females ##REF##11251786##[38]##, whereas FBD occurs in the greater white-toothed shrew ##REF##9061966##[Crocidura russula, 9]##. In a review of sex-biased dispersal in mammals, FBD did not strongly correlate with any particular mating system but was found in taxonomic clusters (e.g. Atelidae). Additionally, some studies have detected FBD in polygynous species where local mate competition is known to be intense [##TAB##1##Table 2## in 18].</p>", "<p>Predictions of a simple association between FBD and monogamy are complicated by increasingly common genetic results indicating substantial extra-pair paternity in putatively monogamous mammals ##UREF##17##[e.g. 52% in swift foxes]##, ##UREF##18##[Vulpes velox, 39, 35% in mountain brushtail possums, Trichosurus cunninghami, 40]##. Although these species may live in male-female pairs that occupy joint territories (i.e. behavioral monogamy), the genetic mating system more closely resembles polygyny where individual males sire offspring with multiple females. Thus, male-biased dispersal due to inbreeding avoidance, LRC or local mate competition between males may occur in these species. Alternatively, LRC among females may be intense enough to favor FBD despite extra-pair paternity comprising an important component of the genetic mating system. FBD is prevalent among birds even though most avian species studied to date exhibit behavioral monogamy and extra-pair paternity in greater than 5% of offspring ##REF##12406233##[112/130 bird species reviewed in 41]##. These results are likely due to the benefits males gain from philopatry by defending a successful breeding territory to attract females. Few tests of sex-biased dispersal have been conducted in behaviorally monogamous mammals without mating fidelity. Alpine marmots exhibit moderate extra-pair paternity ##UREF##19##[19%, 42]## and male-biased dispersal, whereas fat-tailed dwarf lemurs exhibit substantial extra-pair paternity ##UREF##20##[44%, 43]## but no sex bias in dispersal. Additional case studies are clearly needed to determine whether FBD occurs in pair-living mammals with or without extra-pair paternity.</p>", "<p>In this study I use multiple genetic methods to test for FBD and gene flow in the large treeshrew (<italic>Tupaia tana</italic>) in NE Borneo. Large treeshrews form behaviorally monogamous pairs that forage solitarily, potentially as an adaptation to intraspecific foraging competition ##REF##11034017##[dispersed pairs]##, ##UREF##21##[12,44]##. The rate of extra-pair paternity in <italic>T. tana</italic> is one of the highest ever recorded for a mammal (50%), but variance in reproductive success does not vary between males and females ##UREF##22##[45]##. Comparative analysis of testis size also indicates that male <italic>T. tana</italic> are not subject to intense sperm competition ##UREF##22##[45]##, and thus competition among males for mates or resources may not be strong enough to favor male-biased dispersal. Alternatively, FBD may occur in this species due to unique energetic limitations that produce intense competition between females. Treeshrews exhibit an absentee maternal care system that preempts reproduction when resources are scarce ##UREF##23##[46]##. Females deposit their two young in a nest chamber and visit them only once every 48 hours for intensive nursing. In the interim, females devote most of their activity period to foraging ##UREF##23##[10.5 hrs daily, 46]## and travel long daily distances (means = 1.1–1.5 km depending on year and study site) for their body size ##UREF##21##[means = 202–257 g, 44]## to produce and store the required large amounts of milk. Females exhibit sex-specific territorial defense ##UREF##23##[46]##, and competition among females for feeding territories to support their physiologically expensive foraging and maternal behavior may result in FBD.</p>", "<p>I tested the prediction of FBD in <italic>T. tana</italic> by comparing the genetic structure and patterns of relatedness among adult males and females at seven autosomal microsatellite loci. FBD is predicted to produce genotypes with lower population assignment probabilities and pairwise relatedness among adult (i.e. post-natal dispersal) females than among adult males in the population ##REF##12030985##[22]##. I also examined the prediction of female-biased gene flow in <italic>T. tana</italic> by comparing gene flow estimated from bi-parentally inherited microsatellite markers and a maternally inherited mitochondrial DNA (mtDNA) marker. Bayesian methods based on the coalescent ##REF##16317072##[47]## were used to estimate the exchange of migrants between two different <italic>T. tana</italic> populations. If gene flow is female-biased, then the migration rate for mtDNA should substantially exceed the migration rate for bi-parentally inherited microsatellites.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Study sites and genetic sampling</title>", "<p>Large treeshrews are small (200–250 g), diurnal, frugivore-insectivores that inhabit the lowland tropical rainforests of Borneo and Sumatra. I collected ear clips for genetic analyses from 54 <italic>T. tana</italic> individuals at two sites in Sabah, Malaysia (NE Borneo) from 2002–2004 during a larger study on mating systems in treeshrews. The first site (<italic>N</italic> = 39 samples) was located in the Danum Valley Conservation Area (Danum, 4°58′N, 117°48′E) and consisted of undisturbed primary lowland rainforest. The other site (<italic>N = </italic>15 samples) was located 53 km away in the Malua Forest Reserve (5°5′N, 117°38′E). This area was heavily logged in the early 1980's and has yet to recover the multiple closed canopies (typically 10 m and 20–30 m in height) and tall emergent trees (up to 70 m) that characterize lowland rainforests in SE Asia ##UREF##24##[48]##. See Munshi-South <italic>et al.</italic>\n##UREF##21##[44]## for full details of the study sites and trapping methods. This research was approved by the Institutional Animal Care and Use Committee at the University of Maryland, and adhered to all laws governing research in USA and Malaysia.</p>", "<p>I extracted genomic DNA from ear tissue samples using Qiagen DNEasy tissue extraction kits (Qiagen, Valencia, CA). Seven previously-described, unlinked microsatellite loci named JS22, JS132, JS183, JS188, JS196, SKTg19, and SKTg22 were amplified from DNA extracts using the PCR conditions in Munshi-South &amp; Wilkinson ##UREF##25##[49]##. Fluorescently-labeled alleles were separated on an Applied Biosystems 3100 DNA Analyzer and sized and scored using Genotyper 2.5 (Applied Biosystems, Foster City, CA). I also PCR-amplified a 602 bp segment of the mtDNA control region with the primers JMSTbel386 and JMSTbel1110 and PCR conditions described in Munshi-South ##UREF##22##[45]##. PCR products were sequenced using the BigDye Terminator 3.1 and an ABI 3100 DNA Analyzer, and then sequences were edited and aligned using Sequencer 4.1.2 (Gene Codes, Ann Arbor, MI) and Bioedit 7.0.4.1 ##UREF##26##[50]##.</p>", "<p>To examine differences in genetic variability between the primary and logged forest populations, I calculated the number of alleles and allelic richness at each microsatellite locus for each population using FSTAT v. 2.9.3.2 ##UREF##27##[51]##. I also used the log-likelihood G test of genotypic differentiation implemented in FSTAT ##REF##8978076##[10,000 randomizations not assuming Hardy–Weinberg equilibrium, 52]## to examine whether the two populations exhibited significantly different microsatellite allele frequencies. I investigated mtDNA sequence divergence between populations by calculating the number of fixed differences and shared mutations between populations, and the average nucleotide substitutions and number of net substitutions per site between populations ##UREF##28##[Dxy and Da, respectively, with Jukes–Cantor correction, 53]##, using DNASP v. 4.2.4 ##REF##14668244##[54]##. I also conducted a permutation test (10,000 randomizations without alignment gaps) of genetic differentiation using the nearest-neighbor statistic (<italic>S</italic>\n<sub>nn</sub>) implemented in DNASP. <italic>S</italic>\n<sub>nn</sub> measures how often the most similar sequences in a data set (“nearest neighbors”) are from the same population, and produces a powerful test of genetic differentiation for sequence data in nearly all situations ##REF##10924493##[55]##.</p>", "<title>Tests of female-biased dispersal</title>", "<p>To test for FBD, I compared mean corrected assignment indices (<italic>mAI</italic>\n<sub>c</sub>) between adult males and females using the “biased dispersal” module in FSTAT. One-sided <italic>P</italic> values were calculated using 10,000 randomizations. The assignment index is the probability that an individual's genotype occurred by chance in a population ##REF##7663752##[56]##, and Favre et al. ##REF##9061966##[9]## applied a correction that produces mean <italic>AI<sub>c</sub></italic> values of zero for each population. Negative <italic>AI<sub>c</sub></italic> values characterize individuals with genotypes less likely than average to occur in a population sample, and thus lower <italic>mAI</italic>\n<sub>c</sub> values for one sex (females, in this case) implies sex-biased dispersal. This index was chosen because both simulations and real data sets have indicated that this test has high power at detecting moderately intense biases in dispersal ##REF##12030985##[22]##, ##REF##10434424##[57]##. Adult genotypes were used for these analyses, because this test assumes post-dispersal sampling (<italic>N</italic> = 14 females and 20 males).</p>", "<p>I also tested the prediction that pairs of adult females were less related on average than pairs of adult males, because sex-biased dispersal is predicted to influence local relatedness structure among adults ##REF##15548278##[e.g. 25]##, ##UREF##14##[27]##, ##REF##17148391##[58]##. If female <italic>T. tana</italic> disperse more often or farther than males, then fewer closely related pairs of females should occur in the sample. I calculated two estimates of pairwise relatedness, because the performance of different estimators varies depending on population composition ##REF##11412374##[59]##. Two method-of-moment regression estimators, Lynch and Ritland's <italic>r</italic>\n##REF##10430599##[60]## and Queller and Goodnight's <italic>r</italic>\n##REF##28568555##[61]##, were calculated using the program MARK ##UREF##29##[62]##. Simulations indicate that the Lynch and Ritland estimator performs well for most population compositions ##UREF##30##[63]##. The Queller and Goodnight estimator is commonly used in studies of relatedness, and was included to facilitate comparison with other studies.</p>", "<p>Pairwise relatedness estimates from the primary and logged forest populations were pooled to increase sample sizes, but relatedness was calculated only between pairs of individuals from the same population. Using only dyads from the same population gives a better representation of background population-level allele frequencies. For each different estimator, I tested whether mean female relatedness was lower than male relatedness using a two-sample randomization test ##UREF##31##[64]##. Randomization tests were used because relatedness data were generated for dyads of individuals and thus do not represent independent observations. The one-sided <italic>P</italic> value for these tests was calculated by comparing the observed mean difference to the mean differences calculated from 10,000 randomizations of the same sets of relatedness estimates using POPTOOLS 2.6 ##UREF##32##[65]##.</p>", "<title>Tests of female-biased gene flow</title>", "<p>If gene flow among large treeshrews is female-biased, then migration rates calculated for maternally inherited mtDNA should be higher than migration rates calculated for bi-parentally inherited autosomal markers. To test this prediction, I used the Bayesian coalescence approach implemented in MIGRATE 2.1.3 ##UREF##33##[66]## to estimate the effective number of migrants exchanged per generation (<italic>N<sub>e</sub>m</italic>) between the two populations using both the microsatellite and mtDNA sequence data. Bayesian inference may be more accurate and efficient at sampling genealogy space than maximum likelihood approaches for many datasets ##REF##16317072##[47]##. This method produces estimates of Θ (4<italic>N<sub>e</sub>μ</italic>, where <italic>μ</italic> = mutation rate) and <italic>M</italic> (<italic>m</italic>/<italic>μ</italic>) from microsatellite data, equaling 4<italic>N<sub>e</sub>m</italic> when multiplied together. For mtDNA, this method estimates 2<italic>N<sub>f</sub>m</italic> (<italic>N<sub>f</sub></italic>  = effective population size of females). Assuming an equal sex ratio and equal variance in reproductive success among males and females, <italic>N<sub>f</sub></italic> is equivalent to <italic>N<sub>e</sub></italic>/2 calculated from microsatellites. Higher migration rates for mtDNA than for microsatellites should thus indicate female-biased gene flow.</p>", "<p>To estimate the effective number of migrants from microsatellite data, I ran 10 sequential iterations in MIGRATE using a stepwise mutation model with constant mutation rates, an exponential prior distribution (Θ distribution: minimum = 0.0, maximum = 0.1, mean = 0.01; <italic>M</italic> distribution: minimum = 0.000001, maximum = 1000, mean = 100), starting parameters based on <italic>F<sub>st</sub></italic> calculations, burn-in equaling 10,000 trees, five long chains sampling 2,000,000 genealogies, and an adaptive heating scheme (swapping interval = 1; four chains with start temperatures = 1, 1.2, 1.5 and 3). The same analysis was then repeated using the estimates of Θ and <italic>M</italic> obtained from the first analysis as starting parameters. In this second analysis, a search window for the exponential prior distribution was set according to the distribution of parameter estimates from the first analysis (Δ = 0.03 for Θ; Δ = 110 for <italic>M</italic>). For the mtDNA dataset, I used the same analytical strategy with the F84 model of DNA sequence evolution instead of the stepwise microsatellite mutation model. However, I increased the number of sampled genealogies to 10,000,000 to achieve convergence, and used wider windows in the second run (Δ = 0.06 for Θ; Δ = 250 for <italic>M</italic>). These analyses produced values of Θ<italic>M</italic> (4<italic>N<sub>e</sub>m</italic> and 2<italic>N<sub>f</sub>m</italic> for microsatellites and mtDNA, respectively) estimated in each direction between the two populations along with their approximate 95% confidence intervals ##UREF##33##[0.025 and 0.975 posterior distribution values, 66]##. Following Wright et al. ##REF##15773946##[67]##, I then calculated the overall number of migrants per generation (<italic>N<sub>e</sub>m</italic>) by summing Θ<italic>M</italic> in each direction and dividing by four for microsatellites and two for mtDNA.</p>" ]
[ "<title>Results</title>", "<title>Genetic differentiation between primary and logged forest populations</title>", "<p>Microsatellite allelic diversity was moderate in both <italic>T. tana</italic> populations, ranging from two to nine alleles (mean = 6.43) in the primary forest and from two to six alleles (mean = 4.0) in the logged forest (##TAB##0##Table 1##). Allelic richness, a measure of allelic diversity independent of sample size, showed a similar pattern (##TAB##0##Table 1##). Genotypic differentiation between the two populations was highly significant overall (<italic>P</italic>&lt;0.0001), as well as for four out of the seven loci (JS183, JS188, SKTg19, and SKTg22; ##TAB##0##Table 1##). There were zero fixed differences and 14 shared mutations between populations in the 602 bp mtDNA d-loop sequence. The average number of nucleotide substitutions per site between populations was <italic>D</italic>\n<sub>xy</sub>±SD = 0.026±0.007, and the net substitutions per site was <italic>D</italic>\n<sub>a</sub>±SD = 0.0016±0.006. In contrast to the microsatellite genotypes, genetic differentiation in the mtDNA sequence was not significant between the two populations (<italic>S</italic>\n<sub>nn</sub> = 0.66, <italic>P</italic> = 0.16).</p>", "<title>Female-biased dispersal</title>", "<p>In agreement with predictions for FBD, I found significantly lower <italic>mAI</italic>\n<sub>c</sub> for adult females than for adult males (##TAB##1##Table 2##). Mean <italic>AI</italic>\n<sub>c</sub> was negative for females (mean = −0.70) and positive for males (mean = 0.48), indicating that females are more likely to be immigrants than males. Two method-of-moment estimators of relatedness, Lynch and Ritland's <italic>r</italic> (##FIG##0##Figure 1##) and Queller and Goodnight's <italic>r</italic>, also indicated that adult female pairs were significantly less related than adult males (<italic>P</italic>&lt;0.05; ##TAB##1##Table 2##).</p>", "<title>Female-biased gene flow</title>", "<p>Bayesian inference of migration rates produced an estimate for mtDNA of 2<italic>N<sub>f</sub>m</italic> = 8.20 (95<sup>th</sup> percentile = 1.64–24.56) from primary to logged forest and 2<italic>N<sub>f</sub>m</italic> = 3.35 (95<sup>th</sup> percentile = 0.07–13.46) from logged to primary forest. These two estimates produce an overall estimate of <italic>N<sub>f</sub>m</italic> = 5.77. Assuming an equal sex ratio and low variance in male reproductive success, this value is equivalent to <italic>N<sub>e</sub>m</italic> = 11.54 effective migrants exchanged per generation between the two populations.</p>", "<p>Microsatellite estimates of the effective number of migrants were substantially less than mtDNA estimates. Bayesian inference produced an estimate across all seven loci of 4<italic>N<sub>e</sub>m</italic> = 12.26 (95<sup>th</sup> percentile = 5.93–15.27) from primary to logged forest and 4<italic>N<sub>e</sub>m</italic> = 2.04 (95<sup>th</sup> percentile = 1.05–3.40) from logged to primary forest. These estimates correspond to an overall effective number of migrants exchanged per generation of <italic>N<sub>e</sub>m</italic> = 3.58, which is more than three times less than <italic>N<sub>e</sub>m</italic> estimated for mtDNA.</p>" ]
[ "<title>Discussion</title>", "<p>Multiple genetic analyses presented here provide evidence of FBD in large treeshrews. As predicted for FBD, adult females had significantly lower mean values than males for two different tests (<italic>mAI<sub>c</sub></italic> and pairwise relatedness). These methods detect sex-biased dispersal only when adults have been thoroughly sampled and the sex bias is intense ##REF##12030985##[e.g. 80:20 in simulated datasets, 22]##. A sex bias was detected for <italic>T. tana</italic> despite moderate sample sizes and genetic variability at seven microsatellite markers, suggesting that dispersal is substantially female-biased in this species. The magnitude of the difference between males and females in <italic>mAI</italic>\n<sub>c</sub> (1.18) for large treeshrews was similar to values for two other cases where a sex bias was also confirmed using trapping data ##REF##9061966##[mean of 1.82 in white]##-##REF##10434424##[toothed shrews, 9, 1.35 in white–footed mice, 57]##.</p>", "<p>Evidence of FBD in <italic>T. tana</italic> was also provided by significantly lower relatedness values among adult females than among males for two pairwise measures of relatedness. Average male and female relatedness were negative for two method-of-moment regression estimators (##FIG##0##Figure 1##), but negative relatedness values are not unexpected given the high sampling variance of these estimators inherent in all but the largest data sets ##REF##10430599##[60]##, ##UREF##30##[e.g. &gt;40 loci, 63]##. Negative pairwise relatedness results whenever one pair member exhibits the other's alleles at a frequency less than the estimated population frequency ##REF##15525404##[68]##. Female relatedness may thus be negative more often if immigrant females with genotypes that do not reflect overall population allele frequencies are present in the sample. A relatively large proportion of related individuals (e.g. male relatives, as predicted if males disperse less often) in the sample could also contribute to negative relatedness for unrelated females. These methods do not distinguish between biases in the numbers of individuals of each sex dispersing vs. the distances dispersed. This study also did not address whether males are actually philopatric, but male offspring born in one study period were typically not present on their natal territory in the following study period ##UREF##22##[45]##. The differences in <italic>mAI<sub>c</sub></italic> and relatedness for <italic>T. tana</italic> were likely caused by females with uncommon genotypes that immigrated to the study site (i.e. a bias in the dispersal distance) rather than male philopatry.</p>", "<p>The prediction of greater migration (i.e. gene flow) rates for maternally inherited markers than bi-parentally inherited markers was also supported. The overall number of migrants per generation estimated using mtDNA was more than three times higher than the microsatellite estimate. The substantially higher migration rate for mtDNA suggests that historical gene flow in large treeshrews has been female-biased. Recent studies have raised concerns that migration rates and confidence intervals estimated from mtDNA using maximum likelihood coalescence techniques are often not accurate ##REF##15012759##[69]##. However, the Bayesian coalescence approach implemented in this study ameliorates these problems by achieving improved accuracy and more thorough genealogical sampling ##REF##16317072##[47]##. The magnitude of the difference in migration for mtDNA and microsatellite markers may be reduced if <italic>T. tana</italic> samples for this study violate the assumptions of an equal sex ratio and equal variance in male and female reproductive success. However, variance in reproductive success was not different between males and females, and the sex ratio of offspring was equal in these populations ##UREF##22##[45]##, indicating that these assumptions are reasonable for <italic>T. tana</italic>.</p>", "<p>This study is only the second to find genetic evidence of FBD, and the first to report female-biased gene flow, in a behaviorally monogamous mammal. Evidence for FBD is more prevalent among polygynous mammals ##UREF##14##[especially primates]##, ##REF##16615216##[e.g. 27,36]## with social and mating systems characterized by inbreeding avoidance and male kin-cooperation rather than LRC ##REF##17402974##[19 spp., 18]##. The only other genetic evidence of FBD in a behaviorally monogamous species comes from studies on a temperate shrew <italic>C. russula</italic>, which also exhibited lower <italic>mAI<sub>c</sub></italic> values among females than males ##REF##9061966##[9]##. However, behavioral pairs of <italic>C. russula</italic> only persist for less than one breeding season, placing them at the short-term end of the continuum of pair duration in behaviorally monogamous mammals ##UREF##8##[13]##. Large treeshrews represent a unique case study of FBD because they form behaviorally monogamous pairs that persist for several breeding periods ##UREF##23##[and potentially for life, 46]##, but also exhibit substantial extra-pair paternity ##UREF##22##[45]##. Thus, one might predict the opposite sex bias in dispersal due to competition between male treeshrews for extra-pair copulations. Potentially unexpected results such as these from treeshrews highlight the need to identify specific pressures driving FBD in species with contrasting mating systems ##REF##17402974##[18]##.</p>", "<p>Greenwood ##UREF##1##[3]## predicted that monogamy would correlate with FBD because a sexual asymmetry in the costs of resource competition may favor the evolution of these two behavioral patterns. Foraging competition is the most likely driver of the evolution of behavioral monogamy in large treeshrews ##UREF##21##[44]##, and would also be expected to exert evolutionary pressure on dispersal patterns. Treeshrews live in behaviorally monogamous pairs, but forage solitarily and do not share sleeping sites. This dispersed form of behavioral monogamy likely arose through a two-step evolutionary scenario: female avoidance and territoriality due to foraging competition, followed by male defense of a single female's territory to prevent other males feeding in the same area ##UREF##21##[intersexual feeding competition hypothesis]##, ##UREF##34##[44,70]##. Female body condition and reproductive output increase during supra-annual fruit masting events in Borneo, suggesting that fruit abundance is a key factor limiting reproduction in this species ##UREF##21##[44]##, ##UREF##23##[46]##. The unique, energetically-expensive absentee maternal care system of <italic>T. tana</italic> may also limit the ability of females to produce young on poor-quality territories, or during periods of resource scarcity. These physiological and behavioral limitations on reproduction are likely to produce intense competition between females for resources, and may be the main factor driving females to disperse away from their natal territory to settle on a high-quality territory for their own reproduction.</p>", "<p>The costs and benefits influencing the evolution of behavioral monogamy appear to influence dispersal patterns in large treeshrews. The fitness benefits that females gain from dispersal and the proximate factors influencing dispersal rates are fruitful areas for future research that could be addressed using provisioning experiments. Benefits males gain from philopatry, if any and if they are indeed philopatric, also deserve closer examination. The results from this study also indicate that gene flow is ongoing between <italic>T. tana</italic> populations in primary forests and logged forests in Sabah, Malaysia. Southeast Asia has experienced greater rates of deforestation than other tropical regions ##REF##16701328##[71]##, and Sabah is typical in that most of the valuable timber has already been extracted from its lowland rainforests ##UREF##35##[72]##. Most vertebrate species are present after logging, but the connectivity of populations in primary and logged forests is not well understood ##UREF##36##[73]##. I found significant genotypic differentiation at microsatellite loci between the primary and logged forest populations, but gene flow estimated for mtDNA suggests that female migration may be sufficiently high to avoid rapid loss of genetic variation among large treeshrews in Sabah.</p>" ]
[]
[ "<p>Conceived and designed the experiments: JMS. Performed the experiments: JMS. Analyzed the data: JMS. Contributed reagents/materials/analysis tools: JMS. Wrote the paper: JMS.</p>", "<p>Current address: Department of Natural Sciences, Baruch College, City University of New York, New York, New York, United States of America</p>", "<title>Background</title>", "<p>Female-biased dispersal (FBD) is predicted to occur in monogamous species due to local resource competition among females, but evidence for this association in mammals is scarce. The predicted relationship between FBD and monogamy may also be too simplistic, given that many pair-living mammals exhibit substantial extra-pair paternity.</p>", "<title>Methodology/Principal Findings</title>", "<p>I examined whether dispersal and gene flow are female-biased in the large treeshrew (<italic>Tupaia tana</italic>) in Borneo, a behaviorally monogamous species with a genetic mating system characterized by high rates (50%) of extra-pair paternity. Genetic analyses provided evidence of FBD in this species. As predicted for FBD, I found lower mean values for the corrected assignment index for adult females than for males using seven microsatellite loci, indicating that female individuals were more likely to be immigrants. Adult female pairs were also less related than adult male pairs. Furthermore, comparison of Bayesian coalescent-based estimates of migration rates using maternally and bi-parentally inherited genetic markers suggested that gene flow is female-biased in <italic>T. tana</italic>. The effective number of migrants between populations estimated from mitochondrial DNA sequence was three times higher than the number estimated using autosomal microsatellites.</p>", "<title>Conclusions/Significance</title>", "<p>These results provide the first evidence of FBD in a behaviorally monogamous species without mating fidelity. I argue that competition among females for feeding territories creates a sexual asymmetry in the costs and benefits of dispersal in treeshrews.</p>" ]
[]
[ "<p>I am grateful to the Malaysia Economic Planning Unit, Dr. Henry Bernard and Prof. Datin Dr. Maryati Mohamed at the Institute of Tropical Biology and Conservation, University of Malaysia, Sabah, Danum Valley Management Committee, Royal Society Southeast Asian Rainforest Research Programme, Animal Behavior Society, University of Maryland, Cleveland Zoological Society, Sigma Xi, American Society of Mammalogists, and Explorers Club for logistical support and funding. Animal procedures were approved by the University of Maryland's Animal Care and Use Committee.</p>" ]
[ "<fig id=\"pone-0003228-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003228.g001</object-id><label>Figure 1</label><caption><title>Frequencies of pairwise relatedness values (Lynch &amp; Ritland's <italic>r</italic>) for male (gray bars) and female (black bars) large treeshrews.</title></caption></fig>" ]
[ "<table-wrap id=\"pone-0003228-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003228.t001</object-id><label>Table 1</label><caption><title>Number of alleles and allelic richness of seven microsatellite loci among large treeshrews from the primary forest (<italic>N</italic> = 39) and logged forest (<italic>N</italic> = 15) populations.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td colspan=\"3\" align=\"left\" rowspan=\"1\">No. alleles</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">Allelic richness</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Locus</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Primary</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Logged</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Primary</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Logged</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> value</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">JS22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.87</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.11</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">JS132</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.64</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">JS183</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.74</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.93</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.68</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.02</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">JS188</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.86</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.93</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.86</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;0.001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">JS196</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.76</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.57</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.55</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SKTg19</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.48</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SKTg22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.79</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;0.0001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.43</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.96</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003228-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003228.t002</object-id><label>Table 2</label><caption><title>Mean values and tests of FBD based on the corrected assignment index (<italic>mAI</italic>\n<sub>c</sub>) and two relatedness estimators.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Test</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>mAI</italic>\n<sub>c</sub>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.48</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.70</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Lynch-Ritland <italic>r</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.05</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Queller-Goodnight <italic>r</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.04</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;0.05</td></tr></tbody></table></alternatives></table-wrap>" ]
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[ "<table-wrap-foot><fn id=\"nt101\"><label/><p>\n<italic>P</italic> values correspond to 10,000 randomizations of log-likelihood <italic>G</italic> tests of population differentiation for each locus. The test of population differentiation over all loci was highly significant (<italic>P</italic>&lt;0.0001).</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>Institute of Tropical Biology and Conservation, University of Malaysia, Sabah, Danum Valley Management Committee, Royal Society Southeast Asian Rainforest Research Programme, Animal Behavior Society, University of Maryland, Cleveland Zoological Society, Sigma Xi, American Society of Mammalogists, and Explorers Club. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></fn-group>" ]
[ "<graphic id=\"pone-0003228-t001-1\" xlink:href=\"pone.0003228.t001\"/>", "<graphic xlink:href=\"pone.0003228.g001\"/>", "<graphic id=\"pone-0003228-t002-2\" xlink:href=\"pone.0003228.t002\"/>" ]
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{ "acronym": [], "definition": [] }
73
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 17; 3(9):e3228
oa_package/7d/d1/PMC2528962.tar.gz
PMC2528963
18795101
[ "<title>Introduction</title>", "<p>Spike timing in nerve cells is determined by temporal integration of synaptic potentials and intrinsic response properties. However, little is known about the timescale of this integration during functional network activity and how it is affected by synaptic events. In the absence of synaptic input the spike afterhyperpolarization (AHP) determines spike timing during repetitive firing. In motoneurons (MNs), the frequency range of this firing is well suited for force regulation in the muscle fibers they innervate ##REF##17287343##[1]##. This suggests that main role of AHP is temporal filtering that converts the continuous asynchronous synaptic bombardment to a regular output discharge of action potentials. Furthermore, firing maintained by AHP and other slow intrinsic properties is also appealing because it is a metabolically inexpensive way of shaping the spike patterns to suit particular functions, e.g. spinal motor rhythms ##REF##10747207##[2]##–##REF##16112756##[5]##.</p>", "<p>On the other hand, the AHP and other slow intrinsic properties would impede rapidly changing motor responses and it is not known how resilient they are to a noisy background of synaptic activity. Recent evidence suggests that intrinsic response properties may be shunted by synaptic conductance in cortical and sub-cortical networks ##REF##12951566##[6]##–##REF##9620800##[10]##. In the spinal cord of the adult turtle, scratch motor network activity is associated with a dramatic rise in conductance and in fluctuations of the membrane potential (V<sub>m</sub>) in both MNs and interneurons during spiking ##REF##16000621##[11]##, ##REF##3253428##[12]##. This is due to a concurrent intense inhibitory and excitatory synaptic activity ##REF##17234950##[13]##. Under these conditions of high synaptic conductance, the temporal resolution is predicted to be enhanced ##REF##164391##[14]##–##UREF##2##[16]## and the role of slow intrinsic properties becomes less obvious. Surprisingly few experimental studies have explored this interplay between high synaptic conductance, AHP and temporal integration in active networks.</p>", "<p>For this reason, we have conducted experiments on spinal motoneurons embedded in a functionally active network during fictive motor behavior. In earlier studies the conductance increase in motoneurons during fictive locomotor and scratch network activity was first measured in vivo in the cat ##REF##4078609##[17]##, ##REF##12223580##[18]## and in the turtle ##REF##3253428##[12]##. The isolated spinal cord-carapace preparation from the turtle ##REF##6850342##[19]## offers uniquely stable recording conditions in which intrinsic and synaptic conductance changes during network activity can be measured against a background of very low leak conductance ##REF##16000621##[11]##, ##REF##17234950##[13]##, ##UREF##3##[20]##. This allows us for the first time to quantify the relative importance of active and passive intrinsic properties and the dynamics of synaptic input for spike timing during functional network activity.</p>", "<p>We measured the effective integration time in MNs during network activity using a novel statistical approach that quantified the V<sub>m</sub>-fluctuations before and after the action potential. Three temporal features were characterized: the membrane time-constant, <italic>effective synaptic integration time</italic> (eSIT) and the <italic>effective recovery time</italic> (eRT). We define the eSIT as the time it takes to sum up enough synaptic input to cause a spike. The eRT is defined as the time it takes for the V<sub>m</sub>-distribution following a spike to return to the pre-spike condition, i.e. how long it takes the cell to “forget” that a spike has occurred. We report eRT as short as 4 ms during network activity, which is more than a 10-fold decrease compared with quiescent network. Our results show that even prominent intrinsic response properties like the AHP are severely attenuated concurrent with increase in synaptic conductance. For this reason, the contribution of synaptic activity and active membrane properties to network dynamics can only be captured by a conductance-based model ##REF##18385337##[8]##, ##UREF##2##[16]##, ##UREF##4##[21]##, ##UREF##5##[22]## (##FIG##0##Figure 1##). We conclude that a shortening of the recovery time and integration time of motoneurons cause an increase in the temporal resolution of the motor system during activity, which we suggest as a mechanism to facilitate rapidly changing movements.</p>" ]
[ "<title>Materials and Methods</title>", "<p>All the experiments were performed in an integrated spinal cord-carapace preparation from the adult turtle except the heuristic control experiment of NMDA-induced spike frequency adaptation in ##FIG##5##figure 6A##, which was performed in a transverse slice from adult turtle. In the integrated preparation the spinal cord remains in the spinal canal with the tactile sensory nerves from the carapace intact. The motor nerves are carefully transected to avoid muscle movements and dissected out to for electro-neurogram recordings. The scratch reflex was activated by mechanical somato-sensory stimulation of selected regions on the carapace (see below) as described ##REF##16000621##[11]##, ##UREF##3##[20]##.</p>", "<title>Integrated preparation</title>", "<p>Red-eared turtles (<italic>Trachemys scripta elegans</italic>) were placed on crushed ice for 2 hrs to ensure hypothermic anesthesia. Animals were killed by decapitation and blood substituted by perfusion with a Ringer solution containing (mM): 120 NaCl; 5 KCl; 15 NaHCO<sub>3</sub>; 2 MgCl<sub>2</sub>; 3 CaCl<sub>2</sub>; and 20 glucose, saturated with 98% O<sub>2</sub> and 2% CO<sub>2</sub> to obtain pH 7.6. The carapace containing the D4-D10 spinal cord segments was isolated by transverse cuts and removed from the animals, similar to studies published elsewhere ##REF##6850342##[19]##, ##UREF##3##[20]##. The surgical procedures complied with Danish legislation and were approved by the controlling body under the Ministry of Justice.</p>", "<title>Slice preparation</title>", "<p>One mm thick slices of the turtle spinal cord were placed in a chamber for intracellular recording and submerged in and perfused with oxygenated Ringer solution. The pharmacological agent N-methyl-D-aspartate (NMDA) was added to the ringer medium to induce bursting activity (10 µM).</p>", "<title>Recordings</title>", "<p>Intracellular recordings in current-clamp mode were performed with an Axoclamp-2A amplifier (Axon Instruments, Union City, CA). Glass pipettes (part no. 30-0066, Havard Apparatus, UK) were pulled with a electrode puller (model P-87, Sutter instrument co., USA) and filled with a mixture of 0.9 M potassium acetate and 0.1 M KCl. Intracellular recordings were obtained from neurons in segment D10. Recordings were accepted if neurons had a stable membrane potential more negative than −50 mV. Data were sampled at 20 kHz with a 12-bit analog-to-digital converter (Digidata 1200, Axon Instruments, Union City, CA), displayed by means of Axoscope and Clampex software (Axon Instruments, Union City, CA), and stored on a hard disk for later analysis. Hip flexor nerve activity was recorded with a differential amplifier Iso-DAM8 (WPI) using a suction pipette. The bandwidth was 100 Hz–1 kHz.</p>", "<title>Activation of network</title>", "<p>Mechanical stimulation was performed with the fire polished tip of a bent glass rod mounted to the membrane of a loudspeaker in the cutaneous region known to elicit “pocket scratch” ##REF##3253428##[12]## which results in a broad activation of cells ##REF##11495969##[59]##. The duration, frequency, and amplitude of the stimulus were controlled with a function generator (##FIG##1##Figure 2A##). This tactile stimulus induced the scratch-like network activity, which was monitored by the suction electrode nerve recordings from the Hip-flexor nerve (##FIG##1##Figure 2B##).</p>", "<title>Model data</title>", "<p>The data used to illustrate the difference between synaptic-current and synaptic-conductance based fluctuating inputs (##FIG##0##Figure 1##) was based on a one-compartment model simulation ##UREF##5##[22]## supplemented with Ca<sup>2+</sup> conductance and a Ca<sup>2+</sup>-activated K<sup>+</sup>-conductance. The synaptic noise was modeled as white current noise in the current-based regime with the heuristic expression,where the fast conductances of the action potential and Ca<sup>2+</sup>-conductance were not shown here for simplicity (for complete description see ##SUPPL##0##Data and Methods S1##) and the total membrane conductance waswhich contains no synaptic component. In this scheme, the synaptic input was represented as a current, I<sub>syn</sub>. The membrane capacitance is C, and G<sub>leak</sub>, E<sub>leak</sub>, G<sub>AHP</sub>, E<sub>K</sub> are conductance and reversal potential of leak and AHP, respectively.</p>", "<p>In a more realistic regime, the high intensity synaptic input was modeled as a conductance ##UREF##5##[22]##. Here, the AHP was reduced as a consequence of the increase in total input conductance (##FIG##0##Figure 1##). The conductance-based regime applies when the synaptic conductance (G<sub>syn</sub>) is so large that it can no longer be considered small compared with the G<sub>total</sub>\n##REF##12951566##[6]##. Heuristically expressed similar to the synaptic current modelwhere E<sub>syn</sub> is the weighted reversal potential of excitatory and inhibitory synaptic reversal potentials and G<sub>syn</sub> is the sum of both conductances. G<sub>syn</sub> is competing with G<sub>AHP</sub> in controlling inter-spike intervals, and if it is large enough it can render G<sub>AHP</sub> insignificant at steady state:when <italic>G<sub>Syn</sub></italic>≫<italic>G<sub>AHP</sub></italic>. The action potentials were largely unaffected by the conductance increase, since fast Na/K conductances of the action potential were much larger. In a simple Hodgkin-Huxley-model added AHP- and Ca<sup>2+</sup>- conductances and including synaptic input as either current noise (I<sub>syn</sub> = 12 nA, σ<sub>syn</sub> = 3 nA, OU-simulated with time-constant = 1 ms and D = 0.0005) or conductance noise we verified the theoretical importance of synaptic conductance (##FIG##0##Figure 1##). The conductance noise was a mixture between inhibition and excitation balanced at −60 mV (see ##SUPPL##0##Data and Method S1## for details).</p>", "<title>Evolution of V<sub>m</sub>-distribution</title>", "<p>The variable constituting the estimation of effective eSIT and eRT was the membrane potential (V<sub>m</sub>). This variable was stochastic and a sample measurement drawn from an underlying probability distribution function (P<sub>V</sub>), which we assumed had the same statistics in all interspike intervals. The probability distribution depends on time after occurrence of spike and this dependence was a manifestation of intrinsic current generators like SK-channels. Because of the large synaptic fluctuations, it was necessary to look at the distribution P<sub>V</sub> instead of just isolated instances of V<sub>m</sub>. These fluctuations were assumed uncorrelated from trial to trial, so we could estimate P<sub>v</sub> by superimposing spikes.</p>", "<p>The key assumption is, if the distribution at some given point in time, P<sub>v</sub>(t<sub>1</sub>), is different from the distribution at a later point in time, P<sub>v</sub>(t<sub>2</sub>), then there has been a change in the intrinsic current generation (cf. ##FIG##2##Figure 3A and B##). We selected P<sub>v</sub> at one point in time (t = t<sub>template</sub>) as a template distribution, which all distributions P<sub>V</sub>(t≠t<sub>template</sub>) were compared with. The P<sub>V</sub>(t<sub>template</sub>) was chosen more than 10 ms before the spike, since this region constitute a background V<sub>m</sub>, and was compared via the Kolmogorov-Smirnov-2 sample test (KS-test) ##UREF##14##[60]## expressed formally as:\n</p>", "<p>F(V, t) is the empirical cumulative probability distribution function of V<sub>m</sub>. N is the total number of traces (and spikes) used to estimate the distribution at time t (##FIG##2##Figure 3D##). The maximal difference between the cumulatives, D(t), was the measure for rejecting or accepting the null hypothesis. If the difference was larger than a critical value, then we rejected the null hypothesis that V<sub>m</sub>(t) and V<sub>m</sub>(t<sub>template</sub>) were drawn from the same distribution. The binary test outcome was plotted (##FIG##2##Figure 3A and B##) where 1 represented rejection (gray area) and 0 represented no rejection of null hypothesis, at a 5% confidence limit. The p-value of the test was plotted below.</p>", "<title>Effective Recovery time</title>", "<p>The first point in time after the spike, where the KS-test was zero (i. e. no rejection of hypothesis of same distribution) was where we defined the AHP conductance and other transient intrinsic current generators no longer had a significant impact on the V<sub>m</sub> and the passive diffusive spread had reach steady state. We dubbed this period <italic>effective recovery time</italic> (<italic>eRT</italic>, arrow in ##FIG##2##Figure 3B##) in analogy to the effective membrane resistance and effective membrane time constant ##REF##164391##[14]##, ##UREF##2##[16]##, ##UREF##4##[21]##, ##UREF##5##[22]##. This recovery time told us how long time after the spike had occurred that there was still a <italic>memory</italic> of the spike in V<sub>m</sub> (##FIG##2##Figure 3B##).</p>", "<title>Effective synaptic integration time</title>", "<p>Similar to eRT, we could ask how long time prior to the spike, that P<sub>v</sub> was different from the template distribution. This point represented a net depolarization caused either by reduced inhibition or increased excitation. We named this period <italic>effective synaptic integration time</italic> (eSIT) indicating the time prior to the spike where its occurrence can be predicted (arrow in ##FIG##2##Figure 3B##).</p>", "<title>Location of template</title>", "<p>Obviously, the choice of template distribution is important. The template is always chosen prior to the spike. The earlier before the spike we choose the template, the more independent it is. However, there is a trade off, since the inter-spike interval has to be longer than the window between the template distribution and the spike. As a result, the larger the window is, the fewer spikes in a finite dataset will participate in the distribution (##FIG##2##Figure 3D##). Fortunately, the estimation of both eRT and eSIT is largely independent on the window size (##FIG##2##Figure 3C##). We chose to average the values of eRT and eSIT from templates 20 to 40 ms prior the spike, since these locations gave nearly constant values (##FIG##2##Figure 3C##).</p>", "<title>Critique of method</title>", "<p>The above described statistical testing of the evolution of P<sub>v</sub> only accounts for changes that are locked to the occurrence of a single action potential, such as the AHP. Accumulative events that build up over several spikes as e.g. spike frequency adaptation or plateau potentials are not easily accounted for using this statistics. One way to test for slow changes would be to divide the spikes according into several different groups depending on their position in the epoch. These groups could then be compared to evaluate if the distributions have changed. However, we decided this was outside the scope of the present study and it was not necessary since the test of spike frequency adaptation (##FIG##5##Figure 6##) came out negative.</p>", "<title>Inter-spike interval analysis</title>", "<p>The inter-spike intervals were extracted from the intracellular recording during scratch episodes and processed. The auto-correlations were calculated as the normalized covariance function ##UREF##7##[26]##, ##UREF##15##[61]## between ISI<sub>N</sub> and ISI<sub>N+1</sub>, ISI<sub>N+2</sub>, ISI<sub>N+3</sub> etc (##FIG##5##Figure 6d##). The test for spike frequency adaptation (##FIG##5##Figure 6e##) was performed assuming statistical independence of observation of (ISI<sub>N</sub>, ISI<sub>N+1</sub>)-pairs ##UREF##9##[29]## and thus a binomial distribution with chance of 50% above and 50% below the line ISI<sub>N</sub> = ISI<sub>N+1</sub>. If the number of points above was within a standard deviation ofof the 50%-line, as expected from an even binomial distribution ##UREF##15##[61]##, there was no significant spike frequency adaptation. K is the total number of points and p is the probability of a point being above the line, when assuming no correlation.</p>", "<title>Data processing</title>", "<p>All analysis was performed in Matlab (version 7.3, Mathworks). The data was converted from Axoclamp format to matlab and the spikes were identified and superimposed (##FIG##2##Figure 3##) to study the V<sub>m</sub> statistics before and after the spike as described above. KS-testing of the V<sub>m</sub> distributions was done with the matlab procedure “kstest2.m”. The custom made procedures for calculating eRT and eSIT has been uploaded to mathworks code sharing web site (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mathworks.com/matlabcentral/\">http://www.mathworks.com/matlabcentral/</ext-link>) with the name “eRT.m” for the interested reader. The remaining matlab code is available on request.</p>" ]
[ "<title>Results</title>", "<p>The question we ask is how the surge in conductance from intense synaptic bombardment during network activity modifies intrinsic properties exemplified by the spike AHP and shrinks response time and integration time of the neuron. The sensitivity of spike generation and spike pattern to conductance is illustrated in simulations in a simple model (##FIG##0##Figure 1##). In the absence of synaptic input the regular firing during maintained depolarization in the model neuron was determined by the AHP (red trace in ##FIG##0##Figure 1A##). Synaptic input simulated as current noise, i.e. linear summation of synaptic potentials, resulted in greater variability in spike timing, but did not change the averaged AHP (black trace ##FIG##0##Figure 1A, B##). Thus, current-based synaptic input fluctuations changed neither total conductance nor neuronal integration time. However, when synaptic activity was modeled more realistically as a noisy conductance increase, spike timing was strongly influenced by the stochastic fluctuations in V<sub>m</sub> (blue trace, ##FIG##0##Figure 1A, B##), the AHP was severely attenuated (arrow in ##FIG##0##Figure 1B##) and the integration time was shortened ##UREF##5##[22]##, ##REF##10200189##[23]##. Thus, the simple simulation showed that a fluctuating current is not likely to affect the integration time or reduce the importance of the AHP whereas intense and fluctuating synaptic conductance is.</p>", "<p>For the purpose of making these statements applicable to quantitative evaluation of experimental data, we developed statistics to capture the temporal features of V<sub>m</sub> before and after the spike. We define two measures linked to the superposition of spikes (##FIG##0##Figure 1C and D##). First, the <italic>effective synaptic integration time</italic> (eSIT), defined as the time period prior to spikes during which fluctuations in membrane potential showed a significant depolarizing trend compared with a baseline template (##FIG##0##Figure 1D##). Likewise, we define the <italic>effective recovery time</italic> (eRT), as the time it takes to regain pre-spike V<sub>m</sub>-levels. Notice that both eRT and eSIT are absent in current noise model whereas they are present in the conductance noise model (cf. ##FIG##0##Figure 1 C and D##).</p>", "<p>To evaluate whether the neuronal integration time and AHP are substantially affected during intense network activity we measured eSIT and eRT in MN during scratching. The data set consisted of more than 10.000 spikes in 185 scratch epochs in 17 MNs. The analysis is organized as follows. First, we estimated the conductance increase in a sub-sample of MN to confirm that strong synaptic components were present (##FIG##1##Figure 2##). Next, we applied our statistics to measure the eSIT and eRT and estimated the effective membrane time-constant from V<sub>m</sub>-fluctuations at different levels of synaptic activity. Since intensity of synaptic activity may vary among cells, we compared the eRT with indicators of input, i.e. the effective membrane time constant as well as the smallest inter-spike interval (ISI) for each MN. Finally, since the functional expression of AHP accumulation in motoneurons is spike frequency adaptation (SFA) ##UREF##0##[4]##, ##REF##16725251##[24]##, we also tested for adaptation during each cycle of the scratch epochs.</p>", "<title>Synaptic conductance</title>", "<p>A scratch epoch was induced by rhythmic cutaneous stimulation of the skin in the hind-limb pocket and the concurrent synaptic conductance (##FIG##1##Figure 2A, B##) was estimated from the voltage deflections to injected current pulses (##FIG##1##Figure 2C–F##) for a subset of MN. Conductance due to action potentials is a potential source of error in these measurements ##UREF##6##[25]##. We therefore avoided spikes by injecting a steady hyperpolarizing current (typically −2 nA) or selected a part of the scratch episode without spikes. The pair of spikes in ##FIG##1##Figure 2F## was left in for illustration, but the sweep was not included in the final estimate. Even with this conservative approach, the estimated conductance increased 2–5 times during scratching (##TAB##0##table 1## and ##FIG##1##Figure 2G##)##UREF##1##[9]##. We attribute this increase to balanced inhibitory and excitatory synaptic input ##REF##17234950##[13]## and conclude that temporal processing in MNs could be affected by synaptic conductance during network activity.</p>", "<title>Effective synaptic integration time</title>", "<p>We first considered the V<sub>m</sub>-statistics prior to spikes. The time of the earliest statistical sign of depolarization prior to action potentials we dub “the effective synaptic integration time” (eSIT). This depolarization may be caused by a rise in excitatory conductance or a fall in inhibitory conductance. For each cell, the eSIT was estimated by comparing a template distribution of V<sub>m</sub> with distributions of V<sub>m</sub> as a function of time prior to action potentials, as in ##FIG##0##figure 1##. The comparison was attained using a KS-two-sample test at each point in time (method, ##FIG##2##Figure 3##). The template distribution was chosen at a time, t<sub>template</sub>, well before the spikes (arrows ##FIG##2##Figures 3A and B##). The eSIT did not depend systematically on the choice of t<sub>template</sub> as long as it was at least 10 ms prior to the spike (##FIG##2##Figure 3C##) even though the number of samples in the distribution decreased (##FIG##2##Figure 3D##).</p>", "<title>Memory of a spike–eRT</title>", "<p>The same statistical test was used to evaluate the impact of a spike by comparing the V<sub>m</sub>-distribution after action potentials with template distributions well before the spike (arrow ##FIG##2##Figure 3B##). In the graph of the KS-test, the gray area represents rejection of the null hypothesis that the V<sub>m</sub>-distribution before and after the spike was statistically similar to the template distribution. When the V<sub>m</sub>-distribution after the spike was indistinguishable from the template distribution, we considered the impact of a spike to have ceased (graph of P-values, ##FIG##2##Figures 3A and B##). Both eRT and eSIT had no clear dependence on t<sub>template</sub> earlier than ∼20 ms (##FIG##2##Figure 3C##). Since the number of spikes in the distribution decreased with t<sub>template</sub> (##FIG##2##Figure 3D##) the eRT estimate should not be performed with a high t<sub>template</sub>. We chose to calculate the mean eRT over the range 20 ms&lt;t<sub>template</sub>&lt;40 ms.</p>", "<title>Impact of τ<sub>eff</sub> and AHP on recovery time</title>", "<p>Right after the occurrence of an action potential V<sub>m</sub> is hyperpolarized. The time it takes for V<sub>m</sub> to re-polarize back to the level prior to the spike depends on the AHP and on the passive effective time-constant of the membrane, which we refer to as τ<sub>eff</sub>. In our attempt to sort out which part of the re-polarization is due to the passive decay of V<sub>m</sub> and which is due to the cessation of AHP conductance, it is important to estimate τ<sub>eff</sub> during network activity. The increase in total conductance will also diminish the relative importance of the AHP conductance and the AHP will appear shorter. To quantify the vestige of the AHP under different levels of input conductance we define the <italic>effective After-Hyperpolarisation Period</italic> (eAHP). This period represents the time it takes until the AHP ceases to affect the V<sub>m</sub> trajectory. If there is no overlap between the passive membrane decay and the eAHP, the eRT is just the sum of τ<sub>eff</sub> and eAHP, while if there is overlap eRT will be less than the sum:\n</p>", "<p>It is important to emphasize the eRT also represents the upper bound on both eAHP and τ<sub>eff</sub>, i.e. eRT≥eAHP and eRT≥τ<sub>eff</sub>. Hence, it was important to determine τ<sub>eff</sub> for each cell in order to determine the contribution of AHP to the integration process.</p>", "<title>Estimating τ<sub>eff</sub> during activity</title>", "<p>For the spinal motor activity we differentiated three situations: The quiescent state with little or no synaptic input; the on-cycle with motor nerve activity and spike activity in motoneurons; and the off-cycle, which is at the low point in between the on-cycles (##FIG##3##Figure 4A–C##). As expected the effective membrane time constant decreased significantly during scratching. For representative data, τ<sub>eff</sub> was 27 ms in the quiescent state, while it was only 2.8 ms in the On-cycle and 5.2 ms in the off-cycle (##FIG##3##Figure 4C–E##). Membrane time constants during quiescence are ordinarily estimated from V<sub>m</sub>-decay times after injected current pulses (##SUPPL##2##figure S2##). During the dynamic and intense synaptic input in the on-cycle and off-cycle, this method was both difficult and imprecise. Instead, if V<sub>m</sub> is assumed to follow a stochastic process known as Ornstein-Uhlenbeck-process (OU-process), then maximum likelihood estimation would be the proper way to obtain τ<sub>eff</sub>\n##UREF##7##[26]##, ##UREF##8##[27]##. The estimated values of τ<sub>eff</sub> with this technique are listed in Data and Methods 1 in ##SUPPL##0##Data and Methods S1## and see ##SUPPL##1##Figures S1##\n–##SUPPL##3##S3##. It turned out that V<sub>m</sub> did not obey an OU-process (##SUPPL##4##Figure S4##) and these estimates of τ<sub>eff</sub> were systematically much higher than eRT and eSIT. Therefore, τ<sub>eff</sub> was instead estimated empirically by fitting an exponential decay to the initial part of the auto-correlation sequence (##FIG##3##Figure 4F##). It was necessary to fit to the initial part (from 0 to 3 ms, vertical gray line) to assure that the auto-correlation lag was small compared to the total length of the sample (200 ms), since the premise of exponential decay is infinite-length of data trace (see ##SUPPL##0##Data and Method S1##).</p>", "<title>Temporal features for the population</title>", "<p>The effective synaptic integration times, the effective recovery times and the effective membrane time constants across the population of neurons are listed in ##FIG##4##Figure 5A##. The average eSIT was <italic>μ</italic> = 7.7±0.8 ms and the average eRT was <italic>μ</italic> = 7.5±0.7 ms (mean±SE, n = 17). The median of eSIT and eRT were both 7.7 ms. The eRT was assumed to be approximately equal to the sum of τ<sub>eff</sub> and the effective AHP (see above). However, the population average of τ<sub>eff</sub> was <italic>μ</italic> = 9.3±1.4 ms (mean±SE, n = 17) while the median of τ<sub>eff</sub> was 7.0 ms. In some cases (41%, n = 7/17) τ<sub>eff</sub> was longer than the corresponding eRT, which suggests two things. First, the reset potentials were closer to the steady state mean V<sub>m</sub> than the natural fluctuations of V<sub>m</sub> around the mean, so the decay from reset back to the mean was faster than τ<sub>eff</sub>. Secondly, the eAHP was close to zero or no more than a couple of milliseconds. This value of eAHP is a dramatic decrease from the 200 ms AHP duration previously reported during quiescence ##UREF##0##[4]##, ##REF##16725251##[24]##. Further, not only did the population spread in eRT across cells correlate with the population spread in eSIT (R<sup>2</sup> = 0.81), the eRT and the eSIT both had significant correlation with τ<sub>eff</sub> (##FIG##4##Figure 5B##) (R<sub>eSIT</sub>\n<sup>2</sup> = 0.60, p = 0.0007, R<sub>eRT</sub>\n<sup>2</sup> = 0.47, p = 0.005, when ignoring the two outliers, cell 7 and 16). Since eRT is dependent on both τ<sub>eff</sub> and eAHP, while eSIT is only dependent on τ<sub>eff</sub>, these strong correlations also indicate that the contribution of eAHP to eRT must be minor in most cells.</p>", "<p>Hence, the population spread in eRT, eSIT, and τ<sub>eff</sub> probably reflected different levels of synaptic intensity in different cells. Since the eRT is the major contribution to the refractory period, we expected most of the inter-spike intervals (ISI) to be longer than or equal to the eRT. If this is the case, the data points in a plot of the shortest ISI versus eRT in each cell should fall at the 45°-line or below. Indeed, all points were within their error bars or below (##FIG##4##Figure 5C##). In this way, the inverse of the eRT represents an upper bound for the spike frequency during network activity.</p>", "<title>Absence of spike frequency adaptation</title>", "<p>The AHP contributes to spike frequency adaptation (SFA) in motoneurons at rest ##UREF##0##[4]##, ##REF##16725251##[24]##. A classical example of SFA in absence of synaptic input is during bursting induced by N-Methyl-D-Aspartic acid (NMDA). We therefore performed a heuristic comparison between NMDA-bursting in MNs in slices with the bursting induced by synaptic input in functional networks (cf. ##FIG##5##Figures 6A and 6B##). During each burst in the case of NMDA-bursting (##FIG##5##Figure 6Aa##), spike frequency peaked at onset and adapted to a lower level at the end of the burst (##FIG##5##Figure 6Ab##). The histogram of interspike-intervals confirmed similarity in firing rate distribution between NMDA-bursting and network bursting ##REF##17502919##[28]## (cf. ##FIG##5##Figure 6Ac and 6Bc##). ISI<sub>N</sub> plotted against ISI<sub>N+1</sub> displayed a ring-like pattern (##FIG##5##Figure 6Ad##), as expected from adapting spike trains in each successive burst ##UREF##9##[29]##. Note that the vast majority of points (83%/17%, SE = 6%) are above the 45°-line, which is a consequence of SFA, presumably in large part produced by the prominent AHP. Under these conditions neighboring ISIs were significantly correlated (R<sup>2</sup> = 0.17) as expected from AHP-mediated adaptation (##FIG##5##Figure 6Ae##).</p>", "<p>The firing pattern in MNs during scratching was qualitatively different (##FIG##5##Figure 6Ba##). Though the distribution of ISI times resembled the NMDA-bursting (cf. ##FIG##5##Figure 6Bc and 6Ac##), spike timing was irregular (##FIG##5##Figure 6Bb##) and points scattered nearly symmetrically around the 45°-line in the return map (51%/49%, SE = 6%) (##FIG##5##Figure 6Bd##). This shows that <italic>spike frequency acceleration</italic> was as prevalent as <italic>spike frequency adaptation</italic> during bursts ##UREF##9##[29]## and therefore that the AHP and other intrinsic mechanisms for adaptation or acceleration in spike frequency did not have a detectable influence on the firing pattern during network activity. Furthermore, the correlation between ISI<sub>N</sub> and ISI<sub>N+1</sub> was marginal (R<sup>2</sup> = 0.03) (##FIG##5##Figure 6Be##), which shows that spikes were driven by a stochastic process rather than by deterministic intrinsic properties. In all 17 motoneurons SFA was insignificant during depolarizing waves, i.e. the numbers above the line were not significantly higher than 50% (##FIG##6##Figure 7##). Thus, we conclude that the mechanisms causing SFA such as AHP accumulation were not pervasive enough to overcome the increase in synaptic conductance during motor activity.</p>" ]
[ "<title>Discussion</title>", "<p>Spike timing, the principal output of neurons, is determined by interacting synaptic and intrinsic ionic conductances. Recent decades have provided a wealth of information about the intrinsic response properties and their proposed roles in specific cell types in many parts of the nervous system ##REF##16112756##[5]##, ##REF##3059497##[30]##, ##REF##11052929##[31]##. Properties of the AHP in particular have been linked to classification of fast and slow spiking neurons with clear functional connotations ##REF##16112756##[5]##, ##REF##17180162##[32]##. The present study reports up to ten-fold decrease in the membrane time constant (##FIG##3##Figure 4##) and severe reduction in the AHP during functional activity (##FIG##2##Figure 3## and ##FIG##4##5##) which emphasize that assertions about the functional significance of intrinsic response properties must be validated in neurons embedded in an active network. In our experimental paradigm, the role of the AHP in spike timing is essentially eliminated and replaced by rapid temporal integration in the high conductance state provided by intense synaptic input (##FIG##2##Figures 3##, ##FIG##4##5##, ##FIG##5##6##). We therefore propose that motor systems perform rapidly changing movements by letting intense synaptic input transiently supplant slow intrinsic properties when necessary.</p>", "<title>Coincidence detection</title>", "<p>This transition has previously been referred to as a transition from temporal integration to coincidence detection in sensory perception ##REF##8658595##[33]##. Konig and colleagues defined a coincidence detector as a neuron in which the integration time for synaptic potentials is short compared with the average ISI, and a temporal integrator if the reverse is true. Adopting this definition, the motoneurons in the present study were all coincidence detectors during motor activity, since their eSIT and eRT on average are an order of magnitude shorter than their average ISI (cf. ##FIG##4##Figure 5A## and ##FIG##5##6Bc##, and see ##SUPPL##5##Figure S5##). The functional effect and benefit of MNs working as coincidence detectors could be to minimize aberrant firing due to the background barrage of noisy synaptic input ##REF##17502919##[28]##. The potentially undesirable consequence of having such a coincidence detection scheme is irregular spike patterns, which we indeed did observe (##FIG##5##Figure 6##). However, irregular patterns are not likely to be a problem, since muscle fibers are slow integrators, and therefore the exact temporal structure of the MN firing, whether irregular and yet fast coincidence detectors or slow regular temporal integrators, is unimportant for securing a smooth contraction.</p>", "<title>Origins of spike pattern</title>", "<p>The network mechanisms underlying the phasic spike activity in motoneurons during rhythmic motor behaviors are unknown. It has been hypothesized that certain intrinsic properties are crucial mediators of bursting rhythms in spinal networks ##REF##17145498##[3]##, ##REF##16112756##[5]##, ##UREF##10##[34]##. It is also widely accepted that the after-hyperpolarization in MN secures repetitive firing at low rates appropriate for regulation of muscle contraction ##REF##17145498##[3]##, ##UREF##0##[4]##. The high conductance state, however, compromises the ability to fire repetitively during steady depolarization ##REF##16000621##[11]##. This is probably a direct consequence of the reduced slow AHP since the same effect is observed when the AHP is reduced by blocking the underlying ionic current pharmacologically rather than by shunting ##REF##2455803##[35]##. Nevertheless, MNs still fired in a broad range of rates in the high conductance state while driven by rapid fluctuations in membrane potential during motor activity (##FIG##5##Figure 6B##) ##REF##17234950##[13]##. Further evidence for fluctuation driven irregular firing is the fact that the shortest ISIs in the active network are longer than the eRT (and thus eAHP) (##FIG##5##Figure 6C##). This is in contrast to the regular firing and frequency adaptation in motoneurons in slices during NMDA induced bursting (##FIG##5##Figure 6A##). The reduced AHP amplitude, increased spike time variability and the absence of adaptation during locomotor network activity have also been observed in MNs in the decerebrate cat ##REF##1426105##[36]##. In the light of our findings it will be interesting to investigate if the underlying mechanism is modulation of intrinsic properties ##REF##17287343##[1]##, ##REF##16725251##[24]##, ##REF##7925804##[37]## or parallel increase in excitatory and inhibitory synaptic activity ##REF##17234950##[13]##, ##UREF##11##[38]##.</p>", "<p>In conclusion, the rhythm-generation could have two origins: a pattern generating subset of neurons elsewhere in the network in which intrinsic response properties are protected from shunting by intense synaptic bombardment or alternatively, the motor rhythm can be an emergent distributed network phenomenon as suggested for respiratory rhythms ##REF##12062027##[39]##, ##REF##16495944##[40]##, i.e. the network bursting hypothesis. Decisive experimental tests of these hypotheses are still missing.</p>", "<title>Caveats of constant current protocols</title>", "<p>The role of AHPs in repetitive firing in MNs induced by depolarizing current through the recording electrode has been thoroughly investigated ##UREF##0##[4]##, ##REF##16112756##[5]##, ##REF##16725251##[24]##. Based on the linear summation of synaptic potentials under certain experimental conditions ##REF##5972179##[41]##, ##REF##10634890##[42]## this approach takes injected current as a simplifying representation of synaptic input. However, important aspects of synaptic input are overlooked in this approach. First, synaptic variability adds fluctuations as a second moment to V<sub>m</sub>\n##UREF##5##[22]##. Synaptic V<sub>m</sub>-fluctuations may have important computational roles ##REF##12194875##[43]##–##UREF##12##[45]##. Secondly, the effect of surge in conductance from synaptic input is unaccounted for in the constant current protocol (##FIG##0##Figure 1##). Therefore the functional role of the AHP and other intrinsic response properties should be assessed either in functional networks with real synaptic input, as in the present study, or by dynamic clamp analysis in neurons at rest ##REF##16261132##[7]##, ##REF##18385337##[8]##, ##REF##15046881##[46]## if a valid estimate of the temporal structure of the functional synaptic conductances is available.</p>", "<title>Balanced state in the spinal cord</title>", "<p>Though anatomical evidence suggests an approximate balance between inhibitory and excitatory contacts in cat motoneurons ##UREF##0##[4]##, the circuit substrate for variations in the balance of inhibition and excitation during motor activity and its prevalence in other spinal networks is yet unresolved. High-conductance states produced by parallel increase in inhibitory and excitatory synaptic activity is a common occurrence in other functional networks ##REF##12951566##[6]##, ##UREF##11##[38]##, ##REF##9870957##[47]##–##UREF##13##[51]##. In the neocortex, the link between excitation and inhibition is provided by feed-forward and recurrent pathways ##REF##16641233##[48]##, ##REF##14647382##[49]##, ##REF##17515899##[52]##–##REF##15665876##[55]##. In the spinal cord, recurrent inhibition is unlikely to contribute significantly to the high conductance state in MNs since recurrent collaterals are scarce in the turtle ##REF##6520242##[56]##, ##REF##16144619##[57]## and Renshaw inhibition has not been documented. Feed-forward inhibition provided by Ia inhibitory interneurons can produce synaptic conductance that clearly reduces intrinsic response properties in cat MNs <italic>in vivo</italic>\n##REF##17293858##[58]##. However, reciprocal inhibition contributes insignificantly to the high conductance state in MNs during breathing ##UREF##11##[38]## and scratching ##REF##17234950##[13]##. We propose that inhibitory input to MN is mostly due to local feedforward inhibitory connections, but it remains to be seen if balanced spinal motor networks resemble architectures of the much better investigated networks in other parts of the nervous system ##REF##12951566##[6]##, ##UREF##11##[38]##, ##REF##16641233##[48]##, ##UREF##13##[51]##, ##REF##15665876##[55]##.</p>" ]
[]
[ "<p>Conceived and designed the experiments: RWB JH. Performed the experiments: RWB. Analyzed the data: RWB. Contributed reagents/materials/analysis tools: RWB SD. Wrote the paper: RWB.</p>", "<p>In neurons, spike timing is determined by integration of synaptic potentials in delicate concert with intrinsic properties. Although the integration time is functionally crucial, it remains elusive during network activity. While mechanisms of rapid processing are well documented in sensory systems, agility in motor systems has received little attention. Here we analyze how intense synaptic activity affects integration time in spinal motoneurons during functional motor activity and report a 10-fold decrease. As a result, action potentials can only be predicted from the membrane potential within 10 ms of their occurrence and detected for less than 10 ms after their occurrence. Being shorter than the average inter-spike interval, the AHP has little effect on integration time and spike timing, which instead is entirely determined by fluctuations in membrane potential caused by the barrage of inhibitory and excitatory synaptic activity. By shortening the effective integration time, this intense synaptic input may serve to facilitate the generation of rapid changes in movements.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>Thanks to Jens Midtgaard for carefully reading an earlier version of the manuscript.</p>" ]
[ "<fig id=\"pone-0003218-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003218.g001</object-id><label>Figure 1</label><caption><title>Computer simulation to illustrate firing pattern and AHP in current-based and conductance based models of synaptic input during repetitive firing.</title><p>(A). Red top trace: Model with constant current injection, no other input. Black middle trace: Model with added current noise with same mean current as in top trace. Blue bottom trace: Model with same mean injected constant current and noise stemming from fluctuating conductance. (B). Spike triggered superimposed spikes. Red, black (n = 243) and blue (n = 72) traces are averages of spikes from (A). Notice the current-noise average closely overlap the no-noise trace (red) whereas the conductance-noise average (blue) rapidly reach pre-spike V<sub>m</sub>-level (see arrow), because of increase in total conductance. (C) and (D) illustrate the statistical quantification of the evolution of V<sub>m</sub> before and after the spike for traces in (B). A template distribution of V<sub>m</sub> traces is chosen at an arbitrary time prior to the spike (see arrow at t<sub>template</sub>) for which the distribution at the rest of the time points is compared with. The outcome of comparison is shown as the KS-test trace below, 1 represents acceptance and 0 represents rejection of the hypothesis that they are different. Below is shown the P-values for the KS test. The distribution of V<sub>m</sub> in (C) is different everywhere, whereas the distribution in (D) is only different up to and immediately after the spike. The time it takes to regain the pre-spike distribution following the spike is referred to as <italic>effective recovery time</italic> (eRT, arrow), while the endmost time of same distribution before the spike is referred to as <italic>effective synaptic integration time</italic> (eSIT, arrow).</p></caption></fig>", "<fig id=\"pone-0003218-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003218.g002</object-id><label>Figure 2</label><caption><title>Increase in synaptic conductance during a single scratch epoch.</title><p>(A) Cutaneous stimulation via sinusoidal movements of a glass rod on the hind-limb pocket skin. (B) Electro-neurogram from hip flexor nerve. (C) V<sub>m</sub> from intracellular recording. Tick mark indicates V<sub>m</sub> of −100 mV. (D) Current pulses of −0.7 nA from constant current level of −1.0 nA. (E) High-pass filtered V<sub>m</sub> from (C) (cut off is 2 Hz). Transient artifacts removed. (F) left, average voltage deflections from (E) during quiescence (n = 14), right during network activity (n = 14). The average increase in conductance is 340%. Notice the occurrence of two spikes. (G) The membrane conductance as a function of time. The peak conductance during network activity is &gt;800% of the conductance during quiescence.</p></caption></fig>", "<fig id=\"pone-0003218-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003218.g003</object-id><label>Figure 3</label><caption><title>Effective recovery time and synaptic integration time during scratching.</title><p>Peri-spike V<sub>m</sub> distribution in quiescence (A) (n = 50) and during scratching (B) (n = 29). Top, superimposed data traces (gray) with mean V<sub>m</sub> in black. Middle, KS-test. Bottom, P-values for the KS test. The <italic>effective synaptic integration time</italic> (eSIT) and <italic>effective recovery time</italic> (eRT), defined as the time periods before and after the spike, in which the V<sub>m</sub> distribution is significantly different from a template distribution (arrows), i.e. gray areas in the KS-graphs in the middle. P-vaules of the test is plotted below. (C) Estimates of eSIT and eRT versus position of the template distribution relative to the spike from (B) (broken lines represent mean values eSIT = 12.1 ms, eRT = 8.7 ms). (D) The number of traces in template distribution decreases with window length (from same data as in (B) and (C)).</p></caption></fig>", "<fig id=\"pone-0003218-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003218.g004</object-id><label>Figure 4</label><caption><title>Temporal characteristics of sub-threshold V<sub>m</sub>-fluctuation in motoneurons during quiescence and in the on- and off-cycle during scratching.</title><p>(A) Hip-flexor nerve recording during scratch. (B) Concurrent V<sub>m</sub> in MN, spikes avoided with −2.5 nA hyperpolarizing current. Shaded regions mark the selected area of on- and off-cycle illustration below. Sample trace of V<sub>m</sub> in quiescence (C) (note time-course of spontaneous synaptic potentials) and in the on-cycle (D) and off-cycle (E). D and E are from the shaded boxes in B. (F) The auto-correlation sequence of each sample trace. Blue is from quiescent trace (C), gray is from the off-cycle trace (E), and red is the on-cycle trace (D). The effective time constant of each trace is obtained by fitting an exponential decay function (broken lines) to the initial 3 ms (until the vertical gray line). The time constants are τ<sub>eff</sub> = 2.8 ms (on-cycle activity), τ<sub>eff</sub> = 5.2 ms (off-cycle activity) and τ<sub>eff</sub> = 27.0 ms (quiescence). C–E are on the same time scale.</p></caption></fig>", "<fig id=\"pone-0003218-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003218.g005</object-id><label>Figure 5</label><caption><title>Time constants across the population of MNs.</title><p>(A) The average τ<sub>eff</sub> (for ON-cycle), eRT and eSIT (mean±SE) as triplets bars for each cell. Horizontal lines represent population averages, μ = 9.5 ms for τ<sub>eff</sub>, μ = 7.5 ms for eRT, and μ = 7.7 ms for eSIT. Cell 3 is marked with a ★ and the sample cell used in ##FIG##2##figures 3##, ##FIG##3##4##, ##FIG##4##5##. (B) eRT and eSIT plotted against τ<sub>eff</sub> show significant correlation. The lines are linear least square fits. (C) The shortest ISI at zero current injection plotted against the average eRT for each MN. Gray line is where x = y.</p></caption></fig>", "<fig id=\"pone-0003218-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003218.g006</object-id><label>Figure 6</label><caption><title>Presence and absence of spike frequency adaptation (SFA) during bursting.</title><p>(Aa) SFA in intracellular recording from motoneuron in slice during NMDA induced bursting. (Ab) Single burst highlighted in (Aa) show gradual increase in ISI. (Ac) Histogram of ISI. The mean ISI is 55 ms (arrow). (Ad) Plot of ISI<sub>N</sub> against ISI<sub>N+1</sub> shows significantly greater proportion of points above than below the ISI<sub>N</sub> = ISI<sub>N+1</sub> line (83.2% above, total N = 239), which is evidence of spike frequency adaptation. In addition, ISIs are correlated with their neighbors (correlation coefficient = 0.41), as expected when ISI are influenced by AHP conductance and the burst pattern is reproduced after 10 spikes (Ae). Gray area represents the 5% confidence limit (Ba) Recording from a MN in a functional spinal network during rhythmic motor activity. (Bb) A hightligted cycle from (Ba) shows irregular spike times and no SFA. (Bc) Histogram of ISI. The mean ISI is 34 ms (arrow) (Bd) A plot of ISI<sub>N</sub> against ISI<sub>N+1</sub> illustrates no discrepancy of points above and below the ISI<sub>N</sub> = ISI<sub>N+1</sub> line (51.3% above, total N = 362), which demonstrates absence of SFA. Furthermore, there is only a marginal correlation of ISI with neighbors (correlation coefficient = 0.18), as expected with negligible AHP conductance (Be). Gray area represents the 5% confidence limit . Tick marks to the left represent −80 mV (A) and −60 mV (B). Inter-burst-intervals are omitted in analysis.</p></caption></fig>", "<fig id=\"pone-0003218-g007\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003218.g007</object-id><label>Figure 7</label><caption><title>Spike frequency adaptation during NMDA induced bursts but absent during scratching.</title><p>The number of points above the ISI<sub>N</sub> = ISI<sub>N+1</sub> –line in a ISI-return map (Figure. 7Ad and 7Bd) divided by the total number of intervals (×100%) for all MNs including the NMDA induced bursting data from slice experiment for comparison. The network induced bursting have little or no SFA since there is an even amount of points above as below. In contrast, the NMDA-activated bursting has a much larger fraction above the line reflecting the high degree of SFA. Error bars are , where K total number of ISI.</p></caption></fig>" ]
[ "<table-wrap id=\"pone-0003218-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003218.t001</object-id><label>Table 1</label><caption><title>Total input conductance for 5 MN during both quiescence and active network.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Motoneuron number</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">G<sub>quiescence</sub> [nS]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">G<sub>active</sub> [nS]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fraction [100%]</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">73</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">320</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">438</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">181</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">405</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">224</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">314</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">200</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">258</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">470</td></tr></tbody></table></alternatives></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003218.s001\"><label>Data and Method S1</label><caption><p>(0.07 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003218.s002\"><label>Figure S1</label><caption><p>(10.67 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003218.s003\"><label>Figure S2</label><caption><p>(4.21 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003218.s004\"><label>Figure S3</label><caption><p>(10.05 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003218.s005\"><label>Figure S4</label><caption><p>(9.28 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003218.s006\"><label>Figure S5</label><caption><p>(3.40 MB TIF)</p></caption></supplementary-material>" ]
[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>This work was funded by the Carlsberg foundation (RB), the Novo Nordisk foundation (RB), the Lundbeck foundation (SD and JH) and FIST (JH). Role of sponsors: None.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"pone.0003218.s001.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003218.s002.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003218.s003.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003218.s004.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003218.s005.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003218.s006.tif\"><caption><p>Click here for additional data file.</p></caption></media>" ]
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{ "acronym": [], "definition": [] }
61
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 16; 3(9):e3218
oa_package/c0/c3/PMC2528963.tar.gz
PMC2528964
18818727
[ "<title>Introduction</title>", "<p>The G-protein-coupled signal transduction system integrates a wide range of intercellular signals and actuates downstream pathways. G-protein-coupled receptors (GPCRs) are composed of seven α-helices that span the plasma membrane, an extracellular domain that is activated by an agonist and an intracellular domain that binds a guanine nucleotide heterotrimer made up of different α, β, and γ subunit isoforms. This receptor system accounts for 40–50% of modern medicinal drug targets but only 10% of the known receptors are targeted by drugs ##REF##14625380##[1]##. Though the system is physiologically and pharmacologically important, the mechanism by which the system integrates multiple signals is not well understood ##REF##12790797##[2]##.</p>", "<p>We address the G-protein-mediated route to calcium release in RAW264.7 cells. When activated by a specific ligand, the G protein heterotrimer dissociates to free Gα-GTP and Gβγ. Specific Gα and Gβγ isoforms are able to bind specific isoforms of phospholipase C β (PLCβ) and catalyze the synthesis of inositol (1,4,5)-triphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol (4,5)-bisphosphate (PIP2) ##REF##2830256##[3]##,##REF##8389480##[4]##. In addition to its catalytic activity, PLCβ acts as a GTPase for Gα-GTP ##REF##10449728##[5]##. IP3 binds to specific receptor-channels on the membrane of the ER to release Ca<sup>2+</sup> into the cytosol ##REF##15189149##[6]##. DAG and Ca<sup>2+</sup> bind to and activate protein kinase C (PKC) which may phosphorylate and inactivate specific PLCβ isoforms ##REF##12954613##[7]##. G protein receptor kinase (GRK) is activated once it is phosphorylated by PKC ##REF##14499340##[8]## and is localized to the plasma membrane by Gβγ ##REF##7744811##[9]##. Though phosphorylation has not been shown to be necessary for GRK activation, we have assumed so in our model because phosphorylation by PKC may release the inhibition of GRK2 by being bound to calmodulin ##REF##14499340##[8]##. Activated GRK can then phosphorylate specific GPCRs which leads to receptor inactivation—perhaps directly or by arrestin activity ##REF##14499340##[8]##. In this complex signal transduction network, Gα and Gβγ subunits have different patterns of specificity for PLCβ isoforms and calcium is an important cofactor in several important feedback loops ##UREF##0##[10]##.</p>", "<p>The two extracellular signaling ligands we consider here are C5a and UDP. The small peptide C5a is a potent anaphylotoxin and a strong chemoattractant for many immune system components ##REF##15638737##[11]##. The calcium response due to stimulation by C5a is predominantly coupled through Gαi-linked heterotrimers. Macrophage cells and their precursors, monocytes, express several receptors that are specific to extracellular nucleotides and it has been shown that the P2Y6 receptor, which is sensitive to UDP, regulates the production and secretion of the chemokine interleukin 8 (IL-8) in monocytes ##REF##11349132##[12]##. The UDP response is mediated by Gαq-linked heterotrimers, but other receptors in the P2Y family may respond to UDP and couple the signal through other G protein isoforms ##REF##11390975##[13]##.</p>", "<p>Four recent models have sought to explore various aspects of the G protein coupled signal transduction system in detail. Lukas et al. compare measured calcium response over a range of bradykinin doses to their model predictions ##REF##15345523##[14]##. Mishra and Bhalla built a model to investigate the role of IP4 as a signal coincidence detector in the GPCR pathway ##REF##12202356##[15]##. The model by Lemon et al. predicts the calcium response to UTP stimulation and is the closest in focus to our model ##REF##12782119##[16]##. A recent model of calcium dynamics in RAW cells has been proposed that is quite similar to this model, but does not deal with crosstalk between receptors or formal statistical uncertainty in model predictions ##REF##17483189##[17]##,##REF##17483174##[18]##.</p>", "<p>Several hypotheses for the mechanism of crosstalk and synergy among GPCR-mediated pathways have been proposed. Crosstalk among GPCR-mediated pathways is important both physiologically and pharmaceutically. Quitterer et al. propose that crosstalk is mediated by Gβγ exchange between Gαi-coupled and Gαq-coupled receptors ##REF##10485876##[19]##. Zhu et al. speculated that PLC is under either conditional or dual regulation of Gβγ and Gα ##REF##8610126##[20]##. Though these hypothetical mechanisms for crosstalk among G protein coupled receptor systems are conceptually plausible we have not found these or any other of the many competing hypothetical mechanisms tested in the context of a quantitative mathematical model ##REF##12790797##[2]##.</p>", "<p>In this paper Bayesian statistical inference is used to provide a rigorous connection between the mathematical model derived from mass-action kinetics, prior information from in-vitro biochemical studies and heterogeneous experimental data. The prior distribution over the parameters represents our uncertainty before observing a set of experimental data. A broad, high variance, prior distribution means we are quite uncertain and a concentrated, low variance, prior means we are more certain about the parameter a priori. The objective of our inference is the posterior distribution over the parameters because it is an informed estimate of both the value of the parameter and the uncertainty in the parameter value. The posterior distribution over the parameters is then used as a tool for experiment design to estimate the model-based posterior distribution over observable quantities such as the cytosolic calcium concentration and to drive the design of new experiments. This statistical approach is possible in a model of this size because of the abundance and quality of the data collected for this study.</p>" ]
[ "<title>Materials and Methods</title>", "<p>The model equations are given in ##SUPPL##7##Figure S7##. The initial conditions and parameter values are in ##SUPPL##11##Table S1## and ##SUPPL##12##Table S2##, respectively. All the data used in this work and a stand-alone implementation of the model is provided at <ext-link ext-link-type=\"uri\" xlink:href=\"http://genomics.lbl.gov/supplemental/flaherty-gpcr/\">http://genomics.lbl.gov/supplemental/flaherty-gpcr/</ext-link>. The model was simulated using CVODE ##UREF##1##[42]## and the GNU Scientific Library. Further details on materials and methods are available in ##SUPPL##0##Dataset S1##.</p>", "<title>Experimental Methods</title>", "<p>Intracellular free calcium in cultured adherent RAW264.7 cells was measured in a 96-well plate format using the Ca<sup>2+</sup>-sensitive fluorescent dye Fura-2 ##REF##2538708##[43]##,##REF##3838314##[44]##. A Molecular Devices FLEXstation scanning fluorometer was used to measure fluorescence using a bottom read of a 96-well plate. Each well was sampled approximately every 4 seconds. The measurement protocol is described in AfCS experimental protocol ID #PP00000211 (available from <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.signaling-gateway.org\">http://www.signaling-gateway.org</ext-link>). The parameters in ligand concentration model were estimated using FITC solution in the FLEXstation scanning fluorometer as described in Molecular Devices Maxline Application Note #45 and in ##SUPPL##10##Protocol S1## (see also ##SUPPL##5##Figure S5##).</p>", "<title>Statistical Inference</title>", "<p>Twenty of the 84 parameters were chosen to be estimated from data based on relevance to the experimental hypothesis. Only those parameters that related to the knockdown experiments in the dataset were estimated and are denoted with a star in ##SUPPL##12##Table S2##. We used data to estimate only the two forward rate constants in the enzymatic mass-action equations because the forward and reverse rate constants for a given reaction will be highly correlated in the posterior distribution making estimation by Markov chain methods computationally expensive. An analysis of the sensitivity of the model to each parameter is shown in ##SUPPL##9##Figure S9##.</p>", "<p>For each estimated parameter we constructed an independent Gaussian prior on a log scale with a mean chosen based on relevant literature and a standard deviation of 0.25. We found that this prior variance was sufficiently permissive to allow exploration of the space while still constraining the rates to be physically reasonable. The prior distribution over the parameters allows the incorporation of both soft and hard constraints in the parameter estimates. Parameter sets with zero measure are not permitted in the posterior distribution and parameter sets with small measure must be assigned a large likelihood in order to have a large posterior probability.</p>", "<p>The likelihood is a function of the parameters (<italic>θ</italic>) and links the prior distribution with the posterior distribution under Bayes rulewhere <italic>y</italic> denotes the observed data.</p>", "<p>In our model, the likelihood function is a Gaussian distribution according to the non-linear regression equation <italic>y</italic> = <italic>f</italic>(<italic>θ</italic>)+<italic>ε</italic>, <italic>ε</italic>∼<italic>N</italic>(0,<italic>σ</italic>\n<sup>2</sup>), where <italic>f</italic>(<italic>θ</italic>) is the deterministic model prediction. The posterior distribution is of interest because it informs us as to the most probable setting of the parameters as well as the uncertainty in the values.</p>", "<p>The Metropolis-Hastings algorithm ##UREF##2##[45]## was used to estimate the posterior density of the parameters Pr(<italic>θ</italic>|<italic>y</italic>). Three independent chains were simulated from different initial parameter values (see ##SUPPL##1##Figure S1##). To assess convergence of the posterior distribution estimate, we used the Gelman-Rubin potential scale reduction factor (PSRF) ##UREF##3##[46]##. The multivariate PSRF is 2.44 and 95% of the individual PSRFs were less than 1.5. A PSRF value of one indicates that the distribution has converged and values near one are close to converged.</p>", "<p>Posterior prediction confidence intervals were constructed using the percentiles from the predictive distribution approximated with 2000 Monte Carlo samples from Pr(<italic>y</italic>\n<sub>new</sub>|<italic>θ<sub>i</sub></italic>) at each of 100 simple random samples from Pr(<italic>θ</italic>|<italic>y</italic>) obtained fromwhere Pr(<italic>y</italic>\n<sub>new</sub>|<italic>θ<sub>i</sub></italic>)∼<italic>N</italic>(<italic>f</italic>(<italic>θ</italic>),<italic>s</italic>\n<sup>2</sup>) and <italic>s</italic>\n<sup>2</sup> is the pooled variance estimate, which is computed as an average of the variances of all the time points in each of the 29 wild-type experiments. These average variances were weighted by the number of technical replicates in each experiment and then averaged to yield the estimate <italic>s</italic>\n<sup>2</sup>. A small factor of 1 nM<sup>2</sup> was added to each variance estimate to bound variance estimates away from zero.</p>" ]
[ "<title>Results</title>", "<p>There are two main features of the structure of our model, shown in ##FIG##0##Figure 1##, which contribute to crosstalk in the system and produce the key dynamical features in the calcium response: isoform specificity and calcium-dependent feedback. As we will show, by including multiple isoforms of PLCβ and Gα as well as the negative feedback mediated by PKC, GRK and the IP3 receptor itself, we are able to predict the synergistic interaction between C5a and UDP observed in the experimental data.</p>", "<p>Our representation of the G-protein-coupled signal transduction system includes C5a and P2Y6 receptors, Gαi2, Gαq, Gβγ, PLCβ3, PLCβ4, PIP2, DAG, IP3, PKC, GRK2, calcium buffer, a Na<sup>2+</sup>/Ca<sup>2+</sup> exchanger, a sarco(endo)plasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA) pump, IP3 receptors and RGS. The model is composed of 53 coupled ordinary differential equations with 84 parameters and 24 non-zero initial conditions. The complete model equations are shown in ##SUPPL##7##Figure S7## and a more detailed model diagram is shown in ##SUPPL##6##Figure S6##. The parameters and initial conditions are in ##SUPPL##12##Table S2## and ##SUPPL##11##Table S1##, respectively. Where available, we have relied on <italic>in-vitro</italic> or <italic>in-vivo</italic> biochemical experiments for the reactions and parameter values (see Supporting Information). In cases where the biochemical parameter values were not known, we chose physically reasonable values. Twenty of the 84 parameters most relevant to the knock-down and wild-type data were estimated from cytosolic calcium measurements as described in the <xref ref-type=\"sec\" rid=\"s4\">Methods</xref> section. Most reactions were assumed to be governed by mass-action kinetics, but for a few proteins—such as RGS—the mechanism of regulation is not known in enough detail and we have approximated with Michaelis-Menten kinetics or a phenomenological function.</p>", "<p>We briefly discuss the reactions involving the Na<sup>2+</sup>/Ca<sup>2+</sup> exchanger, SERCA pump, IP3 receptors, RGS and calcium buffer because they are important for the faithful representation of the system in our model. Regulators of G protein signaling (RGS) are GTPase proteins that down-regulate the extent of signaling ##REF##9462506##[21]##; RGS2 at least is expressed in RAW264.7 macrophage cells and therefore an RGS activity is included in our model. The mechanism of activation of RGS2 as it relates to Gαi and Gαq signaling is not entirely known and is difficult to assess because antibodies that specifically recognize RGS2 are not widely available ##REF##10966476##[22]##–##REF##11906816##[24]##. We have assumed constitutive activity and expect as more information becomes available a more accurate model of the regulation of RGS2 and other RGS isoforms will be possible. The SERCA pump helps to bring the cytosolic Ca<sup>2+</sup> concentration back to the resting level after stimulation. We have modeled the SERCA pump as in the Keizer and DeYoung model ##REF##1324019##[25]##. The IP3 dependent opening of ER calcium channels was found to be cooperative ##REF##2452482##[26]## and we have used the Meyer and Stryer model for the IP3-gated channel with a Hill coefficient of four ##REF##1324019##[25]##,##REF##2455890##[27]##. Finally, many other proteins such as calmodulin and the fluorescent indicator Fura-2 bind Ca<sup>2+</sup>. Because our measurements reflect these effects, we have included a general buffer for cytosolic calcium.</p>", "<title>Isoform Specificity</title>", "<p>Complement factor 5a activates the C5a receptor which is a Gαi-coupled receptor ##REF##8662841##[28]##. The released Gβγ dimer activates PLCβ2 and PLCβ3 which are lumped and called PLCβ3 in our model because: (i) the activity of Gβγ-activated PLCβ3 has been shown to be greater than Gβγ-activated PLCβ2 in <italic>in-vitro</italic> studies and (ii) Gαq activates both PLCβ2 and PLCβ3 so the structural connections from Gβγ and Gαq to PLCβ2 and PLCβ3 in the model are identical ##REF##8389480##[4]##,##REF##8383116##[29]##. PLCβ1 is activated by Gβγ and Gαq, but RAW264.7 macrophage cells do not express this isoform, so we have not included it in the model. PLCβ3 then catalyzes the hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).</p>", "<p>UDP stimulates the P2Y6 receptor and the associated Gαq-GTP activates both PLCβ3 ##REF##8387502##[30]## and PLCβ4 ##REF##7929227##[31]##. The GTPase rate of Gαq is increased 1000-fold when bound to PLCβ ##REF##10449728##[5]##. Due to this rapid hydrolysis rate, we have assumed, in our model, that PLCβ3 or PLCβ4 bound Gαq-GTP may only hydrolyze one molecule of PIP2 before releasing Gαq-GDP. Additionally, the Gβγ released by the P2Y6 receptor also activates PLCβ3 ##REF##8387502##[30]##, but does not activate PLCβ4 ##REF##8125982##[32]##.</p>", "<p>Our model assumes that PLCβ3 does not simultaneously bind Gβγ and Gαq. Indeed, a biochemical study of PLCβ2 activity in reconstituted membrane fractions strongly argues that Gαq and Gβγ do not simultaneously bind this effector ##REF##9931014##[33]##. While this was specifically demonstrated for PLCβ2, we implicitly assume the same holds for PLCβ3 because we lump the two in our model. This is a mechanistic assumption of our model and an interesting issue for future testing with directed experiments.</p>", "<title>Calcium-Dependent Feedback</title>", "<p>Though important for response specificity, the dynamical control of calcium release is not limited to the forward pathway in this system. Calcium participates in feedback processes that both enhance and inhibit its own release at multiple points in the pathway. There are four main nodes of calcium-dependent feedback control in our model: PLCβ, IP3 receptor, protein kinase C (PKC) and G protein receptor kinase (GRK).</p>", "<p>Calcium enhances its own release by binding to the EF-hand domain on PLCβ and is required for PLCβ to hydrolyze PIP2 into IP3 and DAG ##REF##11395409##[34]##. Because the dissociation constant for PLCβ-Ca<sup>2+</sup> in our model is larger than the basal concentration of cytosolic calcium, as more Ca<sup>2+</sup> is released from the ER, more PLCβ-Ca<sup>2+</sup> becomes available to bind Gαq or Gβγ. This positive feedback mechanism accelerates the release of Ca<sup>2+</sup>.</p>", "<p>In our model, Ca<sup>2+</sup> and IP3 cooperatively open the channel between the ER and the cytosol. It is believed that Ca<sup>2+</sup> initially stimulates the IP3 receptor with maximal stimulatory effect at 100–300 nM ##REF##15189149##[6]##. At higher concentrations, Ca<sup>2+</sup> has an inhibitory effect. We use the IP3 receptor model structure in the Keizer and DeYoung model for this component ##REF##1324019##[25]##.</p>", "<p>Protein kinase C (PKC) has been shown to phosphorylate PLCβ3 which inhibits PLCβ3 activation due to Gαq and Gβγ ##REF##10893237##[35]##,##REF##12587975##[36]##. PKC is activated when bound to DAG and Ca<sup>2+</sup>\n##REF##12954613##[7]##,##REF##15282562##[37]##. Because the preferred order of binding is not entirely known, PKC, DAG and Ca<sup>2+</sup> form a thermodynamic cycle of reversible reaction with only the PKC-DAG-Ca<sup>2+</sup> form active. In our model, the dissociation constant of PKC and Ca<sup>2+</sup> is much greater than the basal Ca<sup>2+</sup> concentration, and upon binding DAG, the PKC-DAG complex has a higher affinity for Ca<sup>2+</sup> making the order of binding preferentially PKC to DAG then PKC-DAG to Ca<sup>2+</sup>. It is not known whether PLCβ4 is also regulated by PKC. We have assumed, in our model, the same mechanism of PKC regulation of PLCβ3 and PLCβ4.</p>", "<p>The final key calcium-dependent feedback loop in our model is mediated by G protein receptor kinase (GRK). GRK2 phosphorylates and inactivates ligand-bound C5a receptors when activated by PKC and Gβγ. In sequence, PKC phosphorylates GRK2 which causes translocation to the plasma membrane ##REF##14499340##[8]##. When properly localized, GRK2 may bind Gβγ and then phosphorylate the C5a-C5a receptor complex to inactivate it ##REF##10508278##[38]##. This simplified representation of the receptor desensitization mechanism does not include arrestin activity, multiple receptor phosphorylation sites and other fine grain or slower biochemical interactions that may be present <italic>in-vivo</italic>.</p>", "<title>\n<xref ref-type=\"sec\" rid=\"s4\">Single Ligand Experiments</xref>\n</title>", "<p>Having specified the structure of our model, we direct our attention to the parameters. We estimate 20 of the 84 parameters in our model using a dataset composed of 96 Fura-2 time series measurements as described in the Materials and <xref ref-type=\"sec\" rid=\"s4\">Methods</xref> section. Each experiment consists of 3–4 samples from different wells in a 96 well plate. There are 15 experiments spanning 9 doses of C5a and 14 experiments spanning 11 doses of UDP on wild-type cells in the dataset (see ##SUPPL##3##Figure S3##). The dataset also contains calcium measurements on 5 different shRNAi knockdown cell lines constructed by lentiviral infection (see ##SUPPL##4##Figure S4##). The time interval between samples is approximately 3–4 seconds and each time series is approximately 100–300 seconds of post-stimulation data. ##TAB##0##Table 1## shows a summary of the knockdown data used for statistical parameter estimation for this model in addition to the wild-type experiments.</p>", "<p>We find that our model is generally quantitatively consistent with the experimental data within measurement uncertainty. Where the model is less consistent with the data – specifically for the GRK knockdown experiment – we find the deviation has a reasonable biological explanation. The summary of the dataset and the fit of the model to each single ligand experiment are available in the Supporting Information. We briefly discuss some issues relating to goodness of fit and the Bayesian parameter estimation here.</p>", "<p>While most optimization procedures produce a point estimate of the parameters that maximize the goodness of fit of the model to the observed data, the Bayesian procedure we have employed here estimates the entire posterior distribution of the parameters given the data. This information is valuable for qualitatively and quantitatively evaluating the precision of the parameters estimates. ##FIG##1##Figure 2## shows, as a qualitative evaluation, that while the a-priori forward and reverse binding rates for the receptors (C5aR and P2YR) are uncorrelated they are correlated in the posterior distribution. The calcium measurements have informed and constrained the posterior estimates of the dissociation constants to be approximately 5 nM and 250 nM for the C5aR and P2YR respectively. We have quantitatively computed marginal highest posterior density (HPD) confidence intervals for each of the twenty parameters we have estimated from the data. Those estimates are shown in ##SUPPL##13##Table S3##. Those parameters with large HPD intervals are not well informed by the measurements and are candidates for directed biochemical experiments.</p>", "<title>Wild-Type Experiments</title>", "<p>The calcium response to C5a adapts and returns to the basal level, but the UDP response has a sustained elevated calcium level that slowly decays. ##FIG##2##Figure 3## shows two representative experiments of the response of the wild-type cell to stimulation with C5a and UDP. We expect that the fit to this data will be good because 20 key model parameters were fit using an experimental dataset that included these experiments – the fit is indeed accurate. The point estimate curve is constructed from the maximum a-posteriori parameters from an MCMC chain. The prediction intervals are estimated by Monte Carlo sampling from the posterior parameter distribution and the measurement error distribution conditional on the parameters. The prediction confidence intervals generally cover the observed data.</p>", "<title>Knockdown Experiments</title>", "<p>Lentiviral infection is used to introduce small hairpin RNAs to interfere with the translation of the key signaling proteins GRK2, Gαi2, Gαq, PLCβ3 and PLCβ4 ##REF##16945906##[39]##. There are three main sources of uncertainty in the knockdown experiment model predictions: parametric uncertainty, measurement uncertainty and knockdown efficiency uncertainty. We have dealt with the first two sources in the previous section on wild-type experiments. Here we address prediction variability due to knockdown efficiency uncertainty by using nominal parameter values.</p>", "<p>\n##FIG##3##Figure 4## shows simulations and experimental data for three representative knockdown experiments. The upper-left panel of ##FIG##3##Figure 4## shows a GRK knockdown line stimulated with 250 nM C5a. Because GRK2 desensitizes the C5a receptor, we expect that by eliminating the feedback mechanism, the calcium peak will be higher and more sustained. The experimental data as well as the model indeed show that effect. Quantitatively, the model prediction shows a greater effect than the experimental data. A likely reason is that the model only considers one isoform of GRK while there are four isoforms expressed in the RAW264.7 cell line (GRK1,2,4,6). If more than one isoform can desensitize the C5a receptor, the effective knockdown in desensitization function will be less than as measured by western blot analysis on GRK2.</p>", "<p>While GRK does not desensitize the P2Y receptor in our model, it is a buffer for Gβγ released from Gαq. Reducing the amount of GRK will shift the equilibrium towards more Gβγ bound to PLCβ3 and thus more calcium release even though GRK does not directly feed back on the P2Y6 receptor. The top-right panel in ##FIG##3##Figure 4## shows that, based on the model, the peak intracellular calcium concentration is expected to be very slightly higher in the GRK2 knockdown line when stimulated by 25 µM UDP. A comparison of the experimental peak heights of the wild-type and GRK knockdown cell line data by t-test cannot reject the null hypothesis that the peak heights are equal (<italic>p</italic> = 0.9963). The effect of the GRK knockdown is expected to be so slight that the effect size is overwhelmed by the measurement error in the data. The effect of the uncertainty in the GRK2 knockdown fraction impacts the range of the confidence intervals of the predicted C5a response much more than the confidence intervals of the predicted UDP response which is consistent with GRK2 being a more significant component of the C5a response.</p>", "<p>Our model structure has PLCβ3 stimulated by either Gβγ or Gαq. Because the C5a response signals only through PLCβ3 the effect of the knockdown is expected to be more pronounced for the C5a response than for the UDP response. The bottom-left panel of ##FIG##3##Figure 4## confirms that the model prediction is consistent with the representative experiment. The UDP response activates PLCβ3 through Gβγ, but also activates PLCβ3 and PLCβ4 with Gαq. Therefore, we expect that the calcium response should be more robust to perturbations in just one of the PLCβ isoforms. The UDP response in the PLCβ3 knockdown line (bottom right panel of ##FIG##3##Figure 4##) shows that our model predicts the knockdown effect to be small relative to the total magnitude of the response in part due to the redundancy in the use of PLCβ isoforms in the UDP response.</p>", "<p>Because this dataset was used for parameter estimation, the fit of model to the data may overstate the accuracy of the model. Nonetheless, the good fit does suggest that the model warrants being tested in truly predictive experiments; we describe such experiments in the following section.</p>", "<title>Double Ligand Experiments</title>", "<p>We examine our model response to a simultaneous stimulation by C5a and UDP because it has been shown experimentally that macrophage cells respond synergistically to such conditions ##REF##16699502##[40]##. To quantify the amount of synergy or non-additivity that is present in the calcium response, a <italic>synergy ratio</italic> is computed for each ligand dose pair. The numerator of the ratio is the peak offset from baseline of the intracellular calcium concentration. The denominator of the ratio is the sum of the peak offsets when the cell or model is stimulated with only one ligand. A synergy is present when the ratio is greater than one implying the peak height is greater than expected from an additive combination of ligand effects. While this is certainly not the only possible measure of synergy it is widely adopted and has been used in previous studies on calcium synergy ##REF##16699502##[40]##.</p>", "<p>The left panel of ##FIG##4##Figure 5## shows the results of model simulations at nominal parameters for a grid of doses of C5a and UDP. In the dose response surface, there is a ridge of synergistic calcium release for a moderate dose of UDP. We tested the model prediction with the experiment design measuring the synergy ratio at the points denoted as black open circles in the left panel of ##FIG##4##Figure 5##. A χ<sup>2</sup> goodness-of fit test comparing the model expected synergy ratio to the observed synergy ratio fails to reject the null hypothesis that the data were generated by the model mechanism (<italic>p</italic>-value≈1.0). The root-mean-squared error (RMSE) deviation between the predicted and actual experimental data is 0.492. By way of comparison, the RMSE between the data and the null model of no synergy is 1.044. We therefore conclude that the model predictions are consistent with the experimental observations. It should be noted that measurements of synergy in RAW cells are noisy and the ridge occurs at low doses of UDP. Notwithstanding, the phenomenon has been reported ##REF##16699502##[40]## and has been observed by us in this cell line.</p>", "<p>The right panel of ##FIG##4##Figure 5## shows the same synergy dose response surface but for a GRK knockdown cell line. The synergy ridge observed in the wild-type cell simulation is changed in the GRK knockdown simulation indicating the C5a receptor desensitization mechanism mediated by GRK is important for the synergistic release of calcium. In the next section we pursue this conclusion in more detail, developing a conceptual explanation of the mechanism of crosstalk and synergy within our model.</p>" ]
[ "<title>Discussion</title>", "<p>G-protein-coupled receptors form a complex network of interacting proteins that generally exhibits the properties of a system in which each receptor signal is buffered from the others. For a minority of ligand combinations, however, crosstalk between pairs of receptors is apparent. Due to the complexity and importance of the system many hypothetical mechanisms have been proposed to explain the crosstalk ##REF##12790797##[2]##. In particular, simultaneous Gβγ and Gαq binding to PLCβ ##REF##8610126##[20]## and Gβγ exchange between Gαi and Gαq-coupled receptors have been proposed as potential mechanisms ##REF##10485876##[19]##. While our model does not eliminate these potential mechanisms, we do show that the mechanism represented in our model is consistent with a full range of experimental data including a variety of doses of C5a and UDP, C5a and UDP stimulation of five different knockdown cell-lines and double-ligand dose response experiments.</p>", "<p>To our knowledge, this is the first multireceptor GPCR model and the first to address the complex phenomenon of crosstalk between GPCR receptor pathways that has been statistically estimated and validated with experimental data. This important phenomenon plays a role in processes as diverse as chemotaxis and perhaps drug interactions. In our model, the primary mechanism of synergy is due to the cooperative opening of the IP3 receptor. The robustness of the synergy is due to the feedback of GRK on the C5a receptor and the specificity of the synergy is due to the interaction patterns between specific Gα isoforms and PLCβ isoforms. The simultaneous binding model ##REF##8610126##[20]## accounts for the specificity of synergy, but not the robustness pattern of the synergy.</p>", "<p>We observe in the model that if the Gαq-PLCβ3-Ca<sup>2+</sup> and Gαq-PLCβ4-Ca<sup>2+</sup> binding reactions are inhibited, the system still exhibits synergy. We conclude from this observation that the crosstalk mechanism is mediated by Gβγ. If the binding reaction of Gβγ to phosphorylated GRK2 is removed, the synergy is eliminated. Furthermore, if the GRK2-mediated phosphorylation of complexed C5a receptors is removed, the double ligand response is additive. We deduce then that the synergy mechanism involves GRK2 phosphorylation of complexed C5a receptors. However, GRK2 phosphorylation does not entirely explain the synergy mechanism.</p>", "<p>In our model, the calcium released from the IP3 receptor is a function of the number of receptor molecules complexed to IP3 raised to the fourth power ##REF##1329108##[41]##. Therefore, for a small range of IP3 concentration, the amount of Ca<sup>2+</sup> released is more than additive (see ##SUPPL##8##Figure S8##). We conclude from our analysis of the model that the synergy ridge in ##FIG##4##Figure 5## arises because the GRK2 mediated mechanism holds the IP3 concentration in this non-additive region for most concentrations of C5a. The UDP response does not have the GRK2 mediated feedback and thus only shows a synergistic response for a small range of UDP concentration. If the GRK2 desensitization is removed from the model, the synergy ridge is removed and synergy is only present at low doses of C5a and UDP (see ##FIG##4##Figure 5##).</p>", "<p>The Bayesian method we have used for this model has several advantages for the estimation of model parameters in complex mechanistic system models. We have used an informative prior to exclude negative rate constants from the permitted parameter space. We have also used the prior distribution to center our a priori expectations of the rate constant at values obtained from <italic>in-vitro</italic> and other biochemical experiments. The Bayesian update rule allowed us to estimate parameters with our best current dataset and then update those estimates as new data became available from the calcium assay. In this way, we were able to iteratively refine and recalibrate our model with the most recent data available during data collection period for this project. The posterior distribution provides not only an estimate of the rate constants, but the entire distribution, from which we can calculate highest posterior confidence intervals and posterior correlations between parameters. For example, the posterior correlation between the binding and unbinding rates for the UDP-P2Y receptor complex were highly correlated, but those two constants were uncorrelated with the corresponding rates for the C5a-C5a receptor complex reaction even though we imposed no correlations a priori. Finally, the algorithmic methods for collecting ensembles of samples from the posterior distribution have improved considerably in recent years in terms of speed and robustness</p>", "<p>We have shown that the signal transduction system as it is represented by our model does not require simultaneous binding of Gαq and Gβγ to PLCβ3 to cause a synergistic Ca<sup>2+</sup> response due to simultaneous stimulation by C5a and UDP. We have shown that our representative model is consistent with this experimental dataset in RAW264.7 macrophage cells, but we have not excluded all other potential mechanisms that may be absent or regulated differently in this cell line compared to other macrophage cell lines. Indeed there are a few examples of statistical discrepancies between the model and experiments in our dataset (##SUPPL##14##Table S4##). These differences are substrate for further experimentation and modeling. The purpose of our model is to provide a quantitative tool to aid in reasoning about such complex interacting systems so that meaningful experiments can be designed to explore and understand the biological mechanism.</p>" ]
[]
[ "<p>Conceived and designed the experiments: PF MIJ APA. Performed the experiments: PF MLR. Analyzed the data: PF TD. Contributed reagents/materials/analysis tools: RAR TIR. Wrote the paper: PF MIJ APA.</p>", "<p>Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Gαi-coupled C5a receptor and the Gαq-coupled UDP receptor signaling systems that results in synergistic calcium release.</p>", "<title>Author Summary</title>", "<p>The G protein signal transduction system transmits a wide variety of extracellular signals including light, odors, and hormones, to intracellular effectors in diverse cell types in eukaryotes. G-protein-coupled receptors are involved in many diseases including inflammation, cardiac dysfunction, and diabetes, and are the targets of 40–50% of modern drugs. Despite the physiological and pharmacological importance of this signal transduction system it is not known how the system buffers and integrates information at a biochemical level. The multiple receptors expressed by every cell pass their signals through a common set of downstream effectors distinguished by multiple isoforms with slightly different specificities and activities. The coupling among these pathways causes interactions among the signals sent by the different classes of receptors. We have developed a mechanistic model of the G protein signal transduction system from the receptor to the central intracellular second-messenger calcium. We have used statistical methods to integrate a diverse set of experimental data into our model and quantify confidence in our model predictions. We used this model, trained on single receptor data, to predict the signal processing of two G-protein-coupled-receptor signals. Validation experiments support our hypothesized mechanism for dual receptor signal processing and the predictions of the model.</p>" ]
[ "<title>Supporting Information</title>" ]
[ "<p>The authors would like to thank Mel Simon and the Alliance for Cellular Signaling for the experimental data used in this study and many informative and interesting conversations.</p>" ]
[ "<fig id=\"pcbi-1000185-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000185.g001</object-id><label>Figure 1</label><caption><title>The model for crosstalk between the Gαi and Gαq pathways depends on both differential specificity and activity for Gαi, Gαq, and Gβγ interactions with PLCβ3 and PLCβ4 to catalyze PIP2 hydrolysis and calcium-dependent feedback control mediated by GRK and PKC.</title><p>Selected model parameters are informed by calcium measurements taken for various ligand doses on wild-type and cell lines with shRNAi knockdowns on the proteins shown in red.</p></caption></fig>", "<fig id=\"pcbi-1000185-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000185.g002</object-id><label>Figure 2</label><caption><title>This figure shows that the single and pairwise marginal posterior distributions for the ligand binding reactions for the P2YR and C5aR receptors.</title><p>The vertical line in the single marginal posterior distributions shows the point estimate that were selected. The posterior distributions show the dissociation constants for the reactions are tightly constrained by the data, while the values of the forward and reverse rates that make up the ratio are not as well constrained by the data. Additionally, as expected the UDP binding rates are not correlated with the C5a binding rates. Marginal posterior distributions for all parameters and a discussion of the point estimate selection can be found in ##SUPPL##2##Figure S2##.</p></caption></fig>", "<fig id=\"pcbi-1000185-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000185.g003</object-id><label>Figure 3</label><caption><title>Model simulations are compared to experimental data.</title><p>The point estimate is computed using the posterior distribution of the parameter as estimated by Markov chain Monte Carlo given the data from 96 experiments on C5a and UDP at various doses in combination with 5 different shRNAi knockdown cell lines. The 95% posterior predictive intervals are estimated by Monte Carlo simulations including both parameter and measurement uncertainty. The measured mean and approximate 95% confidence intervals of four replicates is shown by a black dot and error bar. (A) C5a at 250 nM was introduced at 20s and the experimentally observed pulse in cytosolic calcium concentration is shown. (B) The qualitative shape of the calcium pulse for 25 µM UDP is different than for 250 nM C5a. The pulse does not completely adapt and return to the prestimulated level. For both ligands, the model prediction confidence intervals overlap the data error bars that indicate the model fit is consistent with the data within the measurement uncertainty.</p></caption></fig>", "<fig id=\"pcbi-1000185-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000185.g004</object-id><label>Figure 4</label><caption><title>The model simulation results for GRK and PLCβ3 knockdown cell lines stimulated with C5a and UDP are shown.</title><p>The experimental mean±1 s.d. of 3–4 replicates within one experimental run is shown in black. The knockdown simulation result with nominal knockdown fraction and parameters is shown in red and the wild-type simulation result is shown in green for comparison. Upper and lower model 99% confidence intervals (shown as blue dashed lines) are simulated using the upper and lower knockdown fraction values from ##TAB##0##Table 1##. As expected the Ca<sup>2+</sup> response to C5a in the GRK knockdown line (A) was increased compared to wild-type. The quantitative deviation between the model and data is possibly due to the availability of multiple redundant GRK isoforms. (B) The expected effect of the GRK knockdown on the UDP response is an increase in the cytosolic calcium levels. Because GRK2 does not directly desensitize the P2Y receptor in this model, the effect is likely due to a reduction of sequestration of Gβγ by GRK. (C) The signal transduction of the C5a response is predominantly through the PLCβ3 isoform. The effect of the PLCβ3 knockdown is much greater for C5a than for UDP (D).</p></caption></fig>", "<fig id=\"pcbi-1000185-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000185.g005</object-id><label>Figure 5</label><caption><title>The model is used as a predictive tool to infer the effect of stimulating the cell simultaneously with UDP and C5a that signal through the Gαq and Gαi pathways, respectively.</title><p>Synergy was measured as the ratio of peak height offset from baseline attained from simultaneous stimulation to the peak height offset calculated by the sum of the responses to each ligand individually. (A) Expected synergy ratio as a function of UDP and C5a dose (truncated at 1.5). The simulations show a ridge of synergy at a moderate UDP dose for most C5a doses. The black circles indicate dose combinations points of experiments that were conducted to test the model. (B) Expected synergy ratio as a function of UDP and C5a dose for a simulated GRK2 knockdown cell line. Without the GRK-mediated negative feedback to keep the IP3 generation from the C5a receptor within the non-linear range of calcium release the ridge in the synergy dose response is diminished. The synergy in the GRK knockdown simulation is not entirely eliminated because the shRNAi knockdown of GRK does not constitute a complete loss-of-function and low concentrations of ligand are still able to synergize. Furthermore, the asymmetric synergy dose response surface is more symmetric in the GRK knockdown simulation because the asymmetric calcium-dependent feedback mechanism is reduced.</p></caption></fig>" ]
[ "<table-wrap id=\"pcbi-1000185-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pcbi.1000185.t001</object-id><label>Table 1</label><caption><title>Dataset used for parameter estimation.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cell Line</td><td colspan=\"2\" align=\"left\" rowspan=\"1\">Measured Fraction Knockdown</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">Model Value</td><td colspan=\"6\" align=\"left\" rowspan=\"1\">Sample Size</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td colspan=\"2\" align=\"left\" rowspan=\"1\"/><td colspan=\"3\" align=\"left\" rowspan=\"1\"/><td colspan=\"3\" align=\"left\" rowspan=\"1\">C5a</td><td colspan=\"3\" align=\"left\" rowspan=\"1\">UDP</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Western</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Nominal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Lower</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Upper</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;10 nM</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10–100 nM</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;100 nM</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&lt;1 µM</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1–10 µM</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">&gt;10 µM</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Wild-type</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">GRK2 (2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">90%±7%, <italic>n</italic> = 5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40%±6%, <italic>n</italic> = 6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">58.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gai2 (3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">83%±5%, <italic>n</italic> = 4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">73%±6%, <italic>n</italic> = 5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">73.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">91.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gaq (3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">70%±8%, <italic>n</italic> = 7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">66%±23%, <italic>n</italic> = 2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">66.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">95.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PLCβ3 (1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">83%±15%, <italic>n</italic> = 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">83.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">38.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PLCβ4 (1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">87%±6%, <italic>n</italic> = 5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">87.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">69.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100.0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td></tr></tbody></table></alternatives></table-wrap>" ]
[ "<disp-formula></disp-formula>", "<disp-formula></disp-formula>", "<inline-formula></inline-formula>", "<inline-formula></inline-formula>" ]
[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s001\"><label>Dataset S1</label><caption><p>(0.11 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s002\"><label>Figure S1</label><caption><p>This figure shows exemplar MCMC realizations for parameter k109f (the UDP+P2YR forward binding rate) from three independent chains. The chains have converged to the stationary distribution which is the posterior distribution as measured by the PSRF (see <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>).</p><p>(0.20 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s003\"><label>Figure S2</label><caption><p>Posterior distributions and correlations The first figure shows that the pairwise marginal posterior distributions for the ligand binding reactions for P2YR and C5aR. The posterior distributions show the dissociation constants for the reactions are tightly constrained by the data, while the values of the forward and reverse rates that make up the ratio are not as well constrained by the data. Additionally, the UDP binding rates are not correlated with the C5a binding rates. k108f and k108r are the P2YR forward and reverse rates and k101f and k101r are the C5aR rates. The next two figures show the one-way marginal posterior density estimates from three independent MCMC chains with approximately 30,000 samples. The 20 estimates parameters are along the rows and the independent chains are along the columns. In each plot, the light blue density is the prior density and the green, purple and orange densities are the posterior densities. The vertical line shows the parameter value used in the model simulations in the paper and listed in ##SUPPL##13##Table S3##. All of the densities are plotted on a log scale. Each marginal posterior distribution estimate is constructed from independent MCMC chains. The results from each chain (three of them) are shown in the columns of the second figure below. In some cases the algorithm sampled heavily from one mode that was not explored as heavily by another chain. However, the PSRF criterion used to assay convergence and a visual inspection of overall posterior density correspondence do indicate that the posterior distributions are sufficiently sampled by all three chains in aggregate. Furthermore, the fit of the model to the data as shown in ##SUPPL##3##Figure S3## shows that the model point estimates are effective in fitting the actual calcium measurements.</p><p>(0.55 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s004\"><label>Figure S3</label><caption><p>Peak height dose response. This figure shows the single ligand calcium dose responses for C5a and UDP stimulation.</p><p>(0.21 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s005\"><label>Figure S4</label><caption><p>Knockdown simulations. This figure shows representative simulations and data for each knockdown experiment. A complete set of all 96 experiments is provided in a supplementary folder.</p><p>(0.69 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s006\"><label>Figure S5</label><caption><p>Input model fit. This figure shows the input model (described in <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>) fit to the FITC measurements. The ligand concentration that the cell sees does not transit instantaneously from 0 to the final concentration. The ligand concentration is expected to take an amount of time that is significant on the scale of the measurements made for this study to reach the final concentration.</p><p>(0.12 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s007\"><label>Figure S6</label><caption><p>Large pathway diagram.</p><p>(0.18 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s008\"><label>Figure S7</label><caption><p>System of differential equations. This figure shows the complete set of differential equations used to simulate the model. These equations are also available in the source c code for the model supplied. This system of equations with the initial conditions and nominal parameter values reported in ##SUPPL##11##Table S1## and ##SUPPL##12##Table S2##, respectively, completely define the model and allow for the reproduction of the simulations used in this paper on any platform.</p><p>(1.62 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s009\"><label>Figure S8</label><caption><p>Hill function self-synergy. Consider a Hill function, . is a dimensionless critical concentration <italic>y</italic>*, below which self-synergy will occur. Based on the analysis, we conclude that: (i) <italic>n</italic> must be greater than 1 for self-synergy to occur, (ii) self synergy never occurs if the concentration <italic>x</italic> exceeds equilibrium constant <italic>K</italic> (<italic>y</italic>&gt;1), and (iii) for <italic>n</italic>&gt;2, there is a large range of concentration for self-synergy. In the G protein model, <italic>x</italic>, is the concentration of IP3-IP3R, <italic>H</italic>(<italic>x</italic>) is the rate of change in cytosolic calcium concentration and <italic>n</italic> = 4. We have tested the validity of this self synergy hypothesis by stimulating the cells with both 20 nM UDP and 40 nM UDP (data not shown). Though at such low ligand concentrations, the measurement variability is high, we observed that the synergy ratio, on average was 1.17 compared to a value of 1.25 predicted by the model.</p><p>(0.02 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s010\"><label>Figure S9</label><caption><p>Parameter sensitivity analysis. The parameter of interest is varied by 10% while all other parameters are kept constant. The parameters are grouped according to their functionalities. The sensitivity coefficient is the ratio of the relative change in the peak height to the relative change in the parameter value. The four most sensitive parameters (sensitivity coefficient &gt;2) in the Cacyt category are Vqssk50 (IP3+IP3K_a-&gt;IP4+IP3K_a (Vmax)), Kqssk50 (IP3+IP3K_a-&gt;IP4+IP3K_a (Km)), a1 (Ca leak into the cell from outside), and Kex (Na/Ca exchange activation const). The top 3 most sensitive parameters in the PLCb3 category are: k21bf* (PLCb3_Ca_Gbg_PIP2-&gt;PLCb3_Ca_Gbg+IP3+DAG), k20f (Gbg+PLCb3_Ca-&gt;PLCb3_Ca_Gbg), k21af* (PLCb3_Ca_Gbg+PIP2-&gt;PLCb3_Ca_Gbg_PIP2). A star next to the parameter name indicates it was estimated.</p><p>(0.04 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s011\"><label>Protocol S1</label><caption><p>FITC protocol.</p><p>(0.03 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s012\"><label>Table S1</label><caption><p>Model initial conditions. This table shows the initial conditions used for the model. The model was run for sufficient time for the species states in the model to reach equilibrium before ligand stimulation was added. The number of molecules was calculated using a cell volume of 1 pL.</p><p>(0.05 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s013\"><label>Table S2</label><caption><p>Model parameters. This table shows the nominal parameters used for the model. Parameter distributions that were estimated are shown as shaded rows and with a star next to the parameter name in the table. The prior distribution for each parameter is as described in the <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref> section with mean value specified by the column labeled “prior”.</p><p>(0.14 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s014\"><label>Table S3</label><caption><p>Parameter posterior uncertainty and references. This table shows the HPD intervals as computed by the R CODA library function “hpdinterval”. HPD intervals for each of the three MCMC chains were calculated and the union of those intervals is reported for each parameter in this table. The prior value reported in ##SUPPL##12##Table S2## was set using information from references listed in the appropriate column. The references used to form the basis of the parameter estimates are shown in the last column.</p><p>(0.37 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pcbi.1000185.s015\"><label>Table S4</label><caption><p>Goodness of fit evaluation. We use the mean squared error criterion to evaluate the goodness of our model fit to the data. We have used this data in the estimation procedure and thus does not constitute a true validation. However, we show that in general our model fits the bulk of the data. Those areas of lack-of-fit are usually due to extraordinary experiment-to-experiment variation and in some cases point to unaccounted mechanisms. We elaborate on one such mechanism (multiple GRK isoforms) in the text of the article.</p><p>(0.27 MB DOC)</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"nt101\"><label/><p>Five different cell lines that have a perturbation in the level of a key signal transduction protein were constructed by shRNAi lentiviral infection. The calcium response from these cell lines in addition to the wild-type cell line were used to fit relevant parameters in the model. Because shRNAi does not entirely remove the protein product, the fraction knockdown was estimated by qRT-PCR and by Western blot analysis. The standard error (se) was computed for each estimate and the upper and lower confidence intervals were computed as ±3·se. The knockdown confidence intervals are used in the GPCR model to construct prediction confidence intervals for the calcium response. Where several cell lines were constructed for each knockdown, the best was selected and reported in parenthesis. The sample size for each knockdown-ligand dose combination is shown in the last 6 columns.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p>MLR was supported by a Department of Energy Computational Science Graduate Fellowship (DE-FG02-97ER25308). RAR and TIR were supported by National Institutes of Health (NIH) Grant GM62114. PF, TD, and APA were supported by the Alliance for Cellular Signaling (NIH Grant U54 GM62114). APA would also like to acknowledge support of the Howard Hughes Medical Institute. MIJ was supported by a grant from Microsoft Research and a grant from Yahoo! Research.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"pcbi.1000185.s001.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s002.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s003.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s004.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s005.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s006.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s007.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s008.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s009.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s010.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s011.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s012.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s013.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s014.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pcbi.1000185.s015.doc\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["10"], "element-citation": ["\n"], "surname": ["Berg", "Tymoczko", "Stryer"], "given-names": ["JM", "JL", "L"], "year": ["2002"], "source": ["Biochemistry. 5th ed"], "publisher-loc": ["Bethesda (Maryland)"], "publisher-name": ["W.H. Freeman; National Center for Biotechnology Information"]}, {"label": ["42"], "element-citation": ["\n"], "surname": ["Hindmarsh", "Brown", "Grant", "Lee", "Serban"], "given-names": ["AC", "PN", "KE", "SL", "R"], "year": ["2005"], "article-title": ["SUNDIALS: Suite of Nonlinear and Differential/Algebraic Equation Solvers."], "source": ["ACM Trans Math Softw"], "volume": ["31"], "fpage": ["363"], "lpage": ["396"]}, {"label": ["45"], "element-citation": ["\n"], "surname": ["Robert", "Casella"], "given-names": ["CP", "G"], "year": ["2004"], "source": ["Monte Carlo Statistical Methods"], "publisher-loc": ["New York"], "publisher-name": ["Springer"]}, {"label": ["46"], "element-citation": ["\n"], "surname": ["Gelman", "Rubin"], "given-names": ["A", "DB"], "year": ["1992"], "article-title": ["Inference from iterative simulation using multiple sequences."], "source": ["Stat Sci"], "volume": ["7"], "fpage": ["457"], "lpage": ["472"]}]
{ "acronym": [], "definition": [] }
46
CC0
no
2022-01-13 00:55:15
PLoS Comput Biol. 2008 Sep 26; 4(9):e1000185
oa_package/37/7a/PMC2528964.tar.gz
PMC2528965
18818728
[ "<title>Introduction</title>", "<p>This review aims at outlining the broad features of various frequently used quantitative transmission/disequilibrium tests (quantitative TDTs). We focus on describing the models assumed in these tests and the relationships between the tests. It is impossible in a brief review to describe and compare the great variety of quantitative TDT procedures to be found in the literature and that are available in computer packages, because some of these procedures involve quite complex forms of data and a sophisticated statistical analysis. We have therefore deliberately restricted the scope of this review by considering only one simple form of data—namely, family trios—that is families with a mother, a father, and exactly one child. Further, we consider only the basic features of the procedures that we discuss. Even with these limitations, a number of interesting questions arise, some of which we raise but do not answer. We leave a deeper analysis of the procedures discussed here, and an analysis of the more complex procedures that we do not consider, to another occasion.</p>", "<p>The aim of the original qualitative TDT procedure ##REF##8447318##[1]## was to test for linkage (and linkage disequilibrium) between a marker locus and a disease locus in a way that overcomes problems arising from potential population stratification. We assume the same null hypothesis for the quantitative TDT procedures considered here (see below for details). For convenience, we focus here on quantitative TDT procedures carried out in the frequently used FBAT (<ext-link ext-link-type=\"uri\" xlink:href=\"http://biosun1.harvard.edu/~fbat/fbat.htm\">http://biosun1.harvard.edu/~fbat/fbat.htm</ext-link>) and QTDT (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.sph.umich.edu/csg/abecasis/QTDT/\">http://www.sph.umich.edu/csg/abecasis/QTDT/</ext-link>) packages. Specifically we discuss the “Rabinowitz” ##REF##9391826##[2]## and the “Monks-Kaplan” ##REF##10677318##[3]## procedures, as well as various “Abecasis” ##REF##10631157##[4]## and “Allison” ##REF##9042929##[5]## regression-based procedures, when applied specifically to family trio data. The questions addressed in this review are as follows:</p>", "<p>What are the properties of the various Allison/Abecasis regression-based tests and the Rabinowitz and the Monks-Kaplan tests?</p>", "<p>What is the relation between the regression-based tests and the Rabinowitz and Monks-Kaplan tests?</p>", "<p>What hypotheses are tested by the various quantitative TDT procedures that we describe?</p>", "<p>What testing procedures are best suited to various forms of data?</p>", "<p>Do the regression-based tests that we describe overcome population stratification problems?</p>", "<p>What power considerations arise in the choice of the test to be used?</p>", "<p>We note that some of these issues were previously considered by Lange et al. ##REF##12454799##[6]## from a different viewpoint.</p>" ]
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[ "<title>Conclusions</title>", "<p>These notes indicate that there are several matters that the investigator should keep in mind in his/her data analysis. First, as noted above, all the procedures described here test for changes in the phenotype value <italic>Y</italic> as a function of <italic>W</italic> (or equivalently changes in <italic>W</italic> as a function of <italic>Y</italic>). This implies that these procedures are best suited either to a random sample of data or to data only comprising both low and high values of the phenotype under discussion. If the data relate only to “extremely low” or to “extremely high” values of <italic>Y</italic>, the qualitative TDT procedure is perhaps more appropriate. Second, if the investigator suspects population stratification associated with mating types, careful consideration should be given to the test that is to be used. Third, the regression-based methods are more susceptible to departure from the normality assumption, but the Rabinowitz and Monks-Kaplan procedures are not. We suggest that users be cautious when interpreting results from different tests, especially when the distribution of the trait is non-normal.</p>", "<p>In this brief review there are many topics that we have not covered. On the practical side, we have purposely not tried to recommend particular tests for specific kinds of data. This was not our goal, and in any case, would require considering a very large number of possible situations. Similarly, we have not discussed approaches to handle missing genotypes, although there are standard ways to do this ##REF##16921375##[10]##–##UREF##0##[12]##. On the theoretical side, we have not discussed the statistical theory behind the procedures described above. “Optimal” procedures often use score statistics, but the choice of the appropriate statistic relies on a choice of model that is felt best to describe the data. Next, we have considered only informative mating types, whereas some procedures use data from uninformative mating types, which may cause inflation of type I error rate if the phenotype distributions are different for different mating types. These and other theoretical questions will be taken up elsewhere.</p>" ]
[ "<p>Quantitative trait transmission/disequilibrium tests (quantitative TDTs) are commonly used in family-based genetic association studies of quantitative traits. Despite the availability of various quantitative TDTs, some users are not aware of the properties of these tests and the relationships between them. This review aims at outlining the broad features of the various quantitative TDT procedures carried out in the frequently used QTDT and FBAT packages. Specifically, we discuss the “Rabinowitz” and the “Monks-Kaplan” procedures, as well as the various “Abecasis” and “Allison” regression-based procedures. We focus on the models assumed in these tests and the relationships between them. Moreover, we discuss what hypotheses are tested by the various quantitative TDTs, what testing procedures are best suited to various forms of data, and whether the regression-based tests overcome population stratification problems. Finally, we comment on power considerations in the choice of the test to be used. We hope this brief review will shed light on the similarities and differences of the various quantitative TDTs.</p>" ]
[ "<title>Notation and Data</title>", "<p>The notation used in the various quantitative TDT papers on which our comments are based is not consistent from one author to another, and we adopt a unifying notation that is loosely based on that of these papers. In accordance with standard statistical practice, we use upper case notation for random variables and the corresponding lower case notation for the observed values of these random variables. To focus on the main points in this expository review, we assume a specific (and restricted) form of data. We assume that the data concern a marker locus “<italic>A</italic>,” having two possible alleles, denoted by <italic>A</italic> and <italic>a</italic>, and consist of information on <italic>n</italic> family trios, with complete marker locus genotype information on the two parents and the child in each trio. The value of the quantitative trait of interest is known for the child in each trio but not for the parents. We assume, in line with the original qualitative TDT, that all parental mating types are informative (i.e., contain at least one <italic>Aa</italic> parent). The observed number of <italic>A</italic> alleles in the child in trio <italic>i</italic> is denoted by <italic>x<sub>i</sub></italic> (<italic>i</italic> = 1, 2,…, <italic>n</italic>), and the observed value of the quantitative trait of interest in the child in trio <italic>i</italic> is denoted by <italic>y<sub>i</sub></italic>.</p>", "<p>We do not consider here the extent to which the comments made below carry over to data other than those described above, for example cases where several children are observed in each family, where parental phenotype information is available, and where the data contain families with noninformative mating types. We restrict our analysis in this way so as to highlight the main features of the testing procedures that we discuss without getting into the analyses required for forms of data more complicated than those we consider.</p>", "<p>The null hypothesis tested is “no linkage (or no linkage disequilibrium) between the marker locus and a locus involved with the quantitative trait.” Under this null hypothesis, the mean number of <italic>A</italic> alleles, <italic>X<sub>i</sub></italic>, in the child in trio <italic>i</italic> will depend on the parental mating type, being 0.5 if it is <italic>aa</italic>×<italic>Aa</italic>, 1.0 if it is <italic>Aa</italic>×<italic>Aa</italic>, and 1.5 if it is <italic>AA</italic>×<italic>Aa</italic>. The null hypothesis variance of <italic>X<sub>i</sub></italic> also depends on the parental mating type, being 0.25 if it is <italic>aa</italic>×<italic>Aa</italic> or <italic>AA</italic>×<italic>Aa</italic> and 0.5 if it is <italic>Aa</italic>×<italic>Aa</italic>. We frequently use the convenient Abecasis ##REF##9042929##[5]## notation <italic>W<sub>i</sub></italic> (“within family”) to describe <italic>X<sub>i</sub></italic> minus its null hypothesis mean as computed from the mating type in trio <italic>i</italic>. The null hypothesis mean of <italic>W<sub>i</sub></italic> is zero and the null hypothesis variance of <italic>W<sub>i</sub></italic> is the same as that for <italic>X<sub>i</sub></italic>, and depends on the mating type in trio <italic>i</italic>. When discussing the typical family we drop the suffix <italic>i</italic> and use the generic notation <italic>W</italic>, <italic>w</italic>, <italic>X</italic>, <italic>x</italic>, <italic>Y</italic>, and <italic>y</italic>.</p>", "<title>Quantitative TDT Models</title>", "<p>In this section, we describe in algebraic terms the various quantitative TDT procedures outlined above and address questions raised in the Introduction.</p>", "<title/>", "<title>Properties of various quantitative TDT procedures</title>", "<p>We start with five Allison and Abecasis “regression-based” procedures. (The Abecasis “total” test is not a TDT test [as is indicated in the QTDT package documentation], so we ignore it.) These all assume a regression model where the mean of the phenotype <italic>Y<sub>i</sub></italic> of the child in trio <italic>i</italic> depends on the actual value <italic>w<sub>i</sub></italic> for that child, often along with other information, for example, the parental mating type of trio <italic>i</italic>. More precisely, the five models that we consider are as follows:</p>", "<p>The Abecasis “within only” model, denoted here Ab-Wthn. In this model, <italic>Y</italic> is assumed to depend only on the value of <italic>w</italic>, the observed difference between the number of <italic>A</italic> alleles in any child and the null hypothesis mean of this number, given the parental mating type for this child.</p>", "<p>The Abecasis “orthogonal” model, denoted here Ab-Orth. In this model, <italic>Y</italic> is assumed to depend on <italic>w</italic> and also a linear term describing parental mating type.</p>", "<p>The Abecasis “dominance” model, denoted here by Ab-Dom. This model generalizes Ab-Orth in that <italic>Y</italic> is assumed also to depend on whether the child in any trio is a homozygote or a heterozygote.</p>", "<p>The first Allison model, denoted here Al-Lin. This is a “general/linear” model, where <italic>Y</italic> is assumed to depend on parental mating type in an unspecified way and also on <italic>w</italic>.</p>", "<p>The second Allison model (his TDTQ5), denoted here Al-Quad. This is a “general/quadratic” model, and extends Al-Lin by assuming that <italic>Y</italic> depends also on <italic>w</italic>\n<sup>2</sup>. In algebraic terms, the assumptions for the “full,” or alternative, hypothesis case of these models can be written in terms of the three mating types considered as follows:</p>", "<p>\n<italic>Abecasis</italic> “<italic>within only</italic>” <italic>model</italic> (<italic>Ab-Wthn</italic>).</p>", "<p>For all parental mating types: <italic>Y</italic> = <italic>μ</italic>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>E</italic>.</p>", "<p>\n<italic>Abecasis</italic> “<italic>orthogonal</italic>” <italic>model</italic> (<italic>Ab-Orth</italic>).</p>", "<p>For <italic>aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>E</italic>;</p>", "<p>For <italic>Aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>α</italic>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>E</italic>;</p>", "<p>For <italic>AA</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+2<italic>α</italic>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>E</italic>.</p>", "<p>\n<italic>Abecasis</italic> “<italic>dominance</italic>” <italic>model</italic> (<italic>Ab-Dom</italic>).</p>", "<p>For <italic>aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>γd</italic>+<italic>E</italic>;</p>", "<p>For <italic>Aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>α</italic>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>γd</italic>+<italic>E</italic>;</p>", "<p>For <italic>AA</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+2<italic>α</italic>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>γd</italic>+<italic>E</italic>.</p>", "<p>(In this model, <italic>d</italic> = −1 for a homozygous child and +1 for a heterozygous child, and corresponds to <italic>W<sub>d</sub></italic> in the QTDT package documentation. For the data that we consider, the <italic>B<sub>d</sub></italic> term in QTDT package documentation for this model is a constant across the three mating types, and is thus absorbed into the constant <italic>μ</italic>.)</p>", "<p>\n<italic>Allison</italic> “<italic>general</italic>/<italic>linear</italic>” <italic>model</italic> (<italic>Al-Lin</italic>).</p>", "<p>For <italic>aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>β</italic>\n<sub>1</sub>*<italic>x</italic>+<italic>E</italic>;</p>", "<p>For <italic>Aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>α</italic>\n<sub>1</sub>+<italic>β</italic>\n<sub>1</sub>*<italic>x</italic>+<italic>E</italic>;</p>", "<p>For <italic>AA</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>α</italic>\n<sub>2</sub>+<italic>β</italic>\n<sub>1</sub>*<italic>x</italic>+<italic>E</italic>.</p>", "<p>\n<italic>Allison</italic> “<italic>general</italic>/<italic>quadratic</italic>” <italic>model</italic> (<italic>Al-Quad</italic>).</p>", "<p>For <italic>aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>β</italic>\n<sub>1</sub>*<italic>x</italic>+<italic>β</italic>\n<sub>2</sub>*<italic>x</italic>\n<sup>2</sup>+<italic>E</italic>;</p>", "<p>For <italic>Aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>α</italic>\n<sub>1</sub>+<italic>β</italic>\n<sub>1</sub>*<italic>x</italic>+<italic>β</italic>\n<sub>2</sub>*<italic>x</italic>\n<sup>2</sup>+<italic>E</italic>;</p>", "<p>For <italic>AA</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>+<italic>α</italic>\n<sub>2</sub>+β<sub>1</sub>*<italic>x</italic>+<italic>β</italic>\n<sub>2</sub>*<italic>x</italic>\n<sup>2</sup>+<italic>E</italic>.</p>", "<p>In all four models Greek symbols describe unknown parameters and <italic>E</italic> is a random residual term having mean zero and (unknown) variance . Because of the relations <italic>w</italic> = <italic>x</italic>−1/2 for <italic>Aa</italic>×<italic>aa</italic> matings, <italic>w</italic> = <italic>x</italic>−1 for <italic>Aa</italic>×<italic>Aa</italic> matings, and <italic>w</italic> = <italic>x</italic>−3/2 for <italic>Aa</italic>×<italic>AA</italic> matings, Al-Lin and Al-Quad can be re-written conveniently as:</p>", "<p>\n<italic>Allison</italic> “<italic>general</italic>/<italic>linear</italic>” <italic>model</italic> (<italic>Al-Lin</italic>).</p>", "<p>For <italic>aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>\n<sub>1</sub>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>E</italic>;</p>", "<p>For <italic>Aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>\n<sub>2</sub>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>E</italic>;</p>", "<p>For <italic>AA</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>\n<sub>3</sub>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>E</italic>.</p>", "<p>\n<italic>Allison</italic> “<italic>general</italic>/<italic>quadratic</italic>” <italic>model</italic> (<italic>Al-Quad</italic>).</p>", "<p>For <italic>aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>\n<sub>1</sub>+(<italic>β</italic>\n<sub>1</sub>+<italic>β</italic>\n<sub>2</sub>)<italic>w</italic>+<italic>β</italic>\n<sub>2</sub>\n<italic>w</italic>\n<sup>2</sup>+<italic>E</italic>;</p>", "<p>For <italic>Aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>\n<sub>2</sub>+(<italic>β</italic>\n<sub>1</sub>+2<italic>β</italic>\n<sub>2</sub>)<italic>w</italic>+<italic>β</italic>\n<sub>2</sub>\n<italic>w</italic>\n<sup>2</sup>+<italic>E</italic>;</p>", "<p>For <italic>AA</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>\n<sub>3</sub>+(<italic>β</italic>\n<sub>1</sub>+3<italic>β</italic>\n<sub>2</sub>)<italic>w</italic>+<italic>β</italic>\n<sub>2</sub>\n<italic>w</italic>\n<sup>2</sup>+<italic>E</italic>.</p>", "<p>The null hypothesis tested in the Ab-Wthn, Ab-Orth, and Al-Lin models is <italic>β</italic>\n<sub>1</sub> = 0, and in the Ab-Dom model is <italic>β</italic>\n<sub>1</sub> = <italic>γ</italic> = 0. The null hypothesis tested in the original Al-Quad model is , and this is equivalent to <italic>β</italic>\n<sub>1</sub> = <italic>β</italic>\n<sub>2</sub> = 0 in the re-written version above. The testing procedures in all five cases follow standard multiple regression methods, with mating type membership denoted with indicator variables. The null hypothesis model in each case removes a certain sum of squares for the phenotypic measurements in the children, and the full model removes a larger (or in rare cases, an equal) sum of squares. The difference between these two sums of squares forms the key component of the numerator of the <italic>F</italic> statistic used in all testing methods. This component is divided by the respective “model” degrees of freedom, which is equal to the number of extra parameters in each full model compared to the number in the corresponding null hypothesis model. This number takes the value 1 for Ab-Wthn, Ab-Orth, and Al-Lin and takes the value 2 for Ab-Dom and Al-Quad. This division by 2 tends to lead to smaller <italic>F</italic> ratios for Ab-Dom and Al-Quad, and thus to reduce power, and it is a trade-off against the increased generality of those models. This point is discussed further below.</p>", "<p>The use of the <italic>F</italic> distribution to determine the significance of the observed value of the <italic>F</italic> statistic is appropriate only if the data have a normal distribution. For cases where the data are taken from one extreme tail of some distribution, for example very large values of the quantitative measurement, this might be an unreasonable assumption. This matter is discussed further below.</p>", "<p>The three Abecasis models are nested, with Ab-Wthn being a special case of Ab-Orth, which in turn is a special case of Ab-Dom. Similarly Al-Lin is a special case of Al-Quad. Ab-Wthn and Ab-Orth are also special cases of Al-Lin. The nesting property is reflected in the residual degrees of freedom for the respective models: under the assumptions we have made concerning the data analyzed, the Ab-Wthn model has <italic>n</italic>−2 residual degrees of freedom, the Ab-Orth model has <italic>n</italic>−3 residual degrees of freedom, the Al-Lin and the Ab-Dom models have <italic>n</italic>−4 residual degrees of freedom, and the Al-Quad model has <italic>n</italic>−5 residual degrees of freedom.</p>", "<p>There are regression-based models that are more general than those discussed above. A model more general than Ab-Dom and Al-Lin, and including these as particular cases, is:</p>", "<p>“<italic>General/dominance</italic>” <italic>model</italic>.</p>", "<p>For <italic>aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>\n<sub>1</sub>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>γδ</italic>+<italic>E</italic>;</p>", "<p>For <italic>Aa</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>\n<sub>2</sub>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>γδ</italic>+<italic>E</italic>;</p>", "<p>For <italic>AA</italic>×<italic>Aa</italic> parental mating type: <italic>Y</italic> = <italic>μ</italic>\n<sub>3</sub>+<italic>β</italic>\n<sub>1</sub>\n<italic>w</italic>+<italic>γδ</italic>+<italic>E</italic>.</p>", "<p>This model has <italic>n</italic>−5 residual degrees of freedom. A model more general than this, with <italic>n</italic>−6 residual degrees of freedom, and which includes all regression-based models described above, allows a term in <italic>w</italic>\n<sup>2</sup> as well as those in the “general/dominance” model. These more general models are not considered further here.</p>", "<p>We can also consider testing procedures other than those described above. In particular, we suggest a modification of the Ab-Dom test, in which the null hypothesis is changed from the present <italic>β</italic>\n<sub>1</sub> = <italic>γ</italic> = 0 to simply <italic>β</italic>\n<sub>1</sub> = 0. This is for two reasons. First, it does not seem natural to test simultaneously the two hypotheses that no dominance phenotypic effects exist and that <italic>y</italic> does not depend on the transmission values <italic>w</italic>. Second, in testing the hypothesis <italic>β</italic>\n<sub>1</sub> = 0 instead of <italic>β</italic>\n<sub>1</sub> = <italic>γ</italic> = 0, one only has a single “model” degree of freedom, leading to increased power (compared to Ab-Dom) in testing for transmission effects.</p>", "<p>We now describe the Rabinowitz ##REF##9391826##[2]## and Monks-Kaplan ##REF##10677318##[3]## procedures. In both of these procedures, the phenotype measurements <italic>y</italic>\n<sub>1</sub>, <italic>y</italic>\n<sub>2</sub>,…,<italic>y<sub>n</sub></italic> in the children in the <italic>n</italic> trios are taken as given, and used as weights on the transmission random variables <italic>W</italic>\n<sub>1</sub>, <italic>W</italic>\n<sub>2</sub>,…,<italic>W<sub>n</sub></italic>. This is in direct contrast to the Abecasis ##REF##10631157##[4]## and Allison ##REF##9042929##[5]## regression-based procedures, which take the <italic>w<sub>i</sub></italic> as given and the phenotype measurements <italic>Y</italic>\n<sub>1</sub>, <italic>Y</italic>\n<sub>2</sub>, …, <italic>Y<sub>n</sub></italic> as random variables. It is, however, more in line with the original qualitative TDT, which also uses <italic>W</italic>\n<sub>1</sub>, <italic>W</italic>\n<sub>2</sub>, …,<italic>W<sub>n</sub></italic> as the random variables of interest.</p>", "<p>For the data that we consider, Rabinowitz ##REF##9391826##[2]## defines by , where <italic>y̅</italic> is the average of the <italic>y<sub>i</sub></italic> values taken over the <italic>n</italic> children in the data. Given the observed values <italic>w</italic>\n<sub>1</sub>, <italic>w</italic>\n<sub>2</sub>,…, <italic>w<sub>n</sub></italic> of <italic>W</italic>\n<sub>1</sub>, <italic>W</italic>\n<sub>2</sub>,…, <italic>W<sub>n</sub></italic>, his test statistic <italic>z</italic> isIn this expression, the sum (as with all sums in this article) is taken over <italic>i</italic> = 1, 2, …, <italic>n</italic>, and is as defined above. This statistic is based on the “within family” <italic>w<sub>i</sub></italic> values because if this is done ##REF##9391826##[2]##, the effects of population stratification are overcome. (This parallels a similar observation in the original qualitative TDT ##REF##8447318##[1]##.) The Rabinowitz statistic is written as a <italic>z</italic> rather than a <italic>t</italic> because, with the taken as given, the null hypothesis standard deviation of —the term in the denominator of Equation 1 —is known. Central limit theorem arguments then show that if <italic>n</italic> exceeds about 20, the Rabinowitz statistic has an approximate <italic>N</italic>(0, 1) distribution when the null hypothesis is true.</p>", "<p>The Monks-Kaplan statistic is similar to the Rabinowitz statistic, beingThe numerators in <italic>z</italic> and <italic>t<sub>MK</sub></italic> are the same, but the denominator in the Monks-Kaplan statistic contains a standard deviation estimate rather than a known standard deviation. (This allows generalizations to handle data more complex than the data considered here.) It is written as a <italic>t</italic> statistic because of this fact.</p>", "<title>Relationship between regression-based tests and Rabinowitz and Monks-Kaplan tests</title>", "<p>The Rabinowitz and the Monks-Kaplan procedures differ from the Allison and the Abecasis procedures in various ways, of which we mention two. First, and most important, they regard the <italic>W<sub>i</sub></italic> values as random variables with the <italic>y<sub>i</sub></italic> values taken as given, whereas the Abecasis and Allison procedures regard the <italic>Y<sub>i</sub></italic> values as random variables with the <italic>w<sub>i</sub></italic> values taken as given. Second, unlike the Abecasis and Allison procedures, neither is explicitly based on regression models (see Laird et al. ##REF##11055368##[7]## for more details). Despite these differences, it is interesting to consider the hypothesis testing procedure in a “role-reversal” regression model of the formThis model, when compared to the Allison and Abecasis procedures, reverses the roles of <italic>W</italic> and <italic>Y</italic> in the regression. Because the Rabinowitz and Monks-Kaplan statistics are defined in terms of rather than <italic>y<sub>i</sub></italic>, it is convenient to reformulate Equation 3 equivalently asThe estimate of β in this regression is , and the standard regression <italic>t</italic> statistic testing for departures of <italic>β</italic> from zero iswhere <italic>s</italic> is the usual regression estimate of the standard deviation of . The Rabinowitz statistic (Equation 1) has the same numerator as that in Equation 5 but has, in the denominator, the known null hypothesis standard deviation of rather than a regression-based estimate of this standard deviation. The Monks-Kaplan statistic also has the same numerator as that in Equation 5, but has a standard deviation estimate in the denominator different from that in both Equation 1 and Equation 5.</p>", "<p>Despite this similarity, there are essential differences between the Allison and Abecasis regression procedures and the Rabinowitz and Monks-Kaplan procedures. The Rabinowitz procedure, and in general all FBAT procedures, use “score statistics” based on the alleles transmitted to the children, conditional on the parental genotypes and the offspring phenotype. They are not explicitly based on regression models. Under the score test approach, the null hypothesis distribution of the test statistic is calculated directly from Mendel's laws. The test statistic thus has the correct distribution so long as these laws hold, regardless of any hypothetical model for the mean and variance of the offspring phenotype.</p>", "<title>Hypotheses tested by various quantitative TDT procedures</title>", "<p>There is an important difference between the hypothesis being tested by all the quantitative TDT procedures described above and the original qualitative TDT. The original TDT assesses whether the sum of the <italic>w<sub>i</sub></italic> values differs significantly from zero. By contrast, none of the quantitative TDT procedures described above assess whether the <italic>w<sub>i</sub></italic> values (or their weighted sum in the Rabinowitz and Monks-Kaplan procedures) differ significantly from zero. This can be seen from the fact that they are all unchanged if an arbitrary constant is added to the <italic>w<sub>i</sub></italic> values. What they do test is whether there is significant change in the value of <italic>w</italic> as <italic>y</italic> changes (or of <italic>y</italic> as <italic>w</italic> changes). This is explicit in the regression procedures and also applies for the Rabinowitz and Monks-Kaplan procedures. We discuss this fact below in the context of the data that the investigator is analyzing. By contrast, it is the intercept in the estimated regression (Equation 4), namely <italic>w̅</italic>, that is directly comparable to the qualitative TDT statistic. We now show that, if the Rabinowitz approach of using the known variance of <italic>W<sub>i</sub></italic> is taken, the test of whether this intercept is zero is identical to the original qualitative TDT procedure.</p>", "<p>The standard regression test statistic of the null hypothesis <italic>α</italic> = 0 in the regression model (Equation 4) is the estimated intercept (in this case <italic>w̅</italic>) divided by the estimated standard deviation of <italic>W̅</italic>. In this case, the standard deviation of <italic>W̅</italic> is known, so the appropriate (<italic>z</italic>) test statistic is <italic>w̅</italic> divided by the standard deviation of <italic>W̅</italic>. It is equivalent and more convenient to use <italic>z</italic>\n<sup>2</sup> as test statistic, where <italic>z</italic>\n<sup>2</sup> can be written as divided by the variance of . Under the null hypothesis, <italic>z</italic>\n<sup>2</sup> has an approximate chi-square distribution with one degree of freedom. We now calculate the value of <italic>z</italic>\n<sup>2</sup> in terms of transmission information.</p>", "<p>We consider first the case of those trios having either <italic>Aa</italic>×<italic>AA</italic> or <italic>Aa</italic>×<italic>aa</italic> matings. If in any such trio the heterozygous parent transmits the <italic>A</italic> allele, the value of <italic>w</italic> for that trio is +1/2. We write the number of such trios as <italic>m</italic>\n<sub>1</sub>. If in any such trio the heterozygous parent transmits the <italic>a</italic> allele, the value of <italic>w</italic> for that trio is −1/2. We write the number of such trios as <italic>m</italic>\n<sub>2</sub>. The value of <italic>w</italic> in any trio where the parental mating type is <italic>Aa</italic>×<italic>Aa</italic> is +1 if both parents transmit the <italic>A</italic> allele, 0 if one parent transmits the <italic>a</italic> allele and the other parent transmits the <italic>A</italic> allele, and −1 if both transmit the <italic>a</italic> allele. We write the respective numbers of these trios as <italic>m</italic>\n<sub>3</sub>, <italic>m</italic>\n<sub>4</sub>, and <italic>m</italic>\n<sub>5</sub>. Thus is <italic>m</italic>\n<sub>1</sub>/2−<italic>m</italic>\n<sub>2</sub>/2+<italic>m</italic>\n<sub>3</sub>−<italic>m</italic>\n<sub>5</sub> = <italic>m</italic>\n<sub>1</sub>−<italic>m</italic>\n<sub>2</sub>+2<italic>m</italic>\n<sub>3</sub>−2<italic>m</italic>\n<sub>5</sub>/2. But this is just (<italic>b</italic>−<italic>c</italic>)/2, where <italic>b</italic> is the total number of transmissions of <italic>A</italic> from heterozygous parents and <italic>c</italic> is the total number of transmissions of <italic>a</italic> from heterozygous parents. The numerator in <italic>z</italic>\n<sup>2</sup> is thus (<italic>b</italic>−<italic>c</italic>)<sup>2</sup>/4.</p>", "<p>We now turn to the denominator of <italic>z</italic>\n<sup>2</sup>. Suppose that in the <italic>n</italic> trios, there are exactly <italic>n</italic>\n<sub>1</sub> where the parental mating type is <italic>Aa</italic>×<italic>Aa</italic>. Since the variance of <italic>W</italic> in any such trio is 1/2 and for all other mating types is 1/4, the variance of is <italic>n</italic>\n<sub>1</sub>/2+(<italic>n</italic>−<italic>n</italic>\n<sub>1</sub>)/4 = [2<italic>n</italic>\n<sub>1</sub>+(<italic>n</italic>−<italic>n</italic>\n<sub>1</sub>)]/4. This may be written, using the notation of the preceding paragraph, as (<italic>b</italic>+<italic>c</italic>)/4, because <italic>b</italic>+<italic>c</italic> is the total number of transmissions from heterozygous parents. It follows from the above that <italic>z</italic>\n<sup>2</sup> = (<italic>b</italic>−<italic>c</italic>)<sup>2</sup>/(<italic>b</italic>+<italic>c</italic>), and this is the standard qualitative TDT statistic of Spielman et al. ##REF##8447318##[1]##.</p>", "<p>Following a similar line of reasoning, if the variance of is estimated from the data, (as is done in the qualitative TDT procedure of Martin et al. ##REF##9311750##[8]##, where such estimation is needed for non-trio data) the test of the hypothesis <italic>α</italic> = 0 in the regression (Equation 4) can be shown to be identical to the Martin et al. procedure ##REF##9311750##[8]##.</p>", "<title>Testing procedures best suited to various forms of data</title>", "<p>The above considerations lead to a discussion of the data being analyzed. The original qualitative TDT of Spielman et al. ##REF##8447318##[1]## uses data only from children affected by some disease. Spielman et al. ##REF##8447318##[1]## also discuss an alternative to the qualitative TDT procedure when segregation distortion at the marker locus is suspected. In this alternative procedure, the proportion of transmissions of the <italic>A</italic> allele from heterozygous parents is compared not with 1/2, as in the original “standard” TDT, but with the corresponding proportion in nonaffected individuals. The analogues of “affected and not affected” in the quantitative TDT context might be “extreme and nonextreme phenotype values.” If the <italic>y<sub>i</sub></italic> values in a quantitative TDT procedure are derived from a random sample, and if the null hypothesis is not true, one might expect extreme and nonextreme <italic>y</italic> values to tend to correspond to different <italic>w</italic> values. This might lead to a significant dependence of <italic>w</italic> on <italic>y</italic> (or equivalently of <italic>y</italic> on <italic>w</italic>). Thus for a random sample, a “slope” test such as those carried out by all quantitative TDT procedures described above appears to be appropriate. These procedures are analogous to the alternative qualitative TDT testing procedure of Spielman et al. ##REF##8447318##[1]##.</p>", "<p>Thus if the data analyzed concern either only extremely low or extremely high <italic>y<sub>i</sub></italic> values (but not both), which might be thought of as corresponding to “affected” children, it might be more appropriate to carry out the qualitative TDT test that uses only data from such children. As shown above, this is identical to an “intercept” regression test. One may, if desired, carry out both this procedure and a (slope) quantitative TDT test, since in a regression procedure, the test of the slope and the test of the intercept in a regression line are independent. However, extreme phenotypes might well not have a normal distribution, so that those regression-based procedures that use <italic>F</italic> tests might be unreliable. The Rabinowitz and the Monks-Kaplan procedures are not subject to this problem. The information provided jointly by the qualitative TDT and the Rabinowitz or the Monks-Kaplan tests would show whether there is significant absolute linkage disequilibrium and also a significant change in linkage disequilibrium as the phenotypic value varies.</p>", "<title>Population stratification in regression-based tests</title>", "<p>The aim of the original qualitative TDT was to overcome potential problems arising from population stratification, and this was done by using the transmission values <italic>w<sub>i</sub></italic>. By design, the Rabinowitz and Monks-Kaplan procedures also overcome population stratification problems, using the same approach. The situation is not, however, so straightforward for the regression models.</p>", "<p>The simplest of the regression models considered above, namely Ab-Wthn, is a regression of <italic>Y</italic> on <italic>w</italic>. Once parental mating type information has been factored out of the regression, Ab-Orth, Al-Lin, and Al-Quad also have this property. The same is true of our suggested modification of the Ab-Dom procedure. It is thus tempting to argue that these regression procedures are immune to problems caused by population stratification, using the claim that it is sufficient to overcome stratification problems by using only <italic>w</italic> in the testing procedure. This conclusion is, however, not necessarily correct.</p>", "<p>We illustrate this by considering an extreme case where the parental mating type <italic>Aa</italic>×<italic>aa</italic> occurs only in one stratum in the population, parental mating type <italic>Aa</italic>×<italic>Aa</italic> occurs only in another stratum, and parental mating type <italic>Aa</italic>×<italic>AA</italic> occurs only in a third stratum in the population. Suppose also that for reasons not connected with the marker locus, the null hypothesis mean phenotypes in the three strata are <italic>μ</italic>\n<sub>1</sub> in the first stratum, <italic>μ</italic>\n<sub>2</sub> in the second and <italic>μ</italic>\n<sub>3</sub> in the third. Then of the models considered above, the Al-Lin model most closely reflects this situation. Suppose finally that for reasons having nothing to do with the marker locus, the three means—<italic>μ</italic>\n<sub>1</sub>, <italic>μ</italic>\n<sub>2</sub>,and <italic>μ</italic>\n<sub>3</sub>—are not all equal.</p>", "<p>Suppose that despite this, the investigator uses the Ab-Wthn test, which in effect assumes equality of <italic>μ</italic>\n<sub>1</sub>, <italic>μ</italic>\n<sub>2</sub>, and <italic>μ</italic>\n<sub>3</sub>. The regression sum of squares (used in the numerator of the <italic>F</italic> ratio for this test) isand the residual sum of squares (used in the numerator of the <italic>F</italic> ratio) iswhere <italic>b</italic>\n<sub>1</sub> is the standard regression estimate of <italic>β</italic>\n<sub>1</sub>. If <italic>μ</italic>\n<sub>1</sub> = <italic>μ</italic>\n<sub>2</sub> = <italic>μ</italic>\n<sub>3</sub>, then under the null hypothesis, these sums of squares have expected values <italic>σ</italic>\n<sup>2</sup> and (<italic>n</italic>−2)<italic>σ</italic>\n<sup>2</sup> respectively, the corresponding mean squares both have expected values <italic>σ</italic>\n<sup>2</sup>, and (assuming a normal distribution for the phenotype) the <italic>F</italic> statistic has the <italic>F</italic> distribution. If however <italic>μ</italic>\n<sub>1</sub>, <italic>μ</italic>\n<sub>2</sub>, and <italic>μ</italic>\n<sub>3</sub> are not all equal, the null hypothesis mean values of these two mean squares areandrespectively. In these expressions all sums are taken over the <italic>n</italic> trios, is the value of <italic>μ</italic> (either <italic>μ</italic>\n<sub>1</sub>, <italic>μ</italic>\n<sub>2</sub>, or <italic>μ</italic>\n<sub>3</sub>) appropriate for trio <italic>i</italic>, and is the average of the values. The term in square brackets in the first expression is clearly non-negative, and the term in square brackets in the second expression can be shown, via the Cauchy-Schwartz inequality, also to be non-negative. Thus the <italic>F</italic> statistic does not now have the <italic>F</italic> distribution when the null hypothesis (<italic>β</italic>\n<sub>1</sub> = 0) is true, so that the type I error of the Ab-Wthn procedure will now not be at the assumed value. Simulations also show that it generally exceeds the assumed value. In this sense, and in this example, the Ab-Wthn procedure is not immune to population stratification. A similar observation was also reported by Yu et al. ##REF##16380716##[9]##. This is of course an extreme example, which might seldom arise in practice. It nevertheless shows that caution is needed in assuming that a test based on the <italic>w<sub>i</sub></italic> values only is automatically immune to stratification problems for quantitative traits. It also indicates that the practitioner should make an assessment of which regression model most closely reflects the situation from which the data were obtained and use the testing procedure for that model.</p>", "<title>Power considerations</title>", "<p>Given the various quantitative TDT procedures, it is important to address the power comparisons between them. First it has to be noted that the power comparison between any two tests is only meaningful if both are “anchored” so as to have the same type I error. In the situation described in the previous paragraph, a power comparison between the Ab-Wthn and the Al-Lin test is not meaningful. (The Al-Lin test is valid in this situation, and has the assumed type I error.)</p>", "<p>Suppose on the other hand that there is no population stratification associated with parental mating type and the Ab-Wthn test can be taken as appropriate. Then both the Ab-Wthn and Al-Lin procedures are valid tests, and the <italic>F</italic> ratios in the two tests both have the <italic>F</italic> distribution under the null hypothesis. The Al-Lin test will lose a small amount of power because of an unnecessary decrease in the residual number of degrees of freedom. It follows from all the above that no uniform statement about power can be made, and that the investigator has to use his/her judgment about the most appropriate test to use.</p>" ]
[ "<p>We acknowledge the comments of several colleagues, in particular Gonçalo Abecasis, Rita Cantor, Heather Cordell, Eleanor Feingold, and Nan Laird. However, the views expressed above do not necessarily represent those of these colleagues.</p>" ]
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[ "<fn-group><fn fn-type=\"financial-disclosure\"><p>This research was supported by NIH grant GM081930 (RSS).</p></fn></fn-group>" ]
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[{"label": ["12"], "element-citation": ["\n"], "surname": ["Li", "Ding", "Abecasis"], "given-names": ["Y", "J", "GR"], "year": ["2007"], "article-title": ["Markov model for rapid haplotyping and genotype imputation in genome wide studies."], "source": ["Am J Hum Genet"], "volume": ["S79"], "fpage": ["2290"]}]
{ "acronym": [], "definition": [] }
12
CC BY
no
2022-01-12 23:38:08
PLoS Genet. 2008 Sep 26; 4(9):e1000180
oa_package/73/c5/PMC2528965.tar.gz
PMC2528966
18795102
[ "<title>Introduction</title>", "<p>Fibroproliferative disorders are a major cause of morbidity and mortality ##UREF##0##[1]##. Traditionally parsed into categories based on the target organ afflicted-lung, liver, kidney, heart, vasculature, CNS or integument-biomedical scientists now view the fibroproliferative diseases as sharing a common pathobiology independent of the organ or tissue that scars ##UREF##1##[2]##, ##UREF##2##[3]##. Organs can heal or scar following toxic exposures, with fibrosis predominating when the injurious agent cannot be eradicated by the host defense system, as occurs with certain infections; or is repeatedly introduced over a protracted interval of time as occurs in asbestosis, silicosis or alcohol-induced hepatic cirrhosis. The clinical focus in these situations is specific antimicrobial therapy or prevention. More vexing, however, are the fibroproliferative diseases of unknown cause, which frequently progress to organ dysfunction or death. Currently, while there are a few therapeutic leads ##REF##16160001##[4]##, ##REF##15735062##[5]##, there are no therapies that reproducibly interdict fibrosis.</p>", "<p>For decades, innate and adaptive immunity has served as the focal point for studies of tissue fibrosis. While an unremitting immune response can lead to fibrosis, in many idiopathic fibrotic disorders, immune-suppressive therapy affords limited benefit. This has shifted attention to the primary effector of the fibroproliferative response, the fibroblast itself. Fibroblasts from patients with systemic sclerosis, renal fibrosis, keloids and pulmonary fibrosis display aberrations in processes that govern nearly every aspect of the fibroproliferative response ##UREF##3##[6]##, ##REF##10213870##[7]##, ##REF##11350829##[8]##, ##REF##11465719##[9]##, ##REF##9776848##[10]##, ##REF##16277007##[11]##. These data indicate that fibrotic fibroblasts manifest pathological control of pathways governing proliferation, viability, motility, contractile function and connective tissue production. It is noteworthy that while these differences emerge in the context of exogenous signals from matrix, cytokines, chemokines, morphogens and peptide growth factors; fibrotic fibroblasts appear to retain a distinct cell biology <italic>in vitro</italic>.</p>", "<p>Here we study the fundamental pathobiology of tissue fibrosis by focusing on a lethal respiratory disorder, idiopathic pulmonary fibrosis (IPF), as a prototype fibroproliferative disease. The histological pattern of IPF is usual interstitial pneumonitis (UIP), a patchy fibroproliferative process that spares some respiratory units while affecting others nearby. Progression of fibrosis leads to obliteration of the gas-exchange surface. This pathological respiratory phenotype is the culmination of complex interactions among myofibroblasts, epithelial cells, cytokines, and the surrounding extracellular matrix. Myofibroblast foci, the pathologic hallmark of IPF, are comprised of myofibroblasts embedded in a type I collagen rich matrix ##REF##3750241##[12]##, and the burden of myofibroblast foci found in lung biopsy samples inversely correlates with patient survival ##REF##11587991##[13]##, ##REF##12119229##[14]##, ##REF##16840378##[15]##. Whereas myofibroblasts in healing wounds contract their matrix and undergo apoptosis in a timely manner, myofibroblasts in IPF lesions persist. The mechanism involves aberrant beta 1 integrin signaling in response to type I collagen. This results in defective PTEN function and unrestrained Akt signaling leading to downstream activation of the translation initiation machinery ##REF##18541712##[16]##. How this pathological integrin signaling alters the gene expression pathway of fibrotic myofibroblasts has not been elucidated.</p>", "<p>To answer this question, we took a systems biology approach and examined two key steps in the myofibroblast gene expression pathway genome-wide–transcription and ribosome recruitment. Transcriptional control in IPF has been previously characterized in lung tissue samples ##REF##16128620##[17]##; however, ribosome recruitment pattern-a measure of which transcripts are being translated into protein-has not been examined in tissue or cell lines. We elected to carry out this analysis using primary lung myofibroblasts in 3-dimensional type I collagen gels, an <italic>in vitro</italic> system that surrounds myofibroblasts in type I collagen in a context that lacks exogenous cytokines. To simulate an aberrant, fibrotic environment, we studied cells in type I collagen gels that were fixed to the sides of a tissue culture dish and therefore not allowed to contract (referred to as “non-contractile” matrices); to simulate the environment of physiological healing, we released the type I collagen gels from the sides of the dish and allowed the myofibroblasts to contract their matrices (referred to as “contractile” matrices) ##REF##8106541##[18]##. By examining the gene expression pathway of control and IPF myofibroblasts in both non-contractile and contractile collagen gels, we are able to determine the extent to which matrix type and the tissue of origin accounts for any differences observed at two levels of gene expression regulation.</p>", "<p>Here we show distinct intrinsic differences in the gene expression pathway between control and IPF myofibroblasts in both non-contractile and contractile type I collagen matrices. While differences are present at the transcriptional level, the majority of differences observed are at the level of ribosome recruitment. Importantly, we demonstrate that IPF myofibroblasts manifest a much greater dependence on collagen matrix conditions than do their control counterparts, changing the translational activity of a large set of transcripts. Our data indicate that IPF myofibroblasts are intrinsically pathological cells with fundamental changes in their gene expression pathway primarily at the level of ribosome recruitment regulation.</p>" ]
[ "<title>Methods</title>", "<title>Cell Line Procurement and Characterization</title>", "<p>Human primary myofibroblasts from twelve different donors were utilized (this study was approved by the University of Minnesota Institutional Review Board for Human Subjects Research). These consisted of six control samples (histologically normal lung distant from resected tumor) and six samples from patients with IPF (histologically confirmed UIP). Tissue was obtained at the time of biopsy, autopsy, lung resection or lung transplantation following procedures approved by the University of Minnesota Institutional Review Board for Human Subjects Research. Previous work performing microarray analysis on normal lung tissue##REF##16498083##[19]## has suggested significant differences when comparing samples from men and women and also when comparing patients older than 60 years with patients younger than 40 years of age. Patients in our study were not significantly different in terms of gender (p = 0.43) or age (IPF range 57–68, control range 56–82, p = 0.14) at the time of tissue procurement. Lung tissue explants were cultivated in 35 mm tissue culture dishes in explant medium (DMEM+20% FBS+antibiotics and antimycotics) at 37°C in 95% air, 5% CO2. Outgrowth was evident in 5 to 7 days, and cells filled the dish in 2 to 3 weeks. Cells from each 35 mm dish were released with trypsin-EDTA and placed in 100 mm tissue culture dishes after trypsin was neutralized with fresh explant medium. These cells, designated passage 1, were cultivated in growth medium (DMEM+10% FBS+antibiotics) at 37°C in 95% air, 5%CO2. Medium was replaced twice weekly, and cells were subcultivated weekly at a 1∶4 split ratio. Cells designated myofibroblasts in both IPF and control samples had typical spindle morphology, were vimentin- and alpha smooth muscle actin-positive; and factor VIII- and surfactant C-negative. Cells used in this study were between passage 4 and 9.</p>", "<title>Assessment of Proliferative Uniformity</title>", "<p>Myofibroblasts in log phase growth were released from culture dishes with Trypsin-EDTA, washed, suspended in PBS containing 2.5 µM of the stable vital dye carboxyfluoroscein succinimidyl ester (CFSE) (Sigma) and incubated at 37°C for 10 minutes (shaking every 2 minutes). The reaction was stopped with ice cold PBS. Cells were centrifuged (1000 g) and washed with PBS. The resultant CFSE labeled myofibroblasts were placed into 6 well clusters at 40,000 cells/well in growth medium (DMEM+10% FBS) and cultures continued (37°C, 5% CO2). At the time points indicated, cells were released from the culture dish and fixed (4% formaldehyde) prior to analysis by FACS (day 0 cells were harvested 4 h after seeding).</p>", "<title>Collagen Gel Preparation</title>", "<p>Collagen was obtained from Cohesion Corporation, Palo Alto, CA. Cells were removed from tissue culture plates using trypsin and mixed with DMEM, 10% FBS and collagen (final collagen concentration 0.5 mg/ml). This mixture was polymerized in a water bath at 37° C, aliquoted into 3.5 cm tissue culture dishes, and placed into an incubator at 37° until harvest. Final cell density was approximately 200,000 cells/ml. Tissue culture plates for non-contractile gels had been pre-coated with collagen 100 µg/ml in phosphate-buffered saline (PBS) in order to prevent matrix contraction. Cells in these non-contractile collagen matrices were incubated at 37°C for 6 hours. Contractile matrices were prepared as above; they were allowed to polymerize in uncoated tissue culture dishes for two hours, and then the gel was released by tapping the side of the dish. They were placed in an incubator for four more hours before harvesting (for a total 6-hour incubation time, equal to non-contractile matrices). Degree of collagen matrix contraction was not significantly different between IPF and control myofibroblasts (data not shown).</p>", "<title>Polyribosome Preparation</title>", "<p>Cells were harvested in log phase using trypsin and incorporated into collagen gels as described above. Myofibroblasts were collected from the gels at the predetermined time point using collagenase 5 mg/ml (Sigma, St. Louis, MO) containing cycloheximide (100 µg/ml) and collected by centrifugation. A small portion of non-homogenized cells was retained for Trireagent (Sigma) processing to isolate total cellular RNA (designated “total RNA”) for microarray analysis. The remaining cells were used for polyribosome preparations as described ##REF##12441348##[20]##. Ten 0.5 ml fractions were collected from each sample into tubes containing 50 µl of 10% SDS. RNA from each fraction was processed using Trireagent according to the manufacturer's directions and precipitated with isopropanol. Fractions 7–10, consisting of mRNA with four or more bound ribosomes, designated “heavy”, were pooled for microarray analysis.</p>", "<title>Microarray Hybridization</title>", "<p>Starting with 10 µg of ribosome-bound or total RNA, conversion to labeled cRNA was performed using the One Cycle Target Labeling and Control Reagent Kit according to the manufacturer's directions (Affymetrix Corp., Santa Clara, CA). Labeled fragmented cRNA (20 µg) was submitted to the University of Minnesota Biomedical Genomics Center and probed with Affymetrix U133plus2 microarrays.</p>", "<title>Quantification of mRNA by Real Time PCR</title>", "<p>A new set of polyribosome RNA preparations, different from those used to perform the microarray analysis, was used for real time PCR. We chose one IPF and one control myofibroblast primary cell type for this set of experiments. RNA from each fraction of the sucrose gradient was extracted using Trireagent and quantified. An internal standard, “alien RNA”, was spiked into each sample to control for differences in cDNA conversion efficiency as described in the instructions for the Alien® QRT-PCR Inhibitor Alert kit (Stratagene, LaJolla, CA). cDNA was synthesized from 2.0 µg of each fraction using Taqman Reverse Transcriptase Reagent Kit (Applied Biosystems, Foster City, CA) primed with oligo dT. Primer sequences for selected genes were selected using the DNASTAR program (DNASTAR, Inc., Madison, WI), and the resulting sequences were synthesized in the University of Minnesota microchemical facility. Real time PCR was performed using a LightCycler FastStart DNA Master<sup>PLUS</sup> SYBR Green I Kit (Roche Diagnostics, Indianapolis, IN). 2.5 ul of the cDNA product was used for amplification of each sample. Primer sequences were as follows: CFL2 forward <named-content content-type=\"gene\">5′GGA CCG TTC GAC ACT TGG AGA3′</named-content> CFL2 reverse <named-content content-type=\"gene\">5′AAT GGA CTG AGC TGG AGA AAT GG3′</named-content>; PDCD8 forward <named-content content-type=\"gene\">5′CAG CGA TGG CAT GTT CCT CTA3</named-content>; PDCD8 reverse <named-content content-type=\"gene\">5′ACG CGG CCT TTT TCT GTT TCT3′</named-content>; FUT10 forward <named-content content-type=\"gene\">5′AGC AGC GCG AGA GTA GAA GTG AAT3′</named-content>; FUT10 reverse <named-content content-type=\"gene\">5′CAG TAG ATG CCC CAG ACA GGA GAG3′</named-content>. Samples were quantified at the log-linear portion of the curve using LightCycler analysis software and compared to an external calibration standard curve. Each sample was normalized for cDNA conversion efficiency using the external “alien control”. The total RNA samples were normalized using β-actin. β-actin primer sequences were: forward <named-content content-type=\"gene\">5′ CTG GAA CGG TGA AGG TGA CA 3′</named-content>, reverse <named-content content-type=\"gene\">5′ AAG GGA CTT CCT GTA ACA ATG CA 3′</named-content>.</p>", "<title>Western blotting</title>", "<p>Cells were grown to 70% confluence and serum starved for 48 hr. Cells were released with trypsin and seeded onto 100 mm dishes that were pre-coated for 1 hr with Pur-Col monomeric collagen 100 ug/ml in PBS (Allergan Sales Inc.). Cells were allowed to adhere for 75 min, mechanically released and resuspended in lysis buffer (40 mM tris pH 7.5, 300 mM NaCl, 2 mM EDTA, 100 mM NaF, 2% NP-40, 1% Na Deoxycholate, supplemented with “Complete” protease inhibitor tablets (Roche)). Lysates were kept on ice for 10 min, centrifuged at 12,000G for 10 min with supernatants retained and subjected to electrophoresis and Western blotting with antibodies for Keratin 18, PDCD8 and CFL2 from Cell signaling (Boston, USA); FUT10 from Abcam (Cambridge, USA); and β-actin from Sigma (USA).</p>", "<title>Data analysis</title>", "<p>Totally 12 samples (6 IPF and 6 controls) informed the study. From each sample we obtained the polyribosome bound (4 or more bound ribosomes) and total RNA under the two conditions under study, thus totally 48 hybridizations. The data was normalized using GCRMA and updated probe sets definitions “RefSeq v7” as defined in ##REF##16284200##[21]## as these provide improved precision and accuracy ##REF##17288599##[22]##. We used the Significance Analysis of Microarrays (SAM) algorithm implemented in R “samr” v1.24 to identify differentially expressed genes using an un-paired or paired approach as applicable (thus when comparing within cell lines, non-contractile vs. contractile from the same donor, a paired test was used, otherwise we used a non-paired test). We further used a fixed s<sub>0</sub> = 0.1, a large delta table (400) and a fixed random seed (1,2,3,4,5,6,7,8,9) ##REF##11309499##[23]##. Only genes that were classified as present in at least 6 samples (in the studied comparison) using the present absent algorithm from MAS5 implemented in the “affy” package in R, were used as input as this reduced the noise ##REF##15921534##[24]##. We used GO::Termfinder v0.72 ##REF##15297299##[25]## to identify gene ontologies that were overrepresented in the generated gene lists and considered all with an False Discovery Rate (FDR)&lt;15% significant (using simulation significances implemented in GO::Termfinder).</p>", "<title>Study of pathway activity</title>", "<p>To test whether selected pathways were active in a specific comparison we sought to identify enrichment of genes within a pathway at the extreme ends of a ranked gene list. First all measured genes were ranked according to their transcription and translational “d-scores” (obtained from a SAM run including all available genes) comparison by comparison. We used a “step down” approach to test for enrichment of genes within the pathways from each end of the ranked gene list. First the range of statistics between the highest and lowest 1% d-scores was used to create 40 bins. Exclusion of the extreme 1% avoided outlier statistics from dominating the definition of the bins. For each pathway, we used Fisher's exact test to look for enrichment of genes until a given bin, compared to the total number of genes in the pathway-and the data set (thus first looking at the top 1% and then stepping down bin by bin). The analysis was performed both from the top and bottom of the ranked gene list, thus assessing activity of the pathway in the IPF and the control group. This generated matrixes of p-values and odds ratios for each pathway and from each direction. As many p-values were generated, we corrected for multiple testing using Benjamin &amp; Hochsberg multiple correction (implemented in fdrtool for R) of all p-values, from the pathways included in the analysis. To summarize the data across all pathways and studies in a matrix, we created a discrete output so that [significant overrepresentation from the top] (1), [significant overrepresentation from the bottom] (−1), [no significant overrepresentation] (0) or [significant overrepresentation from both top and bottom] (2) was indicated in each comparison to pathway interaction. Pathways showing an FDR&lt;0.05 were considered significant. We tested all modules at the same time, and therefore the correction for multiple testing accounted for all modules tested. This approach is similar to ##REF##15840702##[26]##.</p>" ]
[ "<title>Results</title>", "<title>Selection of time point for global analysis of transcription and ribosome recruitment</title>", "<p>Three-dimensional collagen gels have been used to simulate tissue repair ##REF##9457911##[27]##. Because type I collagen is the most abundant matrix component in the lung interstitium, collagen gels are a useful <italic>in vitro</italic> model to investigate the molecular pathways regulating lung myofibroblast function. We previously showed that normal lung myofibroblasts cultured in contractile collagen gels undergo apoptosis as they contract the matrix, whereas myofibroblasts within non-contractile matrices remain viable ##REF##11986332##[28]##, ##REF##15905178##[29]##, ##REF##15166238##[30]##, ##REF##16963781##[31]##. To ensure we were studying gene expression in viable cells, we sought a time point when differences in the intrinsic myofibroblast phenotype could be assessed independent of cell death; we used stability of ribosome loading onto RNA as the viability metric. Within 2 hours of seeding myofibroblasts into gels, cells attached and spread (observed by phase contrast microscopy–data not shown). After 4 additional hours, polyribosome tracings from IPF and control myofibroblasts in contractile and non-contractile collagen gels showed no global shift in ribosome loading (##FIG##0##Figure 1##); this time point (6 h post-seeding) was therefore selected for genome-wide assessment of transcription and ribosome recruitment to RNA.</p>", "<title>Genome-wide assessment of transcriptional and translational profiles identifies large changes at the translational level in IPF myofibroblasts</title>", "<p>We carried out a genome-wide analysis of transcript abundance and ribosome recruitment in primary lung myofibroblast lines derived from 6 patients with IPF and 6 patient controls. Transcript abundance was quantified using total RNA while assessment of ribosome recruitment was performed using RNA associated with more than 3 ribosomes (isolated using polyribosome RNA preparations as described previously ##REF##16936314##[32]##, ##REF##17638893##[33]##) genome wide using microarrays. To assess whether there were intrinsic differences between IPF and control myofibroblasts, we analyzed transcript abundance and ribosome recruitment in both contractile and non-contractile collagen matrices. We characterized the extent of differential regulation of transcription (i.e. transcript abundance) and translation (i.e. ribosome recruitment) between control and IPF myofibroblasts by monitoring the cumulative number of genes passing a range of significance thresholds using both the Significance Analysis of Microarrays (SAM) algorithm and Student's t-test. We considered the possibility that the variance in the data sets generated from polyribosome RNA and total RNA might differ (so that data derived from polyribosome RNA would contain more technical noise due to increased sample processing). In this scenario SAM offers the best estimate of the magnitude of regulation since the internal data set variance directly influences the significance estimates through the sample permutation strategy built into this approach. This is in contrast to fold changes in which increased data set variance can randomly produce more extreme fold-changes. We used three different data inputs: i) data derived from total RNA, ii) data derived from the translationally active (i.e. polyribosome-associated) RNA pool and iii) translational data that had been corrected for total RNA abundance by taking the ratio of (transcript abundance in the actively translated pool)/(total transcript abundance) cell line for cell line. At each significance level, more genes differed between IPF and control at a translational than at a transcriptional level in both contractile and non-contractile gels (##FIG##1##Figure 2##). It is important to note that the transcriptionally corrected translational regulation also showed more significant regulation compared to the total RNA analysis. This finding indicates that the difference between the translational and the transcriptional regulation cannot be explained by higher data set variance for the total RNA data sets (as the fold differences in the translational estimate have been normalized to the fold differences in the transcriptional estimate and therefore contain the variance from both of these comparisons). This corrected analysis will underestimate the translational regulation as a result of the added variance from both the transcriptional and translational data. We also directly compared the data set variance and fold change distribution in these data sets (##SUPPL##0##Figure S1##). As expected, the polyribosome data sets showed higher variance (presumably due to the multi-step sample preparation) and also more genes with extreme fold-changes. Since differences in data set variance are controlled in the SAM algorithm, we conclude that there is substantial translational deregulation in IPF fibroblasts compared to controls that cannot be explained by transcriptional regulation.</p>", "<p>To identify a set of differentially expressed genes that we considered significant, we used SAM and collected genes with a False Discovery Rate&lt;15% using transcription data and data derived from the translationally active pool. Differences in transcript abundance between IPF and control were modest among the more than 15,000 measured genes. In non-contractile collagen matrices, 23 named, unique genes displayed statistically significant transcriptional differences; and in contractile collagen matrices 41 named, unique genes differed. In contrast, translation differed sharply between IPF and control. In non-contractile gels we identified 1346 named, unique genes showing significantly different ribosome recruitment, and in contractile gels there were 488 genes that differed (See ##SUPPL##2##Tables S1##, ##SUPPL##3##S2##, ##SUPPL##4##S3##, ##SUPPL##5##S4## for full list of transcriptionally and translationally regulated genes in both contractile and non-contractile condition).</p>", "<p>To compare the regulation patterns of genes found to be differentially expressed in any comparison (contractile, non-contractile, total RNA or polyribosomal RNA), we collected all such genes (not only those genes that were named) and compared both the significance levels and the fold changes (##FIG##2##Figure 3##, and ##SUPPL##6##Table S5##). The analysis indicates that there is a large set of genes that are regulated at the translational level whose differential expression cannot be appreciated at the transcriptional level (see both the q-value analysis and the fold-change analysis in ##FIG##2##Figure 3##) in both the contractile and the non-contractile state. In accord with the data presented in ##FIG##1##Figure 2##, this fraction of genes is larger than the fraction for which regulation at the translational and transcriptional level is congruent (i.e. no translational regulation). These data demonstrate that IPF myofibroblasts differ from control primarily at the level of ribosome recruitment and that these differences are apparent in a non-contractile matrix simulating fibrosis, and persist in a contractile collagen matrix that simulates normal healing.</p>", "<title>Validation of differential ribosome recruitment</title>", "<p>While the combined polyribosome-microarray approach has been used and validated in established cell lines ##REF##16936314##[32]##, ##REF##14580340##[34]##, ##REF##17311107##[35]##, ##REF##15314157##[36]##, we wanted to test its validity in primary cells. For validation of polyribosome microarray data it is common to trace the profile of the gene of interest using quantitative real time PCR (qRT-PCR) across the fractions of the polyribosome gradient. Regulation is identified as a shift towards fractions with more or fewer ribosomes depending on the direction of regulation. To do this, we identified two cell lines, one control and one IPF (new cell lines not part of the initial microarray study); that were available at a sub-cultivation number identical to that used in the microarray experiments and in sufficient quantity to generate enough mRNA for qRT-PCR from each fraction (an independent validation with new cell lines). We randomly selected one gene shown by our analysis to manifest increased ribosome recruitment in IPF (PDCD8), one gene with decreased ribosome recruitment in IPF (CFL2) and one that did not differ between IPF and control (FUT10). We performed qRT- PCR across the polyribosome fractions for these three genes in both IPF and control to assess the translational activity and measured total RNA levels to assess transcriptional regulation. For the regulated genes, the expected shift in polyribosome profile was observed (PDCD8 shifted towards higher fractions in IPF (##FIG##3##Figure 4A##); CFL2 shifted towards lower fractions in IPF (##FIG##3##Figure 4B##); and the negative control (FUT10) displayed a similar pattern in IPF and control (##FIG##3##Figure 4C##)). The total RNA level was similar for each gene in IPF compared to control (##FIG##3##Figure 4D##).</p>", "<p>In general, increased ribosome loading is expected to lead to increased steady state levels of the encoded protein. To assess this relationship between ribosome recruitment and protein level, we performed immunoblot analysis of PDC8, CFL2 and FUT10 from the cells used for polyribosome qRT-PCR validation in ##FIG##3##Figure 4A–D##. In accord with the polyribosome microarray data and the polyribosome qRT-PCR validation, immunoblot analysis showed that IPF myofibroblasts had increased PDC8 protein levels, decreased CFL2 levels and similar FUT10 levels compared to control (##FIG##3##Figure 4E##).</p>", "<p>To expand our validation set, we identified 6 additional primary fibroblast lines (3 IPF and 3 control, which were part of the initial microarray study) that matched the sub-cultivation criteria for the original array analysis and assessed steady state protein levels for PDCD8, CFL2 and FUT10 (this requires substantially fewer cells than is needed for polyribosome preparations) (##FIG##3##Figure 4F##). For the cell lines assessed in ##FIG##3##Figure 4F##, there was a mean increase in PDCD8 protein level of 1.5 fold in IPF. When also including the cell lines validated in ##FIG##3##Figure 4E##, ##FIG##5##6## out of 8 cell lines confirmed that the PDCD8 protein is more abundant in IPF. For CLF2, a similar analysis indicated a 2-fold mean difference in protein level in ##FIG##3##Figure 4F## with all 8 cell lines from ##FIG##3##Figure 4E–F## confirming the direction of regulation (higher in control). For FUT10 there was no mean difference (1.1 fold higher in IPF) in ##FIG##3##Figure 4F##.</p>", "<p>To assess the significance of the validation, we used the binomial distribution in which the expected pattern can either be confirmed or not between a pair of IPF and control cells (using data from 4E–F). For PDCD8 and CFL2, 7 out of 8 theoretical IPF and control pairs followed the expected pattern of regulation with 4 comparisons for CLF2 showing higher levels in control and 3 comparisons for PDCD8 showing higher levels in IPF. One pair for PDCD8 showed an opposite pattern of regulation with the control higher than IPF (note that we have used the most disadvantageous construction of pairs to obtain one failed pair). Using the binomial distribution to calculate the probability of finding 7 or more confirmed patterns of regulation out of 8 (assuming a 0.5 probability for success in each trial) , we observed a successful validation with a p-value of 0.035. If we include the additional validation of Keratin 18 (below), we observed a successful validation with a p-value of 0.006 (10 out of 11). Thus our data reflect authentic differences at the ribosome recruitment step of gene expression regulation which corresponds to changes in protein level.</p>", "<title>Collagen matrix state modulates translational activity</title>", "<p>We next examined the direction of change among the translationally regulated genes. Of the 1346 genes with a significant translational shift in non-contractile collagen matrices, surprisingly only 138, or 10%, were relatively more active in IPF myofibroblasts compared with controls. However, of the 488 unique genes showing significant translational differences in contractile collagen matrices, 348, or 71%, exhibited greater ribosome loading in IPF myofibroblasts than in controls (see ##SUPPL##2##Table S1##, ##SUPPL##3##S2##) These data indicate that collagen matrix state modulates translational activity.</p>", "<p>Our findings also indicate that at least one of the cell types must undergo extensive translational regulation in a matrix-dependent manner. To assess which of the cell types gave rise to this effect, we compared control and IPF myofibroblasts on both matrices. When examining translation in non-contractile vs. contractile collagen matrices, we found more than 3-fold unique, named genes differing at the level of ribosome recruitment in IPF myofibroblasts compared to control myofibroblasts (1753 <italic>vs.</italic> 575). Very few significant differences were found when analyzing transcriptional data in the same manner–just 26 genes differed in the IPF myofibroblasts and no genes differed in controls when comparing non-contractile vs. contractile matrices. This analysis shows that IPF cells undergo large scale translational regulation depending on matrix state, while control myofibroblasts show relative stability at this level of gene regulation.</p>", "<title>Systems analysis of myofibroblast function and origin</title>", "<p>Comparisons of genes that differ between IPF and normal myofibroblasts could give important information about IPF biology. Such an assessment could be done at a single gene level or by studying the activity of groups of genes organized into pathways. When comparing the single gene approach and the “gene set” or “module” approach, modular analysis has the advantage of giving more biological information and providing a far more robust statistical environment. We therefore used both approaches to study the biology of IPF.</p>", "<p>The gene ontology consortium##REF##16381878##[37]## has developed a system for classifying biological information that has been extensively used to categorize and analyze microarray data. We used this organizational scheme to examine genes classified as differentially expressed at the translational level (too few genes were identified at the transcriptional level to provide a meaningful analysis). One comparison (more active in controls in contractile matrices) resulted in no significant functions (FDR&lt;15% was used as a significance threshold). Two other comparisons (more active in IPF in contractile matrices and more active in IPF in non-contractile matrices) resulted in several significant functions. These included membrane and vesicle transport functions in the non-contractile state and membrane and metabolism (primarily carbohydrate, protein and glycoprotein synthesis) in the contractile state (##SUPPL##7##Table S6##, ##SUPPL##8##S7##). In the final analysis of genes that were more active in control compared to IPF myofibroblasts in the non-contractile state, we found a striking enrichment of genes involved in cell cycle regulation (##SUPPL##9##Table S8##). These genes included both positive and negative cell cycle regulators, indicating that translational control of cell cycle regulation differs between IPF and control; a result in accord with the current literature ##REF##16272460##[38]##, ##REF##11350829##[39]##, ##REF##15095276##[40]##, ##REF##12819039##[41]##. Thus this functional analysis indicated that depending on matrix state, different functions differentiated IPF from controls at the translational level.</p>", "<p>One possible explanation for the differences observed regarding cell cycle regulation is that we are studying myofibroblast populations or subpopulations that have begun to enter cellular senescence. This concern emerged from our observation that IPF myofibroblasts showed morphological changes characteristic of senescence ##REF##14315085##[42]## at an earlier passage than did controls (data not shown). While our experiments employed cells several passages before any morphological senescence was observed, we wanted to investigate whether differences between IPF and controls in our analysis could be attributable to IPF cells just beginning to enter senescence. We therefore compared our microarray data to a data set describing a meta-signature from senescent cells apparent across many cell types and species ##REF##16420669##[43]##; we found no indication for senescence in our primary cell lines.</p>", "<p>As another explanation of the differences between IPF and control myofibroblasts, we considered the possibility that a small subpopulation of rapidly proliferating cells could dominate the results. To address this issue, we applied a technique commonly used in immunology to track the proliferation of subpopulations of lymphocytes and labeled our myofibroblasts with the stable vital dye, carboxyfluoroscein succinimidyl ester (CFSE) ##REF##10214853##[44]##. Uniform proliferation on a population level can be observed as Gaussian distribution whose mean CFSE signal is decreased by approximately 50% per cell division. Using this procedure, we determined that cell proliferation was uniform in all IPF and control cell lines, with an approximate doubling time of 1 day. There was neither a rapidly nor a slowly proliferating subpopulation in any of the cell lines studied (##SUPPL##1##Figure S2##).</p>", "<p>The conventional systems approach we employed to analyze our gene lists has important limitations that arise from its dependence on finite lists of individual genes that pass an arbitrary significance threshold. Such lists are dominated by well-expressed genes experiencing large changes. These lists do not take into account biologically important genes that may barely-or even fail to-pass a significance or fold-change threshold, but nevertheless are critical components of a physiological process that my be the crux of the pathobiology under study ##REF##17069515##[45]##. It is therefore important to supplement these conventional analyses with methods that are not dependent on finite lists of significant genes.</p>", "<p>We therefore used a modular approach that has proved useful in prior studies of translational control ##REF##17069515##[45]##. All genes were ordered based on relative transcriptional or translational activity in IPF compared to control myofibroblasts, as described in <xref ref-type=\"sec\" rid=\"s2\">Materials and Methods</xref>. We looked for over-representation of genes belonging to selected pathways at the extreme ends of the ordered list, thus assessing increased or decreased activity of the pathway in IPF compared to control myofibroblasts. It should be noted that these pathways are different than the GO-collection studied above except for 4 modules as indicated in ##FIG##4##Figure 5##; and that the significance levels were corrected for multiple testing taking into account all analyses presented in ##FIG##4##Figure 5##. We selected all published pathways available for cell signaling (found on the web at <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.genome.ad.jp/kegg\">http://www.genome.ad.jp/kegg</ext-link>), and detected significant differences in pathway activities at the level of transcript abundance and ribosome recruitment (##FIG##4##Figure 5##). Some pathways, such as transforming growth factor (TGF) and actin regulation, have established roles in fibrotic processes. Others, such as wingless (Wnt) or calcium signaling, are less commonly associated with fibrosis but may lead to novel insights into the disease process. We also observed regulation of apoptosis and proliferation at both the transcriptional and translational level. The observed differential regulation of proliferation motivated us to further examine cell cycle regulation using data from additional sources and we collected several categories from GO that describe proliferation. However, only the “cell cycle arrest” pathway was more active in IPF compared to normal myofibroblasts. Thus by using curated pathways and genome-wide data, we find support for a pathological myofibroblast phenotype in IPF that persists <italic>in vitro</italic> up to 9 sub-cultivations. It is noteworthy that this phenotype is manifest at both levels of gene regulation examined as well as in several biological pathways.</p>", "<p>One critical question amenable to a systems level approach is the origin of the IPF myofibroblast. The epithelial to mesenchymal transition (EMT) is a key process during embryonic development that has been implicated as a source of pathological myofibroblasts in renal fibrosis and IPF ##REF##15855634##[46]##, ##REF##16924102##[47]##, ##REF##17562716##[48]##. To examine whether there was genome-wide indication for EMT at the level of transcription and/or translation, we manually constructed a module comprised of 111 genes that had at least one published study documenting its participation in EMT, and built 4 sub-modules to represent the 3 major cell surface receptor-mediated pathways triggering EMT (Wnt, TGF-β and integrin-matrix) and a module comprised of EMT-related transcription factors (module gene lists provided in ##SUPPL##10##Table S9##; these modules were not assessed in the “significant gene list analysis” presented above). The global EMT module was not active in IPF compared to control (##FIG##4##Figure 5##). When studying the subgroups the TGF-β -EMT module, the integrin-matrix-EMT module and the transcription factor module distinguished IPF from control–primarily at the level of transcript abundance. The analysis shown in ##FIG##4##Figure 5## was corrected for multiple testing, and thus the corrected significance level (q&lt;0.05) considers all tests performed in this part of our study. As a result, p&lt;0.001 was necessary to reach a corrected significance level of q&lt;0.05. These data support the contention that EMT is involved in the genesis of some IPF myofibroblasts, and show the power of this systems level analysis.</p>", "<p>To test the biological validity of this systems level analysis implicating EMT in the origin of IPF myofibroblasts, we selected the epithelial intermediate filament component, Keratin 18, for further analysis. In our genome-wide analysis of ribosome recruitment, we found that this epithelial gene was dramatically translationally activated in IPF myofibroblasts compared to controls. To verify this we used the same samples and approach as in ##FIG##3##Figure 4A–C## to assess the level of Keratin 18 mRNA across the polyribosome fractions. ##FIG##5##Figure 6A## shows that Keratin 18 was indeed more translationally active in IPF compared to control. Keratin 18 was also marginally increased at the total RNA level (&lt;1.5 fold, not shown). Nonetheless, to rule out transcriptional regulation as a primary source of the translational difference, we corrected the translational profiling data for total RNA levels. This did not influence the shift towards translational activation in IPF (not shown). To assess Keratin 18 expression in a panel of cell lines, we used the same samples as in ##FIG##3##Figure 4F## (i.e. independent samples from 3 IPF and 3 control fibroblast lines which were part of the microarray study) and measured protein abundance by immunoblot (mammary epithelial cells served as a positive control, ##FIG##5##Figure 6B##). In accord with the genome-wide study and the polyribosome qRT-PCR validation, Keratin 18 displayed an increase in protein abundance (##FIG##5##Figure 6B##) in IPF myofibroblasts compared to controls. These data indicate that our approach is biologically valid, and lends further support for the idea that EMT is involved in the origin of IPF fibroblasts.</p>" ]
[ "<title>Discussion</title>", "<p>Myofibroblasts from fibrotic lesions manifest pathological control of proliferation, viability, motility, contractile function and connective tissue production. In a prior report, we provided the first insights into molecular mechanism, showing that aberrant beta 1 integrin signaling results in defective PTEN function, unrestrained Akt signaling and downstream activation of the translation initiation machinery ##REF##18541712##[16]##. Here, we provide the first genome-wide analysis of the consequences of this aberrant signaling. We find that two steps in the flow of genetic information–transcription and ribosome recruitment-are altered in IPF myofibroblasts, and that changes in ribosome recruitment account for the majority of differences between IPF and control myofibroblasts. When comparing gene expression in contractile and non-contractile matrices, IPF myofibroblast gene expression showed large-scale translational changes depending on matrix state, whereas the pattern in control myofibroblasts was relatively stable. In addition, our analysis provides systems level evidence for EMT as a source of some IPF myofibroblasts, providing strong support for the pathological study suggesting an epithelial origin for some IPF myofibroblasts ##REF##16924102##[47]##. Our data do not exclude the possibility that other IPF myofibroblasts may originate from cytokine altered resident fibroblasts or from circulating fibrocytes. These findings do, however, establish the power of systems level genome-wide analysis to provide mechanistic insights into IPF, and point to derangements of translational control downstream of defective integrin signaling as a fundamental component of IPF pathobiology.</p>", "<p>The clinical outcome of IPF is the result of complex interactions among myofibroblasts, epithelial cells, cytokines, and the surrounding extracellular matrix. Significant debate continues as to what degree, or even whether, an abnormality in any individual component contributes to the overall disease process. Part of this uncertainty relates to conflicting results found in studies of IPF myofibroblasts. As an example, investigators comparing proliferation and apoptosis between IPF and control myofibroblasts have reached contradictory conclusions##REF##16272460##[38]##, ##REF##11350829##[39]##, ##REF##15095276##[40]##, ##REF##12819039##[41]##. While many aspects of the debate are not resolved by the results of this study, there is no doubt that these key processes are fundamentally different in IPF and control myofibroblasts.</p>", "<p>Our study demonstrates this fundamental difference in a comprehensive microarray analysis of two levels in the gene expression pathway–transcription and ribosome recruitment. A few previous studies have been performed using microarray technology to compare transcriptional profiles of IPF and control myofibroblasts ##REF##15191918##[49]##, ##REF##15571627##[50]##. Microarray analysis has also been done using whole lung samples from patients with IPF, hypersensitivity pneumonitis (HP), nonspecific interstitial pneumonitis (NSIP) and controls ##REF##16128620##[17]##, ##REF##15117744##[51]##, ##REF##16166619##[52]##. However, all of these investigations focus on differences in mRNA abundance, which correlates poorly with protein levels ##REF##10022859##[53]##, ##REF##9150937##[54]##. Analysis of translation, the next step in the processing of genetic information, reveals more prominent and informative differences between cells, and correlates more closely with protein levels ##REF##16936314##[32]##, ##REF##14580340##[34]##. Consistent with these data, our study demonstrates markedly more genes differing between IPF and control myofibroblasts at the level of ribosome recruitment compared with mRNA abundance. Our study is also the first to perform this analysis using primary cells rather than immortal, established cell lines.</p>", "<p>Analysis of the stability of the transcriptional and translational profiles between IPF and control as a function of matrix state revealed major differences. Control myofibroblasts demonstrated no significant differences in transcript abundance and relatively few differences in ribosome recruitment on non-contractile compared to contractile matrices. In contrast, IPF myofibroblasts displayed extensive changes in transcription and ribosome recruitment as matrix state changed. This analysis illustrates a difference in phenotype between IPF and control myofibroblasts, suggesting that in IPF there is pathologic relaxation of the gene expression control system found in normal cells. Furthermore, these data fit with the idea that IPF myofibroblast pathobiology includes a loss of translational control, analogous to the loss of tumor suppressor function seen in cancer. Experimental precedent for this concept has been provided by studies showing that IPF myofibroblasts have acquired at least one cancer-related property–the ability to grow in an anchorage-independent manner ##REF##8163656##[55]##.</p>", "<p>Given the growing body of experimental work indicating that fibrotic myofibroblasts have a distinct phenotype, one topic that has recently garnered much interest is the source of myofibroblasts in IPF. Published morphological data suggest that myofibroblasts in IPF have an epithelial origin ##REF##16813649##[56]##; and there is direct experimental data implicating EMT as a source of myofibroblasts in a mouse model of lung fibrosis ##REF##16924102##[47]##. Using a systems approach to analyze genome-wide data, we establish that myofibroblasts cultured from the lungs of patients with IPF have an EMT signature. At least 3 pathways can trigger EMT. Here we group genes associated with each of the 3 EMT pathways, and find that two of the pathways (matrix and TGF) are active in IPF myofibroblasts–providing the first systems-level indication regarding mechanism. In accord with this result, recent data indicates that myofibroblast contraction of its extracellular matrix can trigger release of TGF- β from its latent form in the matrix ##REF##18086923##[57]##.</p>", "<p>In this report, we demonstrate a difference in transcript abundance and ribosome recruitment for a number of genes when comparing myofibroblasts from patients with IPF to controls. We chose a systems biology approach rather than a “reductionist” or “cherry picking” method to analyze our data ##REF##17069515##[45]##, ##REF##16738198##[58]##. We show instability in the translational profile of IPF myofibroblasts when they are placed in different matrix environments, and we present genome-wide data that provide indications for EMT as a source of myofibroblasts in IPF. We anticipate these data, along with more intensive investigations of primary cell lines, will yield important information and significantly impact the search for new molecular targets for therapeutics.</p>", "<title>Accession number</title>", "<p>The data set has been deposited at Gene Expression Omnibus (GEO, GSE11196).</p>" ]
[]
[ "<p>Conceived and designed the experiments: OL DD RSN PBB CAH. Performed the experiments: OL DD MP. Analyzed the data: OL DD DF MP. Wrote the paper: OL DD PBB CAH.</p>", "<p>¶ These authors also contributed equally to this work.</p>", "<title>Background</title>", "<p>As a group, fibroproliferative disorders of the lung, liver, kidney, heart, vasculature and integument are common, progressive and refractory to therapy. They can emerge following toxic insults, but are frequently idiopathic. Their enigmatic propensity to resist therapy and progress to organ failure has focused attention on the myofibroblast–the primary effector of the fibroproliferative response. We have recently shown that aberrant beta 1 integrin signaling in fibrotic fibroblasts results in defective PTEN function, unrestrained Akt signaling and subsequent activation of the translation initiation machinery. How this pathological integrin signaling alters the gene expression pathway has not been elucidated.</p>", "<title>Results</title>", "<p>Using a systems approach to study this question in a prototype fibrotic disease, Idiopathic Pulmonary Fibrosis (IPF); here we show organized changes in the gene expression pathway of primary lung myofibroblasts that persist for up to 9 sub-cultivations in vitro. When comparing IPF and control myofibroblasts in a 3-dimensional type I collagen matrix, more genes differed at the level of ribosome recruitment than at the level of transcript abundance, indicating pathological translational control as a major characteristic of IPF myofibroblasts. To determine the effect of matrix state on translational control, myofibroblasts were permitted to contract the matrix. Ribosome recruitment in control myofibroblasts was relatively stable. In contrast, IPF cells manifested large alterations in the ribosome recruitment pattern. Pathological studies suggest an epithelial origin for IPF myofibroblasts through the epithelial to mesenchymal transition (EMT). In accord with this, we found systems-level indications for TGF-β -driven EMT as one source of IPF myofibroblasts.</p>", "<title>Conclusions</title>", "<p>These findings establish the power of systems level genome-wide analysis to provide mechanistic insights into fibrotic disorders such as IPF. Our data point to derangements of translational control downstream of aberrant beta 1 integrin signaling as a fundamental component of IPF pathobiology and indicates that TGF-β -driven EMT is one source for IPF myofibroblasts.</p>" ]
[ "<title>Supporting Information</title>" ]
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[ "<fig id=\"pone-0003220-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003220.g001</object-id><label>Figure 1</label><caption><title>Representative polyribosome tracings from control and IPF myofibroblasts in non-contractile collagen matrices.</title><p>Shown is OD 254 as a function of position in the sucrose gradient. The fractions pooled to yield the heavy polyribosomes and total RNA are designated</p></caption></fig>", "<fig id=\"pone-0003220-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003220.g002</object-id><label>Figure 2</label><caption><title>Number of genes passing significance thresholds.</title><p>Shown are t-test and SAM statistics of the cumulative number of genes passing significance thresholds for transcription (red line), translation (green line), and the transcriptionally corrected translational activity (blue line). ##FIG##1##Figure 2A## represents data from the non-contractile condition, and 2B shows data from the contractile collagen matrices.</p></caption></fig>", "<fig id=\"pone-0003220-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003220.g003</object-id><label>Figure 3</label><caption><title>A comparison of translational and transcriptional regulation.</title><p>All genes that were classified as differentially expressed (translational or transcriptional level) between IPF and control were collected. The differential regulation at the transcriptional (“Total”) or translational (“Heavy”) were compared in the non-contractile (A–C) and contractile (D–F) state. Significance level (log2 q-value (%)) (A, D) and fold changes (log2) (B–C, E–F) were used for comparison. The lines in the comparison of significances (A, D) indicate a q-value of 15% (3.9 on the log2 scale). The lines in the comparisons of fold changes indicate a 1.5 fold change (B, E) and a 2 fold change (C, F). The number of genes what fall within each sector is indicated. Higher fold changes and lower significances indicate differential regulation.</p></caption></fig>", "<fig id=\"pone-0003220-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003220.g004</object-id><label>Figure 4</label><caption><title>Validation of genome-wide data.</title><p>Three genes were selected: Programmed Cell Death 8 (PDCD8) which demonstrated more ribosome loading in IPF; Cofilin 2 (CFL2) which showed more ribosome loading in controls; and Fucosyltransferase 10 (FUT10) which displayed no change in ribosome loading between the two cell types. A–C. Polyribosome shift using qRT-PCR. Shown is the quantity of mRNA normalized to a spike in standard, as a function of position in the sucrose gradient. D. Total RNA levels. The total RNA was normalized to a “spike in” standard and actin. E–F. Steady state protein levels of PDCD8, CFL2 and FUT10. Cells (passage 5) were seeded on type I collagen matrices and examined for protein expression using actin as a loading control E. Western blotting from the same cell lines as in (A–D). F. Steady state levels of 6 additional primary myofibroblast lines (3 IPF; and 3 control).</p></caption></fig>", "<fig id=\"pone-0003220-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003220.g005</object-id><label>Figure 5</label><caption><title>Systems analysis of pathway activities and myofibroblast origin.</title><p>Pathways are shown in rows and conditions are shown in columns. A red box denotes that the pathway is activated in IPF compared with control myofibroblasts; green boxes represent pathways that are activated in controls compared to IPF myofibroblasts. Blue boxes signify a pathway that is activated both in IPF compared to controls and controls compared to IPF, and gray boxes represent pathways with no significant activity. This analysis was performed using cells in non-contractile matrices setting the threshold at a FDR&lt;0.05, (“TF” = transcription factors). Pathways ending with “.go” were obtained from GO. EMT pathways were manually constructed while all other pathways were obtained from KEGG.</p></caption></fig>", "<fig id=\"pone-0003220-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003220.g006</object-id><label>Figure 6</label><caption><title>Keratin 18 is translationally activated in IPF myofibroblasts.</title><p>A. Polyribosome shift using qRT-PCR. Shown is the quantity of mRNA normalized to a “spike in” standard, as a function of position in the sucrose gradient. B. Steady state protein level. Cells (passage 5) were seeded on type I collagen matrices and examined for protein expression using actin as a loading control.</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s001\"><label>Figure S1</label><caption><p>(0.96 MB EPS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s002\"><label>Figure S2</label><caption><p>(0.94 MB EPS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s003\"><label>Table S1</label><caption><p>(0.21 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s004\"><label>Table S2</label><caption><p>(0.45 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s005\"><label>Table S3</label><caption><p>(0.05 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s006\"><label>Table S4</label><caption><p>(0.05 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s007\"><label>Table S5</label><caption><p>(0.52 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s008\"><label>Table S6</label><caption><p>(0.03 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s009\"><label>Table S7</label><caption><p>(0.04 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s010\"><label>Table S8</label><caption><p>(0.05 MB XLS)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003220.s011\"><label>Table S9</label><caption><p>(0.05 MB XLS)</p></caption></supplementary-material>" ]
[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>These studies were supported by ALA grant RT-10053N (to D. Diebold), HL073719, HL076779 (P.B.), and HL 074882 (C.H.). O.L. was supported by a post-doctoral fellowship from the Swedish Research Council during the majority of the study and a fellowship from the Knut and Alice Wallenberg foundation during the completion of the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></fn-group>" ]
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[{"label": ["1"], "element-citation": ["\n"], "surname": ["Bitterman", "Henke"], "given-names": ["PB", "CA"], "year": ["1991"], "article-title": ["Fibroproliferative disorders"], "fpage": ["81S"], "lpage": ["84S"]}, {"label": ["2"], "element-citation": ["\n"], "surname": ["Cotran", "Kumar", "Collins", "Collins"], "given-names": ["R", "V", "T", "T"], "year": ["1999"], "publisher-loc": ["Philadelphia"], "publisher-name": ["W.B. Saunders Company"], "fpage": ["89"], "lpage": ["112"]}, {"label": ["3"], "element-citation": ["\n"], "surname": ["Wynn"], "given-names": ["TA"], "year": ["2004"], "article-title": ["Fibrotic disease and the T(H)1/T(H)2 paradigm"], "fpage": ["583"], "lpage": ["594"]}, {"label": ["6"], "element-citation": ["\n"], "surname": ["Schuttert", "Liu", "Gliem", "Fiedler", "Zopf"], "given-names": ["JB", "MH", "N", "GM", "S"], "year": ["2003"], "article-title": ["Human renal fibroblasts derived from normal and fibrotic kidneys show differences in increase of extracellular matrix synthesis and cell proliferation upon angiotensin II exposure."], "source": ["Pflugers Arch"], "fpage": ["387"], "lpage": ["393"]}]
{ "acronym": [], "definition": [] }
58
CC BY
no
2022-01-13 07:14:34
PLoS One. 2008 Sep 16; 3(9):e3220
oa_package/6c/37/PMC2528966.tar.gz
PMC2529164
18466612
[ "<title>Background</title>", "<p>Recent experiments on gene and enzyme activity at single cell resolution have revealed the inherent randomness of key cellular processes linked to gene expression [##REF##12183631##1##, ####REF##15166317##2##, ##REF##17048983##3####17048983##3##]. The experiments show that populations with identical genotypes present heterogeneous phenotypes and that noise at the molecular level, due to low copy numbers, contributes to population diversity. For mathematical models to capture this variability, a stochastic description is required.</p>", "<p>Stochastic models in Computational Biology are usually based on the Master Equation (ME) of the chemical reaction kinetics [##UREF##0##4##, ####REF##9023339##5##, ##REF##14749823##6####14749823##6##]. Formally, the ME is a differential form of the Chapman-Kolmogorov equation, which gives the time evolution of <italic>P</italic>(<bold>x</bold>, <italic>t</italic>), the probability of the state of the system <bold>x</bold>. Only a handful of analytical solutions of the ME have been found and one must usually resort to approximations or numerical solutions. The most popular numerical procedure is Gillespie's Stochastic Simulation Algorithm (SSA) [##UREF##1##7##,##UREF##2##8##], a kinetic Monte Carlo algorithm that provides exact stochastic realizations of the underlying system of reactions. Each run of the SSA produces a time trace for the system; a collection of independent runs can be used to obtain convergent statistics of the time-dependent solution of the ME. In many situations, one is interested in the steady state properties of the system, i.e., in the stationary distribution of the ME, <italic>π</italic>. Although in principle <italic>π </italic>could be obtained as the first left eigenvector of the transition matrix, this computation is infeasible for most problems of interest due to the combinatorial explosion of the state space [##UREF##3##9##]. To circumvent this problem, it has become customary to sample <italic>π </italic>by running the SSA for a 'very long time', convincing oneself through different heuristics that stationarity has been attained. However, the SSA does not provide guarantees or information about how long the algorithm must run to converge to <italic>π</italic>. In recent years there has been an increased interest in finding algorithms which can address the issue of sampling from stationarity, e.g., a strategy based on forward flux sampling [##REF##17887830##10##].</p>", "<p>In a seminal paper in the field of Markov Chain Monte Carlo, Propp and Wilson introduced the idea of <italic>Coupling From The Past </italic>(CFTP), an ingenious procedure that provides <italic>guaranteed </italic>sampling from the stationary distribution of a Markov chain by running coupled chains from all possible initial conditions from the past [##UREF##4##11##]. Algorithms that guarantee sampling from the stationary distribution of a Markov chain are referred to as <italic>Perfect Sampling </italic>algorithms [##UREF##4##11##, ####UREF##5##12##, ##UREF##6##13##, ##UREF##7##14####7##14##]. Recently [##REF##17468171##15##], we introduced a Perfect Sampling algorithm for the SSA of biochemical networks based on Kendall's Dominated CFTP (DCFTP) [##UREF##6##13##]. This paper expands on our previous work by providing an explicit implementation of the algorithm together with a mathematical proof of its applicability to a class of reactions prevalent in models of gene regulation. We also study its numerical properties through a series of expanded examples drawn from Systems and Synthetic Biology.</p>" ]
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[ "<title>Discussion</title>", "<p>The present work presents a detailed implementation of the DCFTP-SSA that could be integrated with other packages in Computational Systems Biology. We have also provided a mathematical proof of the algorithm with an explicit statement of the limits of its applicability. This detailed description is key to the extension of the algorithm to a wider class of systems. Specifically, the DCFTP-SSA can be applied to conversion reactions of the type <italic>A </italic>→ <italic>B </italic>with the realistic assumption that the monotone propensity function only depends on <italic>n</italic><sub><italic>A</italic></sub>. Unfortunately, the extension to encompass bimolecular reactions of the type <italic>A </italic>+ <italic>B </italic>→ <italic>C </italic>does not seem to be trivial, since the partial ordering used in this paper will not be preserved and there is no dominating process with known stationary distribution readily available. The latter problem can be addressed partially by using the CFTP under the approximation that there is an upper bound on the number of molecules in the state space. If the bound is chosen to be large enough, it can be shown numerically that the error will be negligible. However, this approximate method will not carry the guarantees of stationarity that the DCFTP-SSA provides.</p>", "<p>From the numerical viewpoint, the DCFTP-SSA is guaranteed to converge almost surely in finite time, but there is no upper bound on the coalescence times <italic>T</italic><sub><italic>c</italic></sub>. Our numerics show that the distribution of coalescence times can be long-tailed when the structure of the stationary distribution is complex (Fig. ##FIG##2##3b##).</p>", "<p>If a simulation is interrupted prematurely by an impatient user, the final sample will be biased. An alternative perfect sampling scheme is the FMMR algorithm [##UREF##7##14##], which uses rejection sampling to circumvent this problem. Our experience has shown that typically a small fraction of runs takes a very long time to converge. Being able to remove these would speed up the algorithm significantly. The bimodal example illustrates this point: if we were able to place a cut-off after the first mode, a large portion simulations would be accepted and at the same time there would be a significant save in terms of both CPU time and memory. As indicated by the examples in this paper, it is important to note that the DCFTP-SSA does not present obvious problems with scalability, as the overhead incurred to provide a certificate of stationarity is moderate. Although the computational cost of the algorithm depends on the intrinsic structure of the network, we have applied the DCFTP-SSA to various networks with several tens of variables.</p>", "<p>In addition to producing guaranteed sampling from the stationary distribution, the DCFTP-SSA can be used to provide initial conditions for ordinary SSA runs. Since any Markov process started from stationarity will remain there for all future times, these runs are guaranteed to represent the stationary time-traces of the system. This is important for the numerical characterization of properties such as escape times and autocorrelation times of systems with high variability, e.g., with underlying multi-stable, oscillatory or excitatory behaviour [##REF##17468171##15##].</p>" ]
[ "<title>Conclusion</title>", "<p>The SSA is an exact procedure to sample the time-dependent probability distribution of the ME of general chemical reaction networks at all times [##UREF##1##7##,##UREF##2##8##]. However, it provides no guarantees when the aim is sampling from the stationary distribution. The DCFTP-SSA presented here addresses this problem for a class of networks of relevance to genetic and enzymatic regulation. Our algorithm provides guaranteed stationary sampling and thus removes one of the sources of uncertainty in stochastic simulations. This can aid in the characterization of regulatory circuits and in the testing of model hypotheses for these systems.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>In recent years, stochastic descriptions of biochemical reactions based on the Master Equation (ME) have become widespread. These are especially relevant for models involving gene regulation. Gillespie’s Stochastic Simulation Algorithm (SSA) is the most widely used method for the numerical evaluation of these models. The SSA produces exact samples from the distribution of the ME for finite times. However, if the stationary distribution is of interest, the SSA provides no information about convergence or how long the algorithm needs to be run to sample from the stationary distribution with given accuracy. </p>", "<title>Results</title>", "<p>We present a proof and numerical characterization of a Perfect Sampling algorithm for the ME of networks of biochemical reactions prevalent in gene regulation and enzymatic catalysis. Our algorithm combines the SSA with Dominated Coupling From The Past (DCFTP) techniques to provide guaranteed sampling from the stationary distribution. The resulting DCFTP-SSA is applicable to networks of reactions with uni-molecular stoichiometries and sub-linear, (anti-) monotone propensity functions. We showcase its applicability studying steady-state properties of stochastic regulatory networks of relevance in synthetic and systems biology.</p>", "<title>Conclusion</title>", "<p>The DCFTP-SSA provides an extension to Gillespie’s SSA with guaranteed sampling from the stationary solution of the ME for a broad class of stochastic biochemical networks.</p>" ]
[ "<title>Preliminaries and definitions</title>", "<title>Dominated Coupling From The Past (DCFTP)</title>", "<p>We give here a brief introduction to the CFTP framework (see [##UREF##4##11##, ####UREF##5##12##, ##UREF##6##13####6##13##] for full proofs).</p>", "<p>The central idea behind CFTP is to find a time in the past such that the whole state space is mapped to the same state at the present, for a given sequence of random numbers. When that occurs, the state at the present can be considered to be a sample of the stationary distribution. More formally, consider a Markov process defined by the transition rule <bold>X</bold><sub><italic>t</italic>+1 </sub>= <italic>ϕ</italic>(<bold>X</bold><sub><italic>t</italic></sub>), where <bold>X</bold><sub><italic>t </italic></sub>≡ <bold>x</bold>(<italic>t</italic>) is shorthand for the state of the system at time <italic>t</italic>. Any Markov chain , started from <italic>t </italic>= -∞ will have reached stationarity at time <italic>t </italic>= -<italic>T</italic>. If a chain with an unknown value <bold>X</bold><sub>-<italic>T </italic></sub>is continued to run until <italic>t </italic>= 0, it will attain a value <bold>X</bold><sub>0 </sub>= <italic>ϕ</italic><sup><italic>T </italic></sup>(<bold>X</bold><sub>-<italic>T</italic></sub>), which also comes from the stationary distribution. The CFTP algorithm searches for a time -<italic>T </italic>such that the composite function <italic>ϕ</italic><sup><italic>T </italic></sup>(<bold>X</bold><sub>-<italic>T</italic></sub>) has a unique image <italic>for all </italic>arguments <bold>X</bold><sub>-<italic>T</italic></sub>. This implies that the chain started at -<italic>T </italic>is equivalent to a chain started from <italic>t </italic>= -∞, since it will reach the same state <bold>X</bold><sub>0 </sub>regardless of its value at <italic>t </italic>= -<italic>T</italic>. Hence the sample <bold>X</bold><sub>0 </sub>comes from the stationary distribution. Starting from the past and running into the present might seem counterintuitive and unnecessarily complicated. However, it is key for the algorithm to work and it can be shown that starting at <italic>t </italic>= 0 and coupling into the future will not guarantee that the samples are unbiased.</p>", "<p>For large state spaces it is infeasible to monitor all initial conditions at time -<italic>T</italic>. However, this can be done efficiently if one can find a partial ordering over the state space that is preserved by the transition rule [##UREF##5##12##]:</p>", "<p></p>", "<p>where ≽ denotes the partial order, i.e., a binary relation which is reflexive, anti-symmetric and transitive, although it does not necessarily satisfy total comparability. Under these conditions, the whole state space can be monitored by checking for the coalescence of coupled Markov chains started at the upper and lower extremes of the state space [##UREF##4##11##,##UREF##8##16##].</p>", "<p>Two Markov chains are said to be coupled if they use the same sequence of random numbers and the same transition rule but are started from different initial conditions. Coupled chains that meet at a time <italic>T</italic><sub><italic>c </italic></sub>are said to coalesce and will have identical states for <italic>t </italic>&gt; <italic>T</italic><sub><italic>c</italic></sub>. A necessary (but not sufficient) condition for the preservation of the partial ordering is that the transition function is either monotone or anti-monotone:</p>", "<p></p>", "<p></p>", "<p>for coupled chains <bold>X </bold>and <bold>Y</bold>. If the partial order is preserved, we can monitor only the paths started at the 'extremes' of the state space, since all the paths in between remain bounded by them. We therefore define <italic>upper </italic>and a <italic>lower </italic>coupled Markov chains that enclose all other paths:</p>", "<p></p>", "<p>where , ∀<bold>x</bold>.</p>", "<p>The preservation of the partial order implies two important properties for coupled chains:</p>", "<p><italic>Sandwiching</italic>: all paths started between <bold>L </bold>and <bold>U </bold>will have coalesced by the time <bold>L </bold>and <bold>U </bold>do,</p>", "<p></p>", "<p><italic>Funneling</italic>: all paths will get closer if they are started further back into the past,</p>", "<p></p>", "<p>If the state space is unbounded from above, we need to use Kendall's DCFTP construction. DCFTP works by introducing a time-evolving dominating process <bold>D </bold>with known stationary distribution, which provides a random upper bound to the state space. The original process <bold>X </bold>can then be generated as an adapted functional of the dominating process and a <italic>mark process </italic><bold>M</bold>:</p>", "<p></p>", "<p>The mark process generates a uniform random number each time <bold>D </bold>is changed. These marks are used to update the original process <bold>X </bold>according to the adapted functional (3) in a process that is equivalent to the direct simulation of <bold>X </bold>[##UREF##5##12##]. Heuristically, the DCFTP scheme works as follows. Since the dominating process is started from the stationary distribution at <italic>t </italic>= -<italic>T</italic>, is equivalent to a process started from <italic>t </italic>= -∞. By the funneling property, all chains from the original process started from <italic>t </italic>= -∞ will be beneath the dominating process: . If we set <bold>U</bold><sub>-<italic>T </italic></sub>= <bold>D</bold><sub>-<italic>T </italic></sub>and <bold>L</bold><sub>-<italic>T </italic></sub>= and check that these two extreme paths coalesce, then all chains started from <italic>t </italic>= -<italic>T </italic>map to the same state at <italic>t </italic>= 0, due to the sandwiching property. It then follows that is equivalent to and the sample comes from the stationary distribution of <bold>X</bold>, due to the equivalence of the adapted functional and the original process. Note that if <bold>D </bold>can be chosen to be a constant process equal to the maximal element of the state space, we obtain the CFTP algorithm [##UREF##6##13##].</p>", "<p>These results are summarized in the following theorem for general DCFTP algorithms [##UREF##5##12##,##UREF##6##13##]:</p>", "<p><bold>Theorem 1 (DCFTP) </bold><italic>Consider a stationary dominating process </italic><bold>D</bold>, <italic>for which </italic><italic>is an ergodic atom, and an associated random mark process </italic><bold>M</bold>. <italic>Suppose that the processes </italic><italic>are produced from </italic><bold>D </bold><italic>and </italic><bold>M </bold><italic>according to the adapted functional (3) so that the sandwiching and funneling properties (1)–(2) are fulfilled. Suppose further that </italic><bold>X </bold><italic>converges weakly to an invariant distribution π as t </italic>→ ∞. <italic>Then </italic><bold>L </bold><italic>and </italic><bold>U </bold><italic>will coalesce almost surely in finite time and, if coalescence is achieved</italic>, <bold>L</bold><sub>0 </sub>= <bold>U</bold><sub>0 </sub><italic>is a sample from the stationary distribution π</italic>.</p>", "<p><bold>Proof </bold>See [##UREF##6##13##].</p>", "<title>Stochastic Simulation Algorithm (SSA)</title>", "<p>This section presents briefly the classic Gillespie algorithm (SSA) for the exact simulation of the Master Equation of chemical reaction networks [##UREF##1##7##].</p>", "<p><bold>Definition 2 (Chemical reaction network) </bold><italic>A system of chemical reactions </italic><italic>is fully specified by the tuple </italic>, <italic>where </italic> = {<italic>S</italic><sub>1</sub>,...,<italic>S</italic><sub><italic>m</italic></sub>} <italic>is a set of m different molecular species interacting through r reaction channels </italic> = {<italic>R</italic><sub>1</sub>,...,<italic>R</italic><sub><italic>r</italic></sub>}. <italic>Each reaction R</italic><sub><italic>i </italic></sub><italic>is described by a stoichiometry vector ν</italic><sub><italic>i</italic></sub>, <italic>which gives the change in the number of molecules of all species when reaction R</italic><sub><italic>i </italic></sub><italic>occurs, and a propensity function </italic>Φ<sub><italic>i</italic></sub>(<bold>x</bold>), <italic>which gives the state-dependent probability that reaction R</italic><sub><italic>i </italic></sub><italic>occurs. The state of the system is given by </italic><bold>X</bold><sub><italic>t </italic></sub>≡ <bold>x</bold>(<italic>t</italic>) = (<italic>x</italic><sub>1</sub>(<italic>t</italic>),...,<italic>x</italic><sub><italic>m</italic></sub>(<italic>t</italic>)) ∈ ℕ<sup><italic>m</italic></sup>, <italic>where each component x</italic><sub><italic>i</italic></sub>(<italic>t</italic>) <italic>indicates the number of molecules of S</italic><sub><italic>i </italic></sub><italic>at time t</italic>.</p>", "<p>Under the assumption that the molecules are confined to a well-stirred volume and held at constant temperature, we can formulate a ME governing the evolution of the system [##UREF##1##7##]:</p>", "<p></p>", "<p>The ME is a conservation equation for the probability distribution and the right hand side accounts for the rate of change of the probability of finding the system in state <bold>x</bold>.</p>", "<p>A general procedure to obtain exact realizations of Markov processes first suggested by Doob [##UREF##9##17##] was applied to chemical reactions by Gillespie in his celebrated Stochastic Simulation Algorithm [##UREF##1##7##]:</p>", "<p><bold>Algorithm 3 (SSA) </bold><italic>Given a chemical reaction network </italic>, <italic>as in Definition 2, with initial state </italic><italic>and stopping time T</italic><sub><italic>s</italic></sub>:</p>", "<p>   <italic>k </italic>← 0</p>", "<p>   <bold><italic>loop</italic></bold></p>", "<p>      <italic>k </italic>← <italic>k </italic>+ 1</p>", "<p>      <italic>V</italic><sub><italic>k</italic></sub>, ~ <italic>U</italic>(0, 1)</p>", "<p>      <bold><italic>for </italic></bold><italic>i </italic>= 1 <italic>to r </italic><bold><italic>do</italic></bold></p>", "<p>         </p>", "<p>      <bold><italic>end for</italic></bold></p>", "<p>      <italic>t</italic><sub><italic>k </italic></sub>← <italic>t</italic><sub><italic>k</italic>-1 </sub>- (1/<italic>θ</italic><sub><italic>r</italic></sub>) log <italic>V</italic><sub><italic>k</italic></sub></p>", "<p>      <bold><italic>if </italic></bold><italic>t</italic><sub><italic>k </italic></sub>&gt; <italic>T</italic><sub><italic>s </italic></sub><bold><italic>then</italic></bold></p>", "<p>         <bold><italic>return </italic></bold></p>", "<p>      <bold><italic>else</italic></bold></p>", "<p>         </p>", "<p>      <bold><italic>end if</italic></bold></p>", "<p>   <bold><italic>end loop</italic></bold></p>", "<p>A run of the SSA uses the uniform random numbers <italic>V</italic>, <italic>V' </italic>to generate a random sequence of reactions <bold>ℜ </bold>= , taking place at the random transition times {<italic>t</italic><sub>1</sub>,...,<italic>t</italic><sub><italic>n</italic></sub>} such that <italic>t</italic><sub><italic>n </italic></sub>&lt;<italic>T</italic><sub><italic>s </italic></sub>&lt;<italic>t</italic><sub><italic>n</italic>+1</sub>. The path is an exact stochastic realization of Eq. (4). Note that the sequence of reactions <bold>ℜ </bold>uniquely determines . For convenience, we have committed a slight of abuse of notation when using real valued indices to denote the state and reaction taking place at time <italic>t</italic><sub><italic>k</italic></sub>.</p>", "<p>Henceforth, we represent compactly the SSA Markov process implemented by Algorithm 3 as:</p>", "<p></p>", "<p>For an arbitrary initial state , repeated runs of the SSA will produce convergent estimates (in the Monte Carlo sense) of the distribution <italic>P</italic>(<bold>x</bold>, <italic>t</italic>|, <italic>t</italic><sub>0</sub>), ∀<italic>t </italic>∈ [<italic>t</italic><sub>0</sub>, <italic>T</italic><sub><italic>s</italic></sub>] [##UREF##2##8##]. However, if one is interested in the stationary distribution <italic>π</italic>, running the SSA repeatedly from different initial conditions for a finite time <italic>T</italic><sub><italic>s </italic></sub>does not guarantee that <italic>P</italic>(<bold>x</bold>, <italic>T</italic><sub><italic>s</italic></sub>) will converge to <italic>π</italic>, unless the starting points are themselves drawn from <italic>π</italic>. Our Perfect Sampling algorithm addresses this issue.</p>", "<title>Proof of the DCFTP-SSA for a class of networks of biochemical reactions</title>", "<p>Viewing the SSA as the Markov process described by (5), we have developed a specific DCFTP algorithm that provides guaranteed sampling from the stationary distribution of the corresponding chemical ME [##REF##17468171##15##]. We now provide a rigorous proof and an explicit implementation of the DCFTP-SSA for an important class of biochemical reactions relevant in gene regulation.</p>", "<title>Partial ordering</title>", "<p>We use the Pareto dominance relation, frequently used in economics, which is defined componentwise:</p>", "<p><bold>Lemma 4 (Partial order) </bold><italic>Given </italic><bold>x</bold>, <bold>y </bold>∈ ℕ<sup><italic>m</italic></sup>, <italic>the relation </italic><italic>if x</italic><sub><italic>i </italic></sub>≥ <italic>y</italic><sub><italic>i</italic></sub>, ∀<italic>i </italic><italic>is a partial order</italic>.</p>", "<p><bold>Proof </bold>The proof follows trivially from the properties of natural numbers:</p>", "<p>Reflexivity: ∀<italic>x</italic><sub><italic>i </italic></sub>∈ ℕ, <italic>x</italic><sub><italic>i </italic></sub>≥ <italic>x</italic><sub><italic>i</italic></sub>, whence </p>", "<p>Anti-symmetry: ∀<italic>x</italic><sub><italic>i</italic></sub>, <italic>y</italic><sub><italic>i </italic></sub>∈ ℕ, if <italic>x</italic><sub><italic>i </italic></sub>≥ <italic>y</italic><sub><italic>i </italic></sub>and <italic>y</italic><sub><italic>i </italic></sub>≥ <italic>x</italic><sub><italic>i </italic></sub>then <italic>x</italic><sub><italic>i </italic></sub>= <italic>y</italic><sub><italic>i</italic></sub>. This means that and implies <bold>x </bold>= <bold>y</bold></p>", "<p>Transitivity: ∀<italic>x</italic><sub><italic>i</italic></sub>, <italic>y</italic><sub><italic>i</italic></sub>, <italic>z</italic><sub><italic>i </italic></sub>∈ ℕ, if <italic>x</italic><sub><italic>i </italic></sub>≥ <italic>y</italic><sub><italic>i </italic></sub>and <italic>y</italic><sub><italic>i </italic></sub>≥ <italic>z</italic><sub><italic>i </italic></sub>then <italic>x</italic><sub><italic>i </italic></sub>≥ <italic>z</italic><sub><italic>i</italic></sub>. And the same property applies to the vectors: and implies .   □</p>", "<title>Assumptions on the reaction network</title>", "<p>Consider a system of chemical reactions as given by Definition 2 with state vector <bold>x</bold>(<italic>t</italic>) ∈ ℕ<sup><italic>m</italic></sup>. To guarantee the preservation of the Pareto partial order under the SSA Markov process (5), we restrict ourselves to a class of chemical networks with the following properties:</p>", "<p>(<italic>a</italic>) all reactions are <italic>uni-molecular birth-death </italic>processes with non-zero propensities, i.e., each reaction <italic>R</italic><sub><italic>i </italic></sub>will only modify one species <italic>S</italic><sub><italic>j </italic></sub>by adding or subtracting one molecule. The reactions can be divided into two subsets:</p>", "<p></p>", "<p>(<italic>b</italic>) the system must be <italic>chemically reversible</italic>, i.e., every reaction must be reversible leading to an irreducible Markov process</p>", "<p>(<italic>c</italic>) all death reactions must be linear, i.e.</p>", "<p></p>", "<p>(<italic>d</italic>) all birth reactions must have <italic>(anti-)monotonic, sub-linear propensity functions</italic>, i.e., ∀<italic>i</italic>, <italic>j</italic>, ∀<bold>x</bold>: ∂Φ<sub><italic>i</italic></sub>(<bold>x</bold>)/∂<italic>x</italic><sub><italic>j </italic></sub>does not change sign and Φ<sub><italic>i </italic></sub>can be bounded by a linear function (or a constant).</p>", "<p>As shown below, the last two assumptions are related to domination by a linear network which is required to have a stationary distribution.</p>", "<p>Although assumptions (<italic>a</italic>) – (<italic>d</italic>) might appear restrictive, the specified class of reactions is generic and encompasses the standard equations used in the modelling of genetic and regulatory networks, the cellular circuits where stochasticity is most significant. Note that assumption (<italic>c</italic>) is not unrealistic for models of gene regulatory networks, in which linear death terms due to the cellular environment are prevalent. Birth reactions in these models are usually represented through <italic>nonlinear</italic>, uni-molecular (compound) rate laws that appear from quasi steady-state approximations. These functional forms have been shown to work well in the stochastic setting [##UREF##10##18##]. Our own simulations confirmed that they provide a good approximation in a wide range of parameters (results not shown). These compound rate laws are the key components that encode the positive and negative feedback in gene regulation. Classic examples are the sigmoid functions:</p>", "<p></p>", "<p></p>", "<p>which are sub-linear, (anti-)monotonic functions.</p>", "<title>Dominating process and adapted functionals</title>", "<p>As stated above, assumption (<italic>d</italic>) is related to domination. In general, the state space of chemical reaction networks is unbounded from above; hence we must use the DCFTP construction, which requires a dominating process <bold>D </bold>with known stationary distribution. Fortunately, it has been shown that any network of <italic>linear </italic>first order reactions has a stationary distribution which is multivariate Poisson [##REF##15998488##19##]. Moreover, it can be shown that is an ergodic atom for the multivariate Poisson, as assumed in Theorem 1 [##UREF##6##13##]. It then follows that a dominating process for any reaction network composed of uni-molecular, sub-linear, (anti-)monotonic birth-death processes, as defined above, can be found by 'linearizing' the original network; that is, by constructing a linearized version of this network , with the same reactions and compounds but with linear propensities (<bold>x</bold>) ≥ Φ<sub><italic>i</italic></sub>(<bold>x</bold>), ∀<bold>x</bold>, ∀<italic>i </italic>that bound the original Φ from above. Under conditions of stability, the ME of will have a stationary distribution , given by a multivariate Poisson that can be obtained by solving a system of linear equations [##REF##15998488##19##]. The existence of the stationary distribution of the dominating linear network guarantees the existence of the stationary distribution for the original network of reversible, uni-molecular nonlinear reactions .</p>", "<p>The <italic>dominating process </italic><bold>D </bold>is defined as the stationary SSA process (5) of the linearized network with initial state sampled from :</p>", "<p></p>", "<p>with the sequence of reactions .</p>", "<p>It has been shown [##UREF##6##13##] that a correct realization of the original (nonlinear) SSA process <bold>X </bold>for a network with monotonic propensities can also be obtained through an <italic>adapted functional </italic> defined in terms of the dominating process <bold>D </bold>and a random mark process where ~ <italic>U</italic>(0, 1):</p>", "<p></p>", "<p>The update rule for uses the ratio of the monotonic propensity functions of the original and dominating processes as follows:</p>", "<p></p>", "<p>where and , , correspond to reaction in the reaction sequence .</p>", "<p>The necessary ingredient for the DCFTP is the construction of an order-preserving Markov process for the evolution of two chains <bold>X </bold>and <bold>Y </bold>coupled to the dominating process <bold>D</bold>. For our network , this process is defined as:</p>", "<p></p>", "<p>with transition rule:</p>", "<p></p>", "<p>where the componentwise transition rule is given in Eq. (9). The transition rule incorporates the cross-over scheme in which the processes <bold>X </bold>and <bold>Y </bold>use the state of each other when determining their update, as introduced by Häggström and Nelander to deal with anti-monotonic processes [##UREF##11##20##].</p>", "<title>Proof</title>", "<p>We now show that the partial ordering defined in Lemma 4 is indeed preserved under the evolution given by Eqs. (10)–(11) for the class of reactions specified above.</p>", "<p><bold>Lemma 5 (Preservation of partial ordering) </bold><italic>Consider a chemically reversible reaction network </italic><italic>of uni-molecular, sub-linear, (anti-)monotone birth-death reactions and its associated SSA dominating process </italic><bold>D</bold>, <italic>obtained from the linearized network </italic>, <italic>with the sequence of events </italic>. <italic>Consider two coupled chains </italic><bold>X </bold><italic>and </italic><bold>Y </bold><italic>evolving under (10)–(11), where </italic><italic>is a sequence of random marks. Then </italic>, ∀<italic>s </italic>&gt; <italic>t</italic>.</p>", "<p><bold>Proof </bold>Assume throughout. First consider the case when is monotonic. Then the possible outcomes for <italic>t</italic><sub>0 </sub>&lt;<italic>s </italic>&lt;<italic>t</italic><sub>1 </sub>are:</p>", "<p></p>", "<p>Outcome (<italic>i</italic>) means that neither <bold>X </bold>nor <bold>Y </bold>is modified and the preservation of the partial order is obvious. For (<italic>iii</italic>), both are modified by the same amount and the order is preserved. The interesting case is (<italic>ii</italic>) for which <bold>X </bold>is modified but not <bold>Y</bold>. If , then which also implies order preservation. However, if , then it is possible for the two chains to coalesce if . Note that since, by uni-molecularity, only unit changes of the states are allowed, it is impossible for two paths to cross.</p>", "<p>When is anti-monotone, the outcomes are:</p>", "<p></p>", "<p>As above, outcomes (<italic>iv</italic>) and (<italic>vi</italic>) lead to no change in relative order. For (<italic>v</italic>), again we update <bold>X </bold>but not <bold>Y </bold>due to the crossover scheme. As for the monotone case, if this leads to order preservation, while if it is possible for the two chains to coalesce.</p>", "<p>It thus follows that <bold>X </bold>and <bold>Y </bold>maintain their partial ordering through every update of the (anti-)monotone process. The proof for <italic>s </italic>&gt; <italic>t</italic><sub>1 </sub>follows by induction.   □</p>", "<p>Note that the dominated processes given by Eqs. (9) and (11) become identical when <bold>X </bold>= <bold>Y</bold>. Therefore, after coalescence the dominated process is statistically identical to the original SSA process. Since we have found a dominating process and an adapted functional, we can use Theorem 1 to obtain:</p>", "<p><bold>Theorem 6 (DCFTP-SSA) </bold><italic>Under the assumptions of Lemma 5, Theorem 1 is fulfilled and the DCFTP-SSA described in Algorithm 7 will produce a sample from the stationary distribution of the original process </italic><bold>X </bold><italic>with a coalescence time which will be finite almost surely</italic>.</p>", "<p><bold>Proof </bold>The sandwiching (1) and funneling (2) properties follow from the preservation of partial ordering (Lemma 5) [##UREF##5##12##]. The remainder can be adapted from the general Theorem 1.   □</p>", "<title>Algorithm</title>", "<p>A brief outline of the DCFTP-SSA is as follows:</p>", "<p><bold>Algorithm 7 (DCFTP-SSA) </bold><italic>Given a reversible system of uni-molecular birth-death chemical reactions </italic><italic>with (anti-)monotone, sub-linear propensity functions, obtain its linearized version </italic><italic>with multivariate Poisson stationary distribution </italic>:</p>", "<p>   <italic>T </italic>← 1</p>", "<p>   </p>", "<p>   </p>", "<p>   </p>", "<p>   <bold><italic>loop</italic></bold></p>", "<p>      </p>", "<p>      </p>", "<p>      <bold><italic>if </italic>U</bold><sub>0 </sub>= <bold>L</bold><sub>0 </sub><bold><italic>then</italic></bold></p>", "<p>         <bold><italic>return </italic>L</bold><sub>0</sub></p>", "<p>      <bold><italic>end if</italic></bold></p>", "<p>      <italic>T </italic>← 2<italic>T</italic></p>", "<p>      </p>", "<p>      </p>", "<p>   <bold><italic>end loop</italic></bold></p>", "<p>The function Extend(, <italic>T</italic>/2) runs Algorithm 3 for the linearized network and appends the path to the path . Similarly, the function GenerateMarks appends paths generated from a uniform distribution to extend the mark process. Both the marks and the forward dominating path are then reversed in time by the function Reverse. Extending these processes <italic>backwards </italic>in time in this manner is justified because of their stationarity and reversibility, which allows us to reverse the processes and translate them in time [##UREF##3##9##]. Finally, the Evolve function starts the coupled upper and lower chains from <bold>L</bold><sub>-<italic>T </italic></sub>= and <bold>U</bold><sub>-<italic>T </italic></sub>= <bold>D</bold><sub>-<italic>T </italic></sub>and evolves them forward as described by Eq. (11). Note that the assumption of reversibility of the network ensures that the reverse process will be forward-evolvable. Our requirement that propensities are non-zero also ensures that reactions are not eliminated from the network. If this were to happen, it would effectively make the system irreversible. If <bold>L </bold>and <bold>U </bold>have not coalesced at <italic>t </italic>= 0, <bold>D </bold>and <bold>M </bold>are extended further back in time and <bold>L </bold>and <bold>U </bold>are restarted. Doubling the starting time at each iteration has been shown to be reasonably efficient (see [##UREF##4##11##] for a discussion).</p>", "<title>Applications of the algorithm</title>", "<title>Numerical convergence: First order reaction</title>", "<p>To characterize numerically the convergence properties of the DCFTP-SSA, consider the first order reaction where species <italic>A </italic>is created at a (normalized) constant rate <italic>k </italic>from a source and degraded to a sink:</p>", "<p></p>", "<p>Here <italic>P</italic><sub><italic>j </italic></sub>denotes the probability of having <italic>j </italic>molecules of <italic>A </italic>and and are step operators [##UREF##0##4##]:</p>", "<p><italic>f</italic>(<italic>j</italic>) = <italic>f</italic>(<italic>j </italic>+ 1) and <italic>f</italic>(<italic>j</italic>) = <italic>f</italic>(<italic>j </italic>- 1) acting on a function <italic>f</italic>(<italic>j</italic>). For the usual initial condition with 0 molecules, the time-dependent solution of Eq. (12) is a Poisson distribution with time-dependent parameter <italic>k</italic>(1 - <italic>e</italic><sup>-<italic>t</italic></sup>) [##REF##17468171##15##]. Equation (12) is an instance of the immigration-death process which appears in different settings in the stochastic processes literature.</p>", "<p>If, as a proxy for sampling the stationary distribution <italic>π</italic>, one obtains samples of <italic>P</italic>(<italic>j</italic>, <italic>T</italic><sub><italic>s</italic></sub>|0, 0) from repeated runs of the SSA for a finite time <italic>T</italic><sub><italic>s</italic></sub>, this will lead to a systematic error that will not disappear as the number of samples (and the CPU time) is increased. The use of the DCFTP-SSA eliminates this source of error, as shown in Fig. ##FIG##0##1a##. This figure also shows that the guaranteed convergence of the DCFTP-SSA incurs a modest additional CPU cost. The increased computational cost is twofold: increased memory requirements, since we need to store the history of the dominating process as well as the sequence of random numbers used to update the coupled chains; and longer running times, since we need to extend the process backwards for an indefinite (unbounded) amount of time. Fig. ##FIG##0##1b## presents the statistics of the coalescence times for this reaction. In this simple reaction, the distribution of stopping times is relatively symmetric and concentrated around the mean value, without the long tails that would correspond to long runs started a long time into the past. As the next example shows, the distribution of coalescence times reflects the complexity of the structure of the stationary distribution.</p>", "<title>Multistability: Genetic toggle switch</title>", "<p>The mutual activation and repression of groups of genes in regulatory networks can lead to multi-stability allowing cells to attain different states [##REF##9023339##5##,##REF##17337276##21##]. An important and difficult problem is to find the probabilities of the different states and the expected switching times. Previously [##REF##17468171##15##], we applied the DCFTP-SSA to the standard toggle switch with two Hill-repressed genes [##REF##10659857##22##]. We now apply the algorithm to a more complex model of two mutually activating genes [##REF##17337276##21##] with a complicated activation function which is not of the standard Monod form:</p>", "<p></p>", "<p></p>", "<p>with the activation given by</p>", "<p></p>", "<p>where <italic>n</italic><sub><italic>A </italic></sub>and <italic>n</italic><sub><italic>B </italic></sub>are the number of protein molecules, <italic>γ </italic>is the basal production rate and <italic>κ</italic><sub><italic>ij </italic></sub>are parameters. The functional form of the activation appears as a consequence of particular properties of this system: each transcription site can be occupied by up to four monomers and becomes activated when a tetramer is bound. However, note that <italic>f</italic>(<italic>n</italic>) is monotonic and sub-linear and therefore the DCFTP-SSA is applicable.</p>", "<p>For certain choices of parameters, the stationary distribution of the system is bimodal: the peak located at the origin corresponds to both genes being 'off', while the other mode indicates both genes are 'on' (Fig. ##FIG##1##2a##). The extreme bimodality of this distribution makes its sampling difficult using the standard SSA. As can be seen in Fig. ##FIG##2##3a##, if we start from the initial condition (0, 0), the standard SSA levels off in a similar manner to Fig. ##FIG##0##1a##, highlighting the presence of a systematic error. In contrast, the DCFTP-SSA converges to the stationary distribution at the expected <italic>N</italic><sup>-1/2 </sup>rate.</p>", "<p>Figure ##FIG##1##2b## also shows that the probability sampled with the DCFTP-SSA captures the global structure of the probability distribution even in this extreme example. On the other hand, closer inspection of the SSA simulations started from the (0, 0) reveals that for short stopping times, the process remains at the mode located near the origin (Fig ##FIG##1##2c##). Although simple heuristics on how to choose the initial condition have been suggested to improve the sampling of <italic>π </italic>with the SSA, Figure ##FIG##1##2## shows that similar mis-sampling errors appear if we run the standard SSA from a variety of initial conditions. Fig. ##FIG##1##2d## shows that sampling the initial condition from a uniform grid in state space does not capture the full features of the distribution since this initial condition does not represent a consistent sampling for stationarity. If we use the fixed points of the corresponding deterministic system as initial conditions for the SSA, we would still lack the probability mass associated with each mode. For instance, starting half of the simulations at the origin and the remaining at the other fixed point provides little improvement since almost half of the simulations remain near the origin (Fig. ##FIG##1##2e##). Similar errors appear if we sample a long SSA run at fixed intervals Δ<italic>t </italic>to provide independent samples as intiial conditions (Fig. ##FIG##1##2f##), or even if we use samples drawn from the true stationary distribution as initial conditions for the SSA.</p>", "<p>We can understand why the stationary distribution of this system presents such a challenge for the finite-time SSA by considering the mean first passage times. Figure ##FIG##3##4a## shows the average time to reach all other states from the origin and Fig. ##FIG##3##4b## shows the average time to reach the other mode. To be certain that an SSA run will produce correct samples from the stationary distribution, it must visit each mode several times. For the system considered in Fig. ##FIG##1##2## we need stopping times on the order of 10<sup>7 </sup>to be certain that the simulation has not been stuck in one mode. With our implementation, the DCFTP compares favorably with the SSA wtih <italic>T</italic><sub><italic>s </italic></sub>= 10<sup>7 </sup>(data not shown).</p>", "<p>Figure ##FIG##2##3a## summarizes the CPU times for the different SSA sampling schemes shown in Fig. ##FIG##1##2## compared to the DCFTP-SSA. Again, the DCFTP-SSA introduces a reasonable overhead but provides guarantees that no systematic sampling error exists. To understand how the extreme bimodality of this distribution affects the running time of the DCFTP-SSA, Figure ##FIG##2##3b## shows the statistics of the coalescence times for this system. As compared with Fig. ##FIG##0##1b##, the distribution of coalescence times is bimodal with a second mode at long coalescence times a long tail. This reflects the complex structure of the stationary distribution in state space which induces longer coalescence times to guarantee the correct sampling. As explained in the Discussion section, the numerical performance of the algorithm in situations where long runs are more likely can be improved by the use of rejection sampling schemes.</p>", "<p>This simple example illustrates the potential pitfalls of using the standard SSA for multimodal systems with long switching time-scales. If the SSA is run with too short stopping times, one runs the risk of missing important features of the distribution that could lead to erroneous conclusions about the number and relative weight of possible states. These problems become more acute as the dimensionality of the state space increases.</p>", "<title>Steady-state dynamics: Generalized repressilator</title>", "<p>Although regularity and robustness are important for their reliable operation in time-keeping, circadian and synchronization processes, cellular oscillators have a biochemical basis and are subject to high levels of noise [##REF##10659856##23##,##REF##12432409##24##]. In previous work [##REF##17468171##15##], we studied the stochastic version of the repressilator, a synthetic transcriptional oscillator that consists of three mutually repressing genes in a loop (Fig. ##FIG##4##5a##) and has been implemented in <italic>E. coli </italic>[##REF##10659856##23##]. Experiments on the original repressilator showed that the oscillations are very noisy and stochastic models are required to capture these features.</p>", "<p>Here, we investigate the stochastic properties of the generalized repressilator with an arbitrary number <italic>n </italic>of genes in the loop [##REF##16951971##25##]. Müller <italic>et al </italic>studied the deterministic version and showed that the system oscillates when <italic>n </italic>is odd, as expected by analogy with the ring oscillator in electronic circuits (see Fig. ##FIG##4##5b##). This system allows us to study the dependence of the variability of the oscillations with the number of genes and to showcase the scalibility of our algorithm as the number of variables (and the dimensionality of the state space) increases.</p>", "<p>We now use the DCFTP-SSA to characterize the periodicity of the stochastic oscillations of the generalized repressilator:</p>", "<p></p>", "<p>where the shorthand <italic>P</italic><sub><italic>j </italic></sub>denotes the state and all integers are <italic>i </italic>mod <italic>n</italic>. Here <italic>M</italic><sub><italic>i </italic></sub>are the mRNA levels (with production rate <italic>k</italic><sub><italic>M </italic></sub>and degradation rate <italic>d</italic><sub><italic>M</italic></sub>) and <italic>R</italic><sub><italic>i </italic></sub>are the corresponding proteins (with basal rate <italic>k</italic><sub><italic>B </italic></sub>and linear production rate <italic>k</italic><sub><italic>R</italic></sub>). The repressilator network fulfills the conditions of applicability of the DCFTP-SSA and we have used our algorithm to generate time-series which are guaranteed to be at stationarity. The fact that the system has a persistent, oscillatory dynamics does not preclude it from being stationary. As expected, our DCFTP-SSA simulations show that the stationary distribution <italic>π </italic>conforms to the shape of a circular ridge in 2<italic>n</italic>-dimensional space, which is directly related to the deterministic limit cycle [##UREF##0##4##]. In this case, the probability mass is unimodal along the ridge, which means that sampling from a long time-series is unproblematic since there is no risk of avoiding regions of state space that have high probability.</p>", "<p>As one would expect from the deterministic analysis, the mean period increases linearly with <italic>n </italic>(Figure ##FIG##4##5d##). This follows from the fact that the oscillatory behavior propagates in a wave-like manner around the loop. If we assume that the period is formed as the sum of <italic>n </italic>independent genes rising and falling in sequence, then a circuit with <italic>n </italic>genes will have a period whose mean scales linearly with <italic>n</italic>, as shown in Figure ##FIG##4##5d## in accordance with the deterministic model. However, the shape and moments of the distribution of the periods change significantly as a function of <italic>n</italic>, as shown in Figs. ##FIG##4##5c–d##. The distribution of the period for shorter circuits will necessarily be right-skewed since there is a minimal waiting time, akin to a refractory period, before the gene can rise again. This asymmetry is observed in the case of <italic>n </italic>= 3 but has almost disappeared for <italic>n </italic>= 9, and is captured by the skewness, which decreases towards zero as <italic>n </italic>increases.</p>", "<p>Our numerics also indicate that the relative variability of the period is not constant as the number of genes in the loop increases. Figure ##FIG##4##5d## shows that the variance of the period increases quadratically, which implies that the successive periods are not independent. This implies that, for the set of parameters in Figure ##FIG##4##5##, there is an optimal length of <italic>n</italic>* = 7 genes in the loop, for which the relative fluctuations of the period, as measured by the coefficient of variation, are minimal. Note also that the kurtosis remains almost constant and positive, which indicates that there are fat tails even for longer circuits. Interestingly, the kurtosis also attains a shallow minimum at <italic>n</italic>* = 7, indicating a relative decrease in the dispersion of the distribution. Another important characteristic of an oscillator is the <italic>rise time</italic>, which gives an indication of its precision. Our numerics find no change in the variance of the rise times as the number of genes increases (results not shown). This is expected since the rise time of a single gene is almost independent of the preceding events unlike the period, which is an aggregated quantity and therefore more susceptible to propagated noise. The investigation of the noise characteristics of networks of transcriptional oscillators will be the object of further study.</p>", "<title>Authors' contributions</title>", "<p>MH and MB developed the method, completed the proof and wrote the paper. MH implemented the algorithm for the simulations.</p>" ]
[ "<title>Acknowledgements</title>", "<p>We would like to thank Matias Rantalainen, Vincent Rouilly and Sophia Yaliraki for valuable discussions and feedback. Research funded by the UK EPSRC through the Life Science Interface and the Mathematics Panels.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Convergence of the DCFTP-SSA for the first order reaction (12)</bold>. (<italic>a</italic>) As a function of CPU time, we represent the Euclidean error <italic>∊</italic><sub><italic>E </italic></sub>of the stationary distribution of Eq. (12) with <italic>k </italic>= 5 sampled with the DCFTP-SSA (+) and with the standard SSA with stopping times <italic>T</italic><sub><italic>s </italic></sub>= 2(○), 4(□), 6(◇). For this simple ME, the limiting value of the Euclidean error of the finite-time SSA is , where <italic>α </italic>= 1 - exp(-<italic>T</italic><sub><italic>s</italic></sub>) and <italic>I</italic><sub>0</sub>(<italic>x</italic>) is the modified Bessel function of the first kind [##REF##17468171##15##]. This means that SSA simulations that are run for a time <italic>T</italic><sub><italic>s </italic></sub>will converge to a systematic sampling error, indicated by the dotted lines. This source of error is eliminated when using the DCFTP-SSA, which shows no flooring for <italic>∊</italic><sub><italic>E </italic></sub>and the expected <italic>N</italic><sup>-1/2 </sup>scaling with the number of Monte Carlo samples [##UREF##12##26##]. The guarantees provided by the DCFTP-SSA come at a modest computational cost, which is comparable to that of long SSA runs. (<italic>b</italic>) The distribution of coalescence times <italic>T</italic><sub><italic>c </italic></sub>for the DCFTP-SSA is relatively symmetric and concentrated around the mean with a rapid decay for long times. The data presented corresponds to 6000 runs. This distribution reflects the benign structure of the unimodal stationary distribution of this particular ME, which makes long coalescence times unlikely.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Sampling of the stationary distribution for the bistable gene network (13) using different methods</bold>. (<italic>a</italic>) The 'true' stationary probability distribution <italic>π </italic>for the ME (13) calculated numerically with the approximate eigenvector method [##REF##17468171##15##]. The parameters are <italic>κ</italic><sub><italic>B </italic></sub>= 25, <italic>κ</italic><sub><italic>A </italic></sub>= 12, <italic>κ</italic><sub><italic>A</italic>0 </sub>= <italic>κ</italic><sub><italic>B</italic>0 </sub>= 60, <italic>κ</italic><sub><italic>A</italic>1 </sub>= <italic>κ</italic><sub><italic>B</italic>1 </sub>= 10, <italic>κ</italic><sub><italic>A</italic>2 </sub>= <italic>κ</italic><sub><italic>B</italic>2 </sub>= <italic>κ</italic><sub><italic>A</italic>3 </sub>= <italic>κ</italic><sub><italic>B</italic>3 </sub>= 1, and <italic>γ </italic>= 0.01. The locations of the two modes match the fixed points of the corresponding deterministic system. Note the extreme asymmetry of the bimodal probability distribution. (<italic>b</italic>) The estimate of <italic>π </italic>obtained from 10<sup>4 </sup>samples of the DCFTP-SSA reproduces the presence of both modes and their relative weights. (<italic>c</italic>) Estimate of <italic>π </italic>from 10<sup>4 </sup>samples of the SSA started at (0,0) with <italic>T</italic><sub><italic>s </italic></sub>= 10<sup>3</sup>. (<italic>d</italic>) Estimate of <italic>π </italic>obtained from 10<sup>4 </sup>SSA simulations started from 10<sup>4 </sup>different initial conditions chosen uniformly at random on the 100 × 100 lattice closest to the origin and run for <italic>T</italic><sub><italic>s </italic></sub>= 10<sup>3</sup>. (<italic>e</italic>) Estimate of <italic>π </italic>obtained from 10<sup>4 </sup>SSA simulations, 5000 of them started from the origin and the other 5000 from the other mode and run for <italic>T</italic><sub><italic>s </italic></sub>= 10<sup>3</sup>. (<italic>f</italic>) Estimate of <italic>π </italic>obtained from 10<sup>4 </sup>samples from a long SSA run sampled at interval Δ<italic>t </italic>= 10<sup>3</sup>. Note the different scale on the <italic>z</italic>-axis for (<italic>c</italic>) and (<italic>e</italic>) and how the SSA runs (<italic>c</italic>)-(<italic>f</italic>) do not capture the overall structure of <italic>π</italic>.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Convergence of the DCFTP-SSA for the bistable gene network (13)</bold>. (<italic>a</italic>) As a function of CPU time, we represent <italic>∊</italic><sub><italic>E</italic></sub>, the Euclidean error of the sampled distributions estimated using: the DCFTP-SSA (+), as in Fig. 2 (<italic>b</italic>); the SSA with <italic>T</italic><sub><italic>s </italic></sub>= 1000(○), as in Fig. 2 (<italic>c</italic>); the SSA started from the two modes (*), as in Fig. 2 (<italic>d</italic>); the SSA started from uniform initial conditions (∇), as in Fig. 2 (<italic>e</italic>); and the SSA uniformly sampled from a long run (□), as in Fig. 2 (<italic>f</italic>). For each scheme, we produced <italic>N </italic>= 100, 316, 1000, 3162 and 10000 samples to show how the error improves as the number of samples increases. The DCFTP-SSA converges to the stationary distribution at the expected <italic>N</italic><sup>-1/2 </sup>rate, whereas the approximate estimates obtained using the SSA level off in a similar manner as in Fig. 1<italic>a</italic>. (<italic>b</italic>) The distribution of coalescence times for the DCFTP-SSA for this network is bimodal with a very long tail for the second mode, indicating the likelihood of long coalescence times. The data presented corresponds to 6000 runs.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Mean transition times for the bistable gene network (13)</bold>. (<italic>a</italic>) The mean first passage time <italic>ξ </italic>to reach the origin for the lattice points of the state space close to the origin. The escape time from the mode located away from the origin is 2 × 10<sup>5</sup>. (<italic>b</italic>) The mean first passage time from the origin to the other mode is 3 × 10<sup>3</sup>.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Noise characteristics of the generalized repressilator (14)</bold>. (<italic>a</italic>) Detailed diagram of the reactions in the standard repressilator with three genes involving six chemical species, as implemented with our stochastic algorithm. In the simplified cartoon, each circle represents a gene repressing the subsequent gene in a cycle. The generalized repressilator studied here considers cycles with odd number of genes <italic>n </italic>= 3, 5, 7, 9. (<italic>b</italic>) The top panel shows time series of one of the proteins for the deterministic model of the repressilator with <italic>n </italic>= 3 (filled) and <italic>n </italic>= 9 (dashed) genes with parameters <italic>k</italic><sub><italic>M </italic></sub>= 25, <italic>d</italic><sub><italic>M </italic></sub>= 3, <italic>θ </italic>= 3, <italic>k</italic><sub><italic>R </italic></sub>= 4 and <italic>α </italic>= 2. The lower panel shows the corresponding time series of the SSA started from stationarity, guaranteed by the DCFTP-SSA. For the top panel, the y-axis has units of protein concentration, whereas for the lower panel the <italic>y</italic>-axis has unitos of number of proteins. (<italic>c</italic>) The top panel shows the distribution of the period for the repressilator with <italic>n </italic>= 3 genes, while the bottom panel shows the same distribution for the generalized repressilator with <italic>n </italic>= 9 genes. Note that the distribution for <italic>n </italic>= 3 is skewed with a long right tail, while that of <italic>n </italic>= 9 is more symmetric, but has fatter tails than would be expected for a Gaussian distribution. The histograms were obtained from time-series with 10<sup>4 </sup>periods. (<italic>d</italic>) The top two panels show the dependence of the mean (∘) and variance (□) of the period distribution with <italic>n</italic>. The lines indicate a linear fit for the means and a quadratic fit for the variances. The inset in the top right panel, shows that, for this set of parameters, the relative noise of the period, as measured by the coefficient of variation (*), is minimal for a length of <italic>n </italic>= 7 genes in the loop. The two lower panels show the skewness (∇) and kurtosis (◇) for the period distribution. The skewness decreases to zero as <italic>n </italic>grows, in accordance with the observed decrease of the asymmetry of the distribution. The kurtosis does not disappear as <italic>n </italic>grows indicating the presence of long-tails. Note that the kurtosis also reaches an apparent minimum at <italic>n </italic>= 7.</p></caption></fig>" ]
[]
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<mml:mi>t</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≽</mml:mo>\n <mml:mi>ϕ</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M4\" name=\"1752-0509-2-42-i5\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mi>A</mml:mi>\n <mml:mi>n</mml:mi>\n <mml:mi>t</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mtext>-</mml:mtext>\n <mml:mi>M</mml:mi>\n <mml:mi>o</mml:mi>\n <mml:mi>n</mml:mi>\n <mml:mi>o</mml:mi>\n <mml:mi>t</mml:mi>\n <mml:mi>o</mml:mi>\n <mml:mi>n</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mi>c</mml:mi>\n <mml:mo>:</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n </mml:msub>\n <mml:mo>≽</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n </mml:msub>\n <mml:mo>⇒</mml:mo>\n <mml:mi>ϕ</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≼</mml:mo>\n <mml:mi>ϕ</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M5\" name=\"1752-0509-2-42-i6\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable columnalign=\"left\">\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mtext>Lower path </mml:mtext>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>L</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mtext> started from</mml:mtext>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>L</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msub>\n <mml:mo>=</mml:mo>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mover accent=\"true\">\n <mml:mn>0</mml:mn>\n <mml:mo>^</mml:mo>\n </mml:mover>\n </mml:mstyle>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mtext>Upper path </mml:mtext>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>U</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mtext> started from</mml:mtext>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>U</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msub>\n <mml:mo>=</mml:mo>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mover accent=\"true\">\n <mml:mn>1</mml:mn>\n <mml:mo>^</mml:mo>\n </mml:mover>\n </mml:mstyle>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M6\" name=\"1752-0509-2-42-i7\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mn mathvariant=\"bold-sans-serif\">0</mml:mn><mml:mo>ˆ</mml:mo></mml:mover></mml:mstyle><mml:mo>≼</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi mathvariant=\"bold-sans-serif\">x</mml:mi></mml:mstyle><mml:mo>≼</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mn mathvariant=\"bold-sans-serif\">1</mml:mn><mml:mo>ˆ</mml:mo></mml:mover></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<disp-formula id=\"bmcM1\"><label>(1)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M7\" name=\"1752-0509-2-42-i8\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>L</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>≼</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>≼</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>U</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:mi>t</mml:mi>\n <mml:mo>&gt;</mml:mo>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n <mml:mo>.</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<disp-formula id=\"bmcM2\"><label>(2)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M8\" name=\"1752-0509-2-42-i9\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable columnalign=\"right\">\n <mml:mtr columnalign=\"right\">\n <mml:mtd columnalign=\"right\">\n <mml:mrow>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>L</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>≼</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>L</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mi>S</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>≼</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>U</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mi>S</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>≼</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>U</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"right\">\n <mml:mtd columnalign=\"right\">\n <mml:mrow>\n <mml:mi>t</mml:mi>\n <mml:mo>&gt;</mml:mo>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n <mml:mo>,</mml:mo>\n <mml:mi>S</mml:mi>\n <mml:mo>&gt;</mml:mo>\n <mml:mn>0.</mml:mn>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M9\" name=\"1752-0509-2-42-i10\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">F</mml:mi></mml:semantics></mml:math></inline-formula>", "<disp-formula id=\"bmcM3\"><label>(3)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M10\" name=\"1752-0509-2-42-i11\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>≽</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>=</mml:mo>\n <mml:mi mathvariant=\"script\">F</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mi>s</mml:mi>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>M</mml:mi>\n </mml:mstyle>\n <mml:mi>s</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>,</mml:mo>\n <mml:mo>−</mml:mo>\n <mml:mi>T</mml:mi>\n <mml:mo>≤</mml:mo>\n <mml:mi>s</mml:mi>\n <mml:mo>≤</mml:mo>\n <mml:mi>t</mml:mi>\n <mml:mo>.</mml:mo>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M11\" name=\"1752-0509-2-42-i12\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mi>D</mml:mi><mml:mi>t</mml:mi><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M12\" name=\"1752-0509-2-42-i13\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mi>∞</mml:mi></mml:mrow></mml:msubsup><mml:mo>≼</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>D</mml:mi></mml:mstyle><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mi>∞</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M13\" name=\"1752-0509-2-42-i14\" overflow=\"scroll\"><mml:semantics><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mn>0</mml:mn><mml:mo>^</mml:mo></mml:mover></mml:mstyle></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M14\" name=\"1752-0509-2-42-i15\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mi>X</mml:mi><mml:mn>0</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M15\" name=\"1752-0509-2-42-i16\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mi>X</mml:mi><mml:mn>0</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>∞</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M16\" name=\"1752-0509-2-42-i14\" overflow=\"scroll\"><mml:semantics><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mn>0</mml:mn><mml:mo>^</mml:mo></mml:mover></mml:mstyle></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M17\" name=\"1752-0509-2-42-i17\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>L</mml:mi></mml:mstyle><mml:mo>≼</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mo>≼</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>U</mml:mi></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M18\" name=\"1752-0509-2-42-i18\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">N</mml:mi></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M19\" name=\"1752-0509-2-42-i19\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>=</mml:mo><mml:mo>{</mml:mo><mml:mi mathvariant=\"script\">S</mml:mi><mml:mo>,</mml:mo><mml:mi mathvariant=\"script\">R</mml:mi><mml:mo>,</mml:mo><mml:mi>Φ</mml:mi><mml:mo>,</mml:mo><mml:mi>ν</mml:mi><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M20\" name=\"1752-0509-2-42-i20\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">S</mml:mi></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M21\" name=\"1752-0509-2-42-i21\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">R</mml:mi></mml:semantics></mml:math></inline-formula>", "<disp-formula id=\"bmcM4\"><label>(4)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M22\" name=\"1752-0509-2-42-i22\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mfrac>\n <mml:mrow>\n <mml:mi>d</mml:mi>\n <mml:mi>P</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>x</mml:mi>\n </mml:mstyle>\n <mml:mo>,</mml:mo>\n <mml:mi>t</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n <mml:mrow>\n <mml:mi>d</mml:mi>\n <mml:mi>t</mml:mi>\n </mml:mrow>\n </mml:mfrac>\n <mml:mo>=</mml:mo>\n <mml:mstyle displaystyle=\"true\">\n <mml:munderover>\n <mml:mo>∑</mml:mo>\n <mml:mrow>\n <mml:mi>i</mml:mi>\n <mml:mo>=</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n <mml:mi>r</mml:mi>\n </mml:munderover>\n <mml:mrow>\n <mml:mrow>\n <mml:mo>[</mml:mo>\n <mml:mrow>\n <mml:msub>\n <mml:mi>Φ</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>x</mml:mi>\n </mml:mstyle>\n <mml:mo>−</mml:mo>\n <mml:msub>\n <mml:mi>ν</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mi>P</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>x</mml:mi>\n </mml:mstyle>\n <mml:mo>−</mml:mo>\n <mml:msub>\n <mml:mi>ν</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo>;</mml:mo>\n <mml:mi>t</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>−</mml:mo>\n <mml:msub>\n <mml:mi>Φ</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>x</mml:mi>\n </mml:mstyle>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mi>P</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>x</mml:mi>\n </mml:mstyle>\n <mml:mo>;</mml:mo>\n <mml:mi>t</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n <mml:mo>]</mml:mo>\n </mml:mrow>\n </mml:mrow>\n </mml:mstyle>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M23\" name=\"1752-0509-2-42-i19\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>=</mml:mo><mml:mo>{</mml:mo><mml:mi mathvariant=\"script\">S</mml:mi><mml:mo>,</mml:mo><mml:mi mathvariant=\"script\">R</mml:mi><mml:mo>,</mml:mo><mml:mi>Φ</mml:mi><mml:mo>,</mml:mo><mml:mi>ν</mml:mi><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M24\" name=\"1752-0509-2-42-i23\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M25\" name=\"1752-0509-2-42-i24\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:msup><mml:mi>V</mml:mi><mml:mo>′</mml:mo></mml:msup><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M26\" name=\"1752-0509-2-42-i25\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>θ</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo>←</mml:mo><mml:mstyle displaystyle=\"true\"><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>j</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>i</mml:mi></mml:msubsup><mml:mrow><mml:msub><mml:mi>Φ</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mrow><mml:mi>k</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M27\" name=\"1752-0509-2-42-i26\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mrow><mml:mi>k</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msubsup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M28\" name=\"1752-0509-2-42-i27\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mrow><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub><mml:mo>←</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mrow><mml:mi>k</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ν</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mi>R</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub><mml:mo>←</mml:mo><mml:msub><mml:mi>R</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mi>θ</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mi>θ</mml:mi><mml:mi>r</mml:mi></mml:msub></mml:mrow></mml:mfrac><mml:mo>&lt;</mml:mo><mml:msub><mml:msup><mml:mi>V</mml:mi><mml:mo>′</mml:mo></mml:msup><mml:mi>k</mml:mi></mml:msub><mml:mo>&lt;</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi>θ</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mi>θ</mml:mi><mml:mi>r</mml:mi></mml:msub></mml:mrow></mml:mfrac></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M29\" name=\"1752-0509-2-42-i28\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mo>{</mml:mo><mml:msub><mml:mi>R</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mn>...</mml:mn><mml:mo>,</mml:mo><mml:msub><mml:mi>R</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>n</mml:mi></mml:msub></mml:mrow></mml:msub><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M30\" name=\"1752-0509-2-42-i29\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>T</mml:mi><mml:mi>s</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msubsup><mml:mo>≡</mml:mo><mml:mo>{</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mn>...</mml:mn><mml:mo>,</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>n</mml:mi></mml:msub></mml:mrow></mml:msub><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M31\" name=\"1752-0509-2-42-i30\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>T</mml:mi><mml:mi>s</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msubsup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M32\" name=\"1752-0509-2-42-i31\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M33\" name=\"1752-0509-2-42-i32\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>R</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<disp-formula id=\"bmcM5\"><label>(5)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M34\" name=\"1752-0509-2-42-i33\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>T</mml:mi>\n <mml:mi>s</mml:mi>\n </mml:msub>\n </mml:mrow>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>=</mml:mo>\n <mml:msub>\n <mml:mi mathvariant=\"script\">G</mml:mi>\n <mml:mrow>\n <mml:mtext>SSA</mml:mtext>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi mathvariant=\"script\">N</mml:mi>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>T</mml:mi>\n <mml:mi>s</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>.</mml:mo>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M35\" name=\"1752-0509-2-42-i23\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M36\" name=\"1752-0509-2-42-i23\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M37\" name=\"1752-0509-2-42-i23\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M38\" name=\"1752-0509-2-42-i34\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>x</mml:mi></mml:mstyle><mml:mo>≽</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>y</mml:mi></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M39\" name=\"1752-0509-2-42-i35\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>x</mml:mi></mml:mstyle><mml:mo>≽</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>x</mml:mi></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M40\" name=\"1752-0509-2-42-i34\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>x</mml:mi></mml:mstyle><mml:mo>≽</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>y</mml:mi></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M41\" name=\"1752-0509-2-42-i36\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>y</mml:mi></mml:mstyle><mml:mo>≽</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>x</mml:mi></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M42\" name=\"1752-0509-2-42-i34\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>x</mml:mi></mml:mstyle><mml:mo>≽</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>y</mml:mi></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M43\" name=\"1752-0509-2-42-i37\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>y</mml:mi></mml:mstyle><mml:mo>≽</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>z</mml:mi></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M44\" name=\"1752-0509-2-42-i38\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>x</mml:mi></mml:mstyle><mml:mo>≽</mml:mo><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>z</mml:mi></mml:mstyle></mml:mrow></mml:semantics></mml:math></inline-formula>", "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M45\" name=\"1752-0509-2-42-i39\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable columnalign=\"left\">\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mtext>Birth</mml:mtext>\n <mml:mo>:</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:msup>\n <mml:mi mathvariant=\"script\">R</mml:mi>\n <mml:mo>+</mml:mo>\n </mml:msup>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>=</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo>{</mml:mo>\n <mml:msub>\n <mml:mi>R</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo>|</mml:mo>\n <mml:msub>\n <mml:mi>ν</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo>=</mml:mo>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mn>0</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>...</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>0</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mo>+</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>0</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>...</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>0</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>⇒</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>x</mml:mi>\n </mml:mstyle>\n <mml:mo>+</mml:mo>\n <mml:msub>\n <mml:mi>ν</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo>≽</mml:mo>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>x</mml:mi>\n </mml:mstyle>\n <mml:mo>}</mml:mo>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mtext>Death</mml:mtext>\n <mml:mo>:</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:msup>\n <mml:mi mathvariant=\"script\">R</mml:mi>\n <mml:mo>−</mml:mo>\n </mml:msup>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>=</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo>{</mml:mo>\n <mml:msub>\n <mml:mi>R</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo>|</mml:mo>\n <mml:msub>\n <mml:mi>ν</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo>=</mml:mo>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mn>0</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>...</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>0</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>0</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>...</mml:mn>\n <mml:mo>,</mml:mo>\n <mml:mn>0</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>⇒</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>x</mml:mi>\n </mml:mstyle>\n <mml:mo>+</mml:mo>\n <mml:msub>\n <mml:mi>ν</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo>≼</mml:mo>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>x</mml:mi>\n </mml:mstyle>\n <mml:mo>}</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<disp-formula>Φ<sub><italic>i </italic></sub>= <italic>k</italic><sub><italic>j</italic></sub><italic>x</italic><sub><italic>j</italic></sub>   for   <italic>R</italic><sub><italic>i </italic></sub>≡ <italic>X</italic><sub><italic>j </italic></sub>→ ∅</disp-formula>", "<disp-formula id=\"bmcM6\"><label>(6)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M46\" name=\"1752-0509-2-42-i40\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mtext>Monod positive feedback</mml:mtext>\n <mml:mo>:</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:mi>m</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi>x</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>=</mml:mo>\n <mml:mfrac>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:msup>\n <mml:mi>x</mml:mi>\n <mml:mi>α</mml:mi>\n </mml:msup>\n </mml:mrow>\n <mml:mrow>\n <mml:msup>\n <mml:mi>θ</mml:mi>\n <mml:mi>α</mml:mi>\n </mml:msup>\n <mml:mo>+</mml:mo>\n <mml:msup>\n <mml:mi>x</mml:mi>\n <mml:mi>α</mml:mi>\n </mml:msup>\n </mml:mrow>\n </mml:mfrac>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<disp-formula id=\"bmcM7\"><label>(7)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M47\" name=\"1752-0509-2-42-i41\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mtext>Hill negative feedback</mml:mtext>\n <mml:mo>:</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:mi>h</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi>x</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>=</mml:mo>\n <mml:mfrac>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:msup>\n <mml:mi>θ</mml:mi>\n <mml:mi>α</mml:mi>\n </mml:msup>\n </mml:mrow>\n <mml:mrow>\n <mml:msup>\n <mml:mi>θ</mml:mi>\n <mml:mi>α</mml:mi>\n </mml:msup>\n <mml:mo>+</mml:mo>\n <mml:msup>\n <mml:mi>x</mml:mi>\n <mml:mi>α</mml:mi>\n </mml:msup>\n </mml:mrow>\n </mml:mfrac>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M48\" name=\"1752-0509-2-42-i14\" overflow=\"scroll\"><mml:semantics><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mn>0</mml:mn><mml:mo>^</mml:mo></mml:mover></mml:mstyle></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M49\" name=\"1752-0509-2-42-i19\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>=</mml:mo><mml:mo>{</mml:mo><mml:mi mathvariant=\"script\">S</mml:mi><mml:mo>,</mml:mo><mml:mi mathvariant=\"script\">R</mml:mi><mml:mo>,</mml:mo><mml:mi>Φ</mml:mi><mml:mo>,</mml:mo><mml:mi>ν</mml:mi><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M50\" name=\"1752-0509-2-42-i42\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mover accent=\"true\"><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mo>=</mml:mo><mml:mo>{</mml:mo><mml:mi mathvariant=\"script\">S</mml:mi><mml:mo>,</mml:mo><mml:mi mathvariant=\"script\">R</mml:mi><mml:mo>,</mml:mo><mml:mover accent=\"true\"><mml:mi>Φ</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mo>,</mml:mo><mml:mi>ν</mml:mi><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M51\" name=\"1752-0509-2-42-i43\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>Φ</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M52\" name=\"1752-0509-2-42-i44\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M53\" name=\"1752-0509-2-42-i45\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi>π</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M54\" name=\"1752-0509-2-42-i44\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M55\" name=\"1752-0509-2-42-i18\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">N</mml:mi></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M56\" name=\"1752-0509-2-42-i44\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M57\" name=\"1752-0509-2-42-i45\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi>π</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<disp-formula id=\"bmcM8\"><label>(8)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M58\" name=\"1752-0509-2-42-i46\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mi>T</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>=</mml:mo>\n <mml:msub>\n <mml:mi mathvariant=\"script\">G</mml:mi>\n <mml:mrow>\n <mml:mtext>SSA</mml:mtext>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mover accent=\"true\">\n <mml:mi mathvariant=\"script\">N</mml:mi>\n <mml:mo>˜</mml:mo>\n </mml:mover>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:mi>T</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>~</mml:mo>\n <mml:mover accent=\"true\">\n <mml:mi>π</mml:mi>\n <mml:mo>˜</mml:mo>\n </mml:mover>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M59\" name=\"1752-0509-2-42-i47\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mover accent=\"true\"><mml:mi>ℜ</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mo>=</mml:mo><mml:mo>{</mml:mo><mml:msub><mml:mover accent=\"true\"><mml:mi>R</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mn>...</mml:mn><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M60\" name=\"1752-0509-2-42-i18\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">N</mml:mi></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M61\" name=\"1752-0509-2-42-i10\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">F</mml:mi></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M62\" name=\"1752-0509-2-42-i48\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mi mathvariant=\"fraktur\">M</mml:mi><mml:mo>=</mml:mo><mml:mo>{</mml:mo><mml:msub><mml:mi>M</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mn>...</mml:mn><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M63\" name=\"1752-0509-2-42-i49\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>M</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M64\" name=\"1752-0509-2-42-i50\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mi>T</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>=</mml:mo>\n <mml:mi mathvariant=\"script\">F</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mi>T</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>,</mml:mo>\n <mml:mi mathvariant=\"fraktur\">M</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>.</mml:mo>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M65\" name=\"1752-0509-2-42-i10\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">F</mml:mi></mml:semantics></mml:math></inline-formula>", "<disp-formula id=\"bmcM9\"><label>(9)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M66\" name=\"1752-0509-2-42-i51\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable columnalign=\"left\">\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>=</mml:mo>\n <mml:msub>\n <mml:mi>ϕ</mml:mi>\n <mml:mi mathvariant=\"script\">F</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo>=</mml:mo>\n <mml:mrow>\n <mml:mo>{</mml:mo>\n <mml:mrow>\n <mml:mtable columnalign=\"left\">\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>+</mml:mo>\n <mml:msub>\n <mml:mi>ν</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mtext>if </mml:mtext>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>&lt;</mml:mo>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mtext>otherwise</mml:mtext>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n </mml:mrow>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M67\" name=\"1752-0509-2-42-i52\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>Ψ</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mrow><mml:mi>k</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>D</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mrow><mml:mi>k</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>≡</mml:mo><mml:msub><mml:mi>Φ</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mrow><mml:mi>k</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>/</mml:mo><mml:msub><mml:mover accent=\"true\"><mml:mi>Φ</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>D</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mrow><mml:mi>k</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M68\" name=\"1752-0509-2-42-i53\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>ν</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M69\" name=\"1752-0509-2-42-i54\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>Φ</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M70\" name=\"1752-0509-2-42-i55\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>Φ</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M71\" name=\"1752-0509-2-42-i56\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>R</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M72\" name=\"1752-0509-2-42-i57\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi>ℜ</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M73\" name=\"1752-0509-2-42-i18\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">N</mml:mi></mml:semantics></mml:math></inline-formula>", "<disp-formula id=\"bmcM10\"><label>(10)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M74\" name=\"1752-0509-2-42-i58\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mi>T</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>,</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mi>T</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>=</mml:mo>\n <mml:mover accent=\"true\">\n <mml:mi mathvariant=\"script\">F</mml:mi>\n <mml:mo>^</mml:mo>\n </mml:mover>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msubsup>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mi>T</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>,</mml:mo>\n <mml:mi mathvariant=\"fraktur\">M</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<disp-formula id=\"bmcM11\"><label>(11)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M75\" name=\"1752-0509-2-42-i59\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable columnalign=\"left\">\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>=</mml:mo>\n <mml:msub>\n <mml:mi>ϕ</mml:mi>\n <mml:mover accent=\"true\">\n <mml:mi mathvariant=\"script\">F</mml:mi>\n <mml:mo>^</mml:mo>\n </mml:mover>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo>=</mml:mo>\n <mml:mrow>\n <mml:mo>{</mml:mo>\n <mml:mrow>\n <mml:mtable columnalign=\"left\">\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mtable columnalign=\"left\">\n <mml:mtr>\n <mml:mtd>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi>ϕ</mml:mi>\n <mml:mi mathvariant=\"script\">F</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>ϕ</mml:mi>\n <mml:mi mathvariant=\"script\">F</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr>\n <mml:mtd>\n <mml:mtext>if </mml:mtext>\n <mml:msub>\n <mml:mover accent=\"true\">\n <mml:mi>R</mml:mi>\n <mml:mo>˜</mml:mo>\n </mml:mover>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mtext> is monotone</mml:mtext>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mtable columnalign=\"left\">\n <mml:mtr>\n <mml:mtd>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi>ϕ</mml:mi>\n <mml:mi mathvariant=\"script\">F</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>ϕ</mml:mi>\n <mml:mi mathvariant=\"script\">F</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr>\n <mml:mtd>\n <mml:mtext>if </mml:mtext>\n <mml:msub>\n <mml:mover accent=\"true\">\n <mml:mi>R</mml:mi>\n <mml:mo>˜</mml:mo>\n </mml:mover>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mi>k</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mtext> is anti-monotone</mml:mtext>\n <mml:mo>,</mml:mo>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n </mml:mrow>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M76\" name=\"1752-0509-2-42-i60\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>ϕ</mml:mi><mml:mover accent=\"true\"><mml:mi mathvariant=\"script\">F</mml:mi><mml:mo>^</mml:mo></mml:mover></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M77\" name=\"1752-0509-2-42-i18\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">N</mml:mi></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M78\" name=\"1752-0509-2-42-i44\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M79\" name=\"1752-0509-2-42-i47\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mover accent=\"true\"><mml:mi>ℜ</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mo>=</mml:mo><mml:mo>{</mml:mo><mml:msub><mml:mover accent=\"true\"><mml:mi>R</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mn>...</mml:mn><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M80\" name=\"1752-0509-2-42-i48\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mi mathvariant=\"fraktur\">M</mml:mi><mml:mo>=</mml:mo><mml:mo>{</mml:mo><mml:msub><mml:mi>M</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mn>...</mml:mn><mml:mo>}</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M81\" name=\"1752-0509-2-42-i61\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mi>t</mml:mi></mml:msub><mml:mo>≽</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>Y</mml:mi></mml:mstyle><mml:mi>t</mml:mi></mml:msub><mml:mo>⇒</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mi>s</mml:mi></mml:msub><mml:mo>≽</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>Y</mml:mi></mml:mstyle><mml:mi>s</mml:mi></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M82\" name=\"1752-0509-2-42-i62\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>≽</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>Y</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M83\" name=\"1752-0509-2-42-i63\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>R</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M84\" name=\"1752-0509-2-42-i64\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi>i</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≤</mml:mo>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≤</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi>i</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≤</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>&lt;</mml:mo>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi>i</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>&lt;</mml:mo>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≤</mml:mo>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>.</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M85\" name=\"1752-0509-2-42-i65\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>ν</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M86\" name=\"1752-0509-2-42-i66\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>R</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>∈</mml:mo><mml:msup><mml:mi mathvariant=\"script\">R</mml:mi><mml:mo>+</mml:mo></mml:msup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M87\" name=\"1752-0509-2-42-i67\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>≽</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>≽</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>Y</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>Y</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M88\" name=\"1752-0509-2-42-i68\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>R</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>∈</mml:mo><mml:msup><mml:mi mathvariant=\"script\">R</mml:mi><mml:mo>−</mml:mo></mml:msup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M89\" name=\"1752-0509-2-42-i69\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>X</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ν</mml:mi><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>Y</mml:mi></mml:mstyle><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M90\" name=\"1752-0509-2-42-i63\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>R</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>", "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M91\" name=\"1752-0509-2-42-i70\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi>i</mml:mi>\n <mml:mi>v</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≤</mml:mo>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≤</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi>v</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≤</mml:mo>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>&lt;</mml:mo>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi>v</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd>\n <mml:mrow>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>&lt;</mml:mo>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>X</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>≤</mml:mo>\n <mml:msub>\n <mml:mi>Ψ</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>Y</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mstyle mathvariant=\"bold\" mathsize=\"normal\">\n <mml:mi>D</mml:mi>\n </mml:mstyle>\n <mml:mrow>\n <mml:msub>\n <mml:mi>t</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>.</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M92\" name=\"1752-0509-2-42-i66\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>R</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>∈</mml:mo><mml:msup><mml:mi mathvariant=\"script\">R</mml:mi><mml:mo>+</mml:mo></mml:msup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M93\" name=\"1752-0509-2-42-i68\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>R</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mrow><mml:msub><mml:mi>t</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mo>∈</mml:mo><mml:msup><mml:mi mathvariant=\"script\">R</mml:mi><mml:mo>−</mml:mo></mml:msup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M94\" name=\"1752-0509-2-42-i18\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"script\">N</mml:mi></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M95\" name=\"1752-0509-2-42-i44\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M96\" name=\"1752-0509-2-42-i45\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi>π</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M97\" name=\"1752-0509-2-42-i71\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>D</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mn>0</mml:mn><mml:mn>0</mml:mn></mml:msubsup><mml:mo>~</mml:mo><mml:mover accent=\"true\"><mml:mi>π</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mo>,</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>M</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mn>0</mml:mn><mml:mn>0</mml:mn></mml:msubsup><mml:mo>~</mml:mo><mml:mi>U</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>0</mml:mn><mml:mo>,</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M98\" name=\"1752-0509-2-42-i72\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>D</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mi>T</mml:mi><mml:mn>0</mml:mn></mml:msubsup><mml:mo>←</mml:mo><mml:mtext>Extend</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>D</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mn>0</mml:mn><mml:mn>0</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:mi>T</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M99\" name=\"1752-0509-2-42-i73\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>M</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mi>T</mml:mi><mml:mn>0</mml:mn></mml:msubsup><mml:mo>←</mml:mo><mml:mtext>GenerateMarks</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>M</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mn>0</mml:mn><mml:mn>0</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:mi>T</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M100\" name=\"1752-0509-2-42-i74\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>D</mml:mi></mml:mstyle><mml:mn>0</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow></mml:msubsup><mml:mo>,</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>M</mml:mi></mml:mstyle><mml:mn>0</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow></mml:msubsup><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>←</mml:mo><mml:mtext>Reverse</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>D</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mi>T</mml:mi><mml:mn>0</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>M</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mi>T</mml:mi><mml:mn>0</mml:mn></mml:msubsup><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M101\" name=\"1752-0509-2-42-i75\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>L</mml:mi></mml:mstyle><mml:mn>0</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow></mml:msubsup><mml:mo>,</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>U</mml:mi></mml:mstyle><mml:mn>0</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow></mml:msubsup><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>←</mml:mo><mml:mtext>Evolve</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>D</mml:mi></mml:mstyle><mml:mn>0</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow></mml:msubsup><mml:mo>,</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mi>M</mml:mi></mml:mstyle><mml:mn>0</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>T</mml:mi></mml:mrow></mml:msubsup><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M102\" name=\"1752-0509-2-42-i76\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>D</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mi>T</mml:mi><mml:mn>0</mml:mn></mml:msubsup><mml:mo>←</mml:mo><mml:mtext>Extend</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>D</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mrow><mml:mi>T</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:mrow><mml:mn>0</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:mi>T</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M103\" name=\"1752-0509-2-42-i77\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>M</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mi>T</mml:mi><mml:mn>0</mml:mn></mml:msubsup><mml:mo>←</mml:mo><mml:mtext>GenerateMarks</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>M</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mrow><mml:mi>T</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:mrow><mml:mn>0</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:mi>T</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M104\" name=\"1752-0509-2-42-i78\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>D</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mrow><mml:mi>T</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:mrow><mml:mn>0</mml:mn></mml:msubsup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M105\" name=\"1752-0509-2-42-i44\" overflow=\"scroll\"><mml:semantics><mml:mover accent=\"true\"><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>˜</mml:mo></mml:mover></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M106\" name=\"1752-0509-2-42-i79\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi mathvariant=\"script\">G</mml:mi><mml:mrow><mml:mi>S</mml:mi><mml:mi>S</mml:mi><mml:mi>A</mml:mi></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mover accent=\"true\"><mml:mi mathvariant=\"script\">N</mml:mi><mml:mo>˜</mml:mo></mml:mover><mml:mo>,</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>D</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mrow><mml:mi>T</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mi>T</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M107\" name=\"1752-0509-2-42-i78\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msubsup><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mi>D</mml:mi><mml:mo>⌣</mml:mo></mml:mover></mml:mstyle><mml:mrow><mml:mi>T</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:mrow><mml:mn>0</mml:mn></mml:msubsup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M108\" name=\"1752-0509-2-42-i14\" overflow=\"scroll\"><mml:semantics><mml:mstyle mathvariant=\"bold\" mathsize=\"normal\"><mml:mover accent=\"true\"><mml:mn>0</mml:mn><mml:mo>^</mml:mo></mml:mover></mml:mstyle></mml:semantics></mml:math></inline-formula>", "<disp-formula id=\"bmcM12\"><label>(12)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M109\" name=\"1752-0509-2-42-i80\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable columnalign=\"left\">\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo>∅</mml:mo>\n <mml:munderover>\n <mml:mo>→</mml:mo>\n <mml:mrow/>\n <mml:mi>k</mml:mi>\n </mml:munderover>\n <mml:mi>A</mml:mi>\n <mml:munderover>\n <mml:mo>→</mml:mo>\n <mml:mrow/>\n <mml:mn>1</mml:mn>\n </mml:munderover>\n <mml:mo>∅</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:msub>\n <mml:mover accent=\"true\">\n <mml:mi>P</mml:mi>\n <mml:mo>˙</mml:mo>\n </mml:mover>\n <mml:mi>j</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>=</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mi>k</mml:mi>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mrow>\n <mml:mi>j</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n <mml:mo>−</mml:mo>\n <mml:mi>k</mml:mi>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mi>j</mml:mi>\n </mml:msub>\n <mml:mo>+</mml:mo>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi>j</mml:mi>\n <mml:mo>+</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mrow>\n <mml:mi>j</mml:mi>\n <mml:mo>+</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n <mml:mo>−</mml:mo>\n <mml:mi>j</mml:mi>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mi>j</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>≡</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msup>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msup>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mi>k</mml:mi>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mi>j</mml:mi>\n </mml:msub>\n <mml:mo>+</mml:mo>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mi>j</mml:mi>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mi>j</mml:mi>\n </mml:msub>\n <mml:mo>.</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M110\" name=\"1752-0509-2-42-i81\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"double-struck\">E</mml:mi></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M111\" name=\"1752-0509-2-42-i82\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msup><mml:mi mathvariant=\"double-struck\">E</mml:mi><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M112\" name=\"1752-0509-2-42-i81\" overflow=\"scroll\"><mml:semantics><mml:mi mathvariant=\"double-struck\">E</mml:mi></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M113\" name=\"1752-0509-2-42-i82\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msup><mml:mi mathvariant=\"double-struck\">E</mml:mi><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M114\" name=\"1752-0509-2-42-i83\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>∊</mml:mi><mml:mi>E</mml:mi></mml:msub><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>T</mml:mi><mml:mi>s</mml:mi></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:mstyle displaystyle=\"true\"><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>j</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>∞</mml:mi></mml:msubsup><mml:mrow><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>π</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>P</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>T</mml:mi><mml:mi>s</mml:mi></mml:msub><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:msub><mml:mi>I</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:mstyle><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>2</mml:mn><mml:mi>k</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:msup><mml:mi>e</mml:mi><mml:mrow><mml:mo>−</mml:mo><mml:mn>2</mml:mn><mml:mi>k</mml:mi></mml:mrow></mml:msup><mml:mo>−</mml:mo><mml:mn>2</mml:mn><mml:msub><mml:mi>I</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>2</mml:mn><mml:mi>k</mml:mi><mml:msqrt><mml:mi>α</mml:mi></mml:msqrt><mml:mo stretchy=\"false\">)</mml:mo><mml:msup><mml:mi>e</mml:mi><mml:mrow><mml:mo>−</mml:mo><mml:mi>k</mml:mi><mml:mo>−</mml:mo><mml:mi>α</mml:mi></mml:mrow></mml:msup><mml:mo>+</mml:mo><mml:msub><mml:mi>I</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>2</mml:mn><mml:mi>k</mml:mi><mml:mi>α</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:msup><mml:mi>e</mml:mi><mml:mrow><mml:mo>−</mml:mo><mml:mn>2</mml:mn><mml:mi>α</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:semantics></mml:math></inline-formula>", "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M115\" name=\"1752-0509-2-42-i84\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mo>∅</mml:mo>\n <mml:mover>\n <mml:mo>→</mml:mo>\n <mml:mrow>\n <mml:mi>f</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mover>\n <mml:mi>A</mml:mi>\n <mml:mover>\n <mml:mo>→</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mover>\n <mml:mo>∅</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr>\n <mml:mtd> \n <mml:mo>⤪</mml:mo> \n </mml:mtd>\n </mml:mtr>\n <mml:mtr>\n <mml:mtd>\n <mml:mrow>\n <mml:mo>∅</mml:mo>\n <mml:munder>\n <mml:mo>→</mml:mo>\n <mml:mrow>\n <mml:mi>f</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>A</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:munder>\n <mml:mi>B</mml:mi>\n <mml:munder>\n <mml:mo>→</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:munder>\n <mml:mo>∅</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<disp-formula id=\"bmcM13\"><label>(13)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M116\" name=\"1752-0509-2-42-i85\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable columnalign=\"right\">\n <mml:mtr columnalign=\"right\">\n <mml:mtd columnalign=\"right\">\n <mml:mrow>\n <mml:msub>\n <mml:mover accent=\"true\">\n <mml:mi>P</mml:mi>\n <mml:mo>˙</mml:mo>\n </mml:mover>\n <mml:mrow>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>A</mml:mi>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>=</mml:mo>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msubsup>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mi>A</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mi>f</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>A</mml:mi>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"right\">\n <mml:mtd columnalign=\"right\">\n <mml:mrow>\n <mml:mo>+</mml:mo>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mi>A</mml:mi>\n </mml:msub>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>A</mml:mi>\n </mml:msub>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>A</mml:mi>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"right\">\n <mml:mtd columnalign=\"right\">\n <mml:mrow>\n <mml:mo>+</mml:mo>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msubsup>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mi>B</mml:mi>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mi>f</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>A</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>A</mml:mi>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"right\">\n <mml:mtd columnalign=\"right\">\n <mml:mrow>\n <mml:mo>+</mml:mo>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>A</mml:mi>\n </mml:msub>\n <mml:mo>,</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M117\" name=\"1752-0509-2-42-i86\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mi>f</mml:mi>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo>=</mml:mo>\n <mml:mi>γ</mml:mi>\n <mml:mo>+</mml:mo>\n <mml:mfrac>\n <mml:mrow>\n <mml:msub>\n <mml:mi>κ</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:msubsup>\n <mml:mi>n</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mn>4</mml:mn>\n </mml:msubsup>\n </mml:mrow>\n <mml:mrow>\n <mml:msub>\n <mml:mi>κ</mml:mi>\n <mml:mrow>\n <mml:mi>i</mml:mi>\n <mml:mn>0</mml:mn>\n </mml:mrow>\n </mml:msub>\n <mml:mo>+</mml:mo>\n <mml:msub>\n <mml:mi>κ</mml:mi>\n <mml:mrow>\n <mml:mi>i</mml:mi>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msub>\n <mml:msub>\n <mml:mi>n</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo>+</mml:mo>\n <mml:msub>\n <mml:mi>κ</mml:mi>\n <mml:mrow>\n <mml:mi>i</mml:mi>\n <mml:mn>2</mml:mn>\n </mml:mrow>\n </mml:msub>\n <mml:msubsup>\n <mml:mi>n</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mn>2</mml:mn>\n </mml:msubsup>\n <mml:mo>+</mml:mo>\n <mml:msub>\n <mml:mi>κ</mml:mi>\n <mml:mrow>\n <mml:mi>i</mml:mi>\n <mml:mn>3</mml:mn>\n </mml:mrow>\n </mml:msub>\n <mml:msubsup>\n <mml:mi>n</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mn>3</mml:mn>\n </mml:msubsup>\n <mml:mo>+</mml:mo>\n <mml:msubsup>\n <mml:mi>n</mml:mi>\n <mml:mi>i</mml:mi>\n <mml:mn>4</mml:mn>\n </mml:msubsup>\n </mml:mrow>\n </mml:mfrac>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<disp-formula id=\"bmcM14\"><label>(14)</label><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M118\" name=\"1752-0509-2-42-i87\" overflow=\"scroll\">\n <mml:semantics>\n <mml:mrow>\n <mml:mtable columnalign=\"left\">\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:msub>\n <mml:mover accent=\"true\">\n <mml:mi>P</mml:mi>\n <mml:mo>˙</mml:mo>\n </mml:mover>\n <mml:mi>j</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>=</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mstyle displaystyle=\"true\">\n <mml:munderover>\n <mml:mo>∑</mml:mo>\n <mml:mrow>\n <mml:mi>i</mml:mi>\n <mml:mo>=</mml:mo>\n <mml:mn>0</mml:mn>\n </mml:mrow>\n <mml:mi>n</mml:mi>\n </mml:munderover>\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msubsup>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n </mml:mrow>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mfrac>\n <mml:mrow>\n <mml:msub>\n <mml:mi>k</mml:mi>\n <mml:mi>M</mml:mi>\n </mml:msub>\n </mml:mrow>\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo>+</mml:mo>\n <mml:msubsup>\n <mml:mi>R</mml:mi>\n <mml:mrow>\n <mml:mi>i</mml:mi>\n <mml:mo>+</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n <mml:mi>α</mml:mi>\n </mml:msubsup>\n <mml:mo stretchy=\"false\">)</mml:mo>\n </mml:mrow>\n </mml:mfrac>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mi>j</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:mstyle>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>+</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:msub>\n <mml:mi>d</mml:mi>\n <mml:mi>M</mml:mi>\n </mml:msub>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mi>j</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>+</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msubsup>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>R</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n </mml:mrow>\n <mml:mrow>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n </mml:mrow>\n </mml:msubsup>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi>k</mml:mi>\n <mml:mi>B</mml:mi>\n </mml:msub>\n <mml:mo>+</mml:mo>\n <mml:msub>\n <mml:mi>k</mml:mi>\n <mml:mi>R</mml:mi>\n </mml:msub>\n <mml:msub>\n <mml:mi>M</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mi>j</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n <mml:mtr columnalign=\"left\">\n <mml:mtd columnalign=\"left\">\n <mml:mrow/>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mo>+</mml:mo>\n </mml:mtd>\n <mml:mtd columnalign=\"left\">\n <mml:mrow>\n <mml:mo stretchy=\"false\">(</mml:mo>\n <mml:msub>\n <mml:mi mathvariant=\"double-struck\">E</mml:mi>\n <mml:mrow>\n <mml:msub>\n <mml:mi>R</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n </mml:mrow>\n </mml:msub>\n <mml:mo>−</mml:mo>\n <mml:mn>1</mml:mn>\n <mml:mo stretchy=\"false\">)</mml:mo>\n <mml:msub>\n <mml:mi>R</mml:mi>\n <mml:mi>i</mml:mi>\n </mml:msub>\n <mml:msub>\n <mml:mi>P</mml:mi>\n <mml:mi>j</mml:mi>\n </mml:msub>\n <mml:mo>,</mml:mo>\n </mml:mrow>\n </mml:mtd>\n </mml:mtr>\n </mml:mtable>\n </mml:mrow>\n \n </mml:semantics>\n </mml:math></disp-formula>", "<inline-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M119\" name=\"1752-0509-2-42-i88\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:msub><mml:mi>P</mml:mi><mml:mrow><mml:msub><mml:mi>M</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:mo>,</mml:mo><mml:mn>...</mml:mn><mml:mo>,</mml:mo><mml:msub><mml:mi>M</mml:mi><mml:mi>n</mml:mi></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mi>R</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:mo>,</mml:mo><mml:mn>...</mml:mn><mml:mo>,</mml:mo><mml:msub><mml:mi>R</mml:mi><mml:mi>n</mml:mi></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:semantics></mml:math></inline-formula>" ]
[]
[]
[]
[]
[]
[]
[ "<graphic xlink:href=\"1752-0509-2-42-1\"/>", "<graphic xlink:href=\"1752-0509-2-42-2\"/>", "<graphic xlink:href=\"1752-0509-2-42-3\"/>", "<graphic xlink:href=\"1752-0509-2-42-4\"/>", "<graphic xlink:href=\"1752-0509-2-42-5\"/>" ]
[]
[{"surname": ["van Kampen"], "given-names": ["NG"], "source": ["Stochastic processes in physics and chemistry"], "year": ["1992"], "edition": ["2"], "publisher-name": ["Elsevier"]}, {"surname": ["Gillespie"], "given-names": ["DT"], "article-title": ["A general method for numerically simulating the stochastic time evolution of coupled chemical reactions"], "source": ["Journal of Computational Physics"], "year": ["1976"], "volume": ["22"], "fpage": ["403"], "lpage": ["434"]}, {"surname": ["Gillespie"], "given-names": ["DT"], "article-title": ["A rigorous derivation of the chemical master equation"], "source": ["Physica A"], "year": ["1992"], "volume": ["188"], "fpage": ["404"], "lpage": ["425"]}, {"surname": ["Norris"], "given-names": ["JR"], "source": ["Markov chains"], "year": ["1999"], "publisher-name": ["Cambridge University Press"]}, {"surname": ["Propp", "Wilson"], "given-names": ["JG", "DB"], "article-title": ["Exact sampling with coupled Markov chains and applications to statistical mechanics"], "source": ["Random structures and algorithms"], "year": ["1996"], "volume": ["9"], "fpage": ["223"], "lpage": ["252"]}, {"surname": ["Th\u00f6nnes", "Mecke KR, Stoyan D"], "given-names": ["E"], "article-title": ["A primer on perfect simulation"], "source": ["Springer Lecture Notes in Physics, Springer"], "year": ["2000"], "volume": ["554"], "fpage": ["349"], "lpage": ["378"]}, {"surname": ["Kendall", "M\u00f8ller"], "given-names": ["WS", "J"], "article-title": ["Perfect simulation using dominating processes on ordered spaces with application to locally stable point processes"], "source": ["Advances in Applied Probability"], "year": ["2000"], "volume": ["32"], "fpage": ["844"], "lpage": ["865"]}, {"surname": ["Fill", "Machida", "Murdoch", "Rosenthal"], "given-names": ["JA", "M", "DJ", "JS"], "article-title": ["Extension of Fill's perfect rejection sampling algorithm to general chains"], "source": ["Random Structures and Algorithms"], "year": ["2000"], "volume": ["9"], "fpage": ["223"], "lpage": ["252"]}, {"surname": ["Lindvall"], "given-names": ["T"], "source": ["Lectures on the coupling method"], "year": ["1992"], "publisher-name": ["John Wiley & Sons, Inc"]}, {"surname": ["Doob"], "given-names": ["JL"], "article-title": ["Markoff chains \u2013 Denumerable case"], "source": ["Transactions of the American Mathematical Society"], "year": ["1945"], "volume": ["58"], "fpage": ["455"], "lpage": ["473"]}, {"surname": ["Rao", "Arkin"], "given-names": ["CV", "AP"], "article-title": ["Stochastic chemical kinetics and the quasi steady-state assumption: Applications to the Gillespie algorithm"], "source": ["Journal of Chemical Physics"], "year": ["2003"], "volume": ["118"], "fpage": ["4999"], "lpage": ["5011"]}, {"surname": ["H\u00e4ggstr\u00f6m", "Nelander"], "given-names": ["O", "K"], "article-title": ["Exact sampling from anti-monotone systems"], "source": ["Statistica Neerlandica"], "year": ["1998"], "volume": ["52"], "fpage": ["360"], "lpage": ["380"]}, {"surname": ["Mitzenmacher", "Upfal"], "given-names": ["M", "E"], "source": ["Probability and computing: randomized algorithms and probabilistic analysis"], "year": ["2005"], "publisher-name": ["Cambridge University Press"]}]
{ "acronym": [], "definition": [] }
26
CC BY
no
2022-01-12 14:47:35
BMC Syst Biol. 2008 May 8; 2:42
oa_package/0e/6c/PMC2529164.tar.gz
PMC2529258
18667094
[ "<title>Background</title>", "<p>Maternal health is one of the main global health challenges and reducing the maternal mortality ratio by three-quarters by 2015 is the target for the fifth Millennium Development Goal. However, this goal is the one towards which the least progress has been made [##REF##16195261##1##]. Of all maternal deaths, those related to unsafe abortions are the most severely underestimated and yet at the same time also the most easily preventable. The case fatality rate associated with unsafe abortion is estimated to be 367 deaths per 100,000 unsafe abortions, which is hundreds of times higher than that for safe and legal abortions in developed nations [##REF##17126724##2##].</p>", "<p>In spite of the compelling logic of preventing future unplanned pregnancies by providing postabortion contraceptive services to women admitted with abortion complications, this component remains one of the weakest parts of postabortion care [##UREF##0##3##,##UREF##1##4##]. This irrespective of important evidence from Burkina Faso [##UREF##2##5##], El Salvador [##REF##14636938##6##], Kenya [##REF##10216893##7##], Mexico [##REF##14519587##8##, ####UREF##3##9##, ##UREF##4##10####4##10##], Peru [##UREF##5##11##], Russia [##UREF##6##12##], and Senegal [##UREF##7##13##] demonstrating that it is possible to increase contraceptive acceptance among women who have experienced abortion. One of the concerns in postabortion contraceptive service delivery is women's medium- and long-term adherence. A Zimbabwean study has revealed that women who received ward-based postabortion contraceptive services had higher contraceptive use for at least one year after hospital discharge, as well as fewer unplanned pregnancies and abortions [##REF##12132639##14##]. In addition, it has been documented that postabortion contraceptive service delivery involving training in counseling skills increases contraceptive use up to 12 months after abortion [##UREF##6##12##]. The two mentioned studies did, however, not make any distinctions between women having experienced an unsafe abortion and women having experienced a spontaneous abortion. This in spite of the fact that these two groups of women may be expected to have quite different reproductive priorities and concerns which may affect their contraceptive acceptance as well as their contraceptive adherence.</p>", "<p>To better understand the acceptance of postabortion contraceptive services, the present study focuses on women having experienced an unsafely induced abortion and describes these women's long-term contraceptive adherence and their experienced barriers.</p>" ]
[ "<title>Methods</title>", "<title>The quantitative study</title>", "<p>The study was conducted in Tanzania, which is a country where abortion is only legally available if the pregnancy is a threat to the woman's life [##UREF##8##15##]. Data were collected among women who attended Temeke Municipal Hospital (TMH) in Dar es Salaam with an abortion-related diagnosis in the period July 2001 to July 2002. The study population is presented in Figure ##FIG##0##1##, 824 women were diagnosed as having abortion complications and 760 of these women were approached and interviewed by using the previously described empathetic approach [##REF##10897196##16##]. According to this approach, a woman is classified as having had an unsafe abortion if she, through an empathic dialogue, reveals that she has had an unwanted pregnancy, which she had terminated by an abortion. In all 392 women (52 percent) were classified as having had unsafe abortions and 347 of these women accepted contraceptive services and were thus included in the study.</p>", "<title>Contraceptive adherence</title>", "<p>The women were counselled in detail about contraception and how to avoid becoming infected with HIV and thereafter offered ward-based contraceptive service. Follow-up data were obtained 12 months after the women had had the abortion by a combination of hospital-based and home-based interviews [##REF##15144340##17##]. In relation to the 12 months' interviews detailed information about the women's present contraceptive use, their possible motives for not using contraception, and pregnancies experienced during the observation period was obtained.</p>", "<title>Follow-up</title>", "<p>The women eligible for follow-up comprised 325 women, 191 of these women (59 percent) contributed with follow-up information; 90 returned spontaneously to the hospital for follow-up, whereas 101 were visited at home and interviewed. Among the 134 women who were lost to follow-up, 38 refused to be visited at home. Attempts were made to make home visits to the remaining 96 women among whom 32 had provided an unspecific address, 64 had, according to information from their neighbours or relatives, moved or were away from home for a longer period due to either work or family obligations.</p>", "<title>Statistics</title>", "<p>Data were entered twice by two different operators using the software Epi Info version 6.04 from Epidemiology and Disease surveillance from the CDC, Atlanta, USA. The two sets were compared and the questionable entries were reconciled. Statistical analyses were carried out by the Statistical Package for the Social Sciences (SPSS, version 12.0).</p>", "<title>The qualitative study</title>", "<p>The qualitative part of the study was designed to shed light on the women's concerns related to contraceptive use, their possible motives for not using contraception, their perceived HIV risk and their motives for using or not using condoms.</p>", "<p>In-depth interviews were carried out 6–12 months after discharge with 10 married women and 10 single women. A thematically structured interview guideline with open-ended questions was developed. The interviewer used the guideline questions to focus the discussion, but was encouraged to probe respondents whenever it was found relevant. The interviews took 30–40 minutes and were carried out in Kiswahili. All interviews were tape recorded, transcribed and later translated into English.</p>", "<p>The strategy used to classify and evaluate the data was predetermined by the guideline; e.g. contraceptive use, motives for not using contraception, perceived HIV risk and condom use. From the transcribed conversations these themes were listed together and all data that related to the classified themes were identified. In the next step of the analyses the identified themes were combined and catalogued according to the women's marital situation.</p>", "<title>Ethics</title>", "<p>All the women were informed that participation in the study was voluntary and that it would have no consequences for their further treatment whether they participated or not. Informed consent was obtained. All women who participated in the study were asked whether they would accept to be visited at home and only women who accepted this were attempted visited. Further, in relation to the home visits the women's abortion procedure was kept strictly confidential, the interviews only focused on the women's contraceptive use and no references were made to the fact that they had had an unsafe abortion. The study was ethically approved by the National Institute of Medical Research in Tanzania and by the Scientific-Ethical Committee at Karolinska Institute in Sweden.</p>" ]
[ "<title>Results</title>", "<title>The quantitative study</title>", "<p>The demographic characteristics of the cohort are presented in table ##TAB##0##1##. The majority (55 percent) of the women were below 25 years and many were single (50 percent). Women aged 19 years or below were more likely to be lost to follow-up. Likewise, less follow-up information was obtained among single women and women who had not given birth previously.</p>", "<title>Contraceptive acceptance</title>", "<p>The 392 women who had experienced an unsafe abortion were offered contraceptive counselling and service, 347 (89 percent) accepted the service offered and left the ward with a contraceptive method according to their own choice.</p>", "<title>Contraceptive use before and after the abortion</title>", "<p>Contraceptive use before the abortion and 12 months after the abortion was assessed among women who accepted postabortion contraceptive services (table ##TAB##1##2##). Before the abortion, 18 percent of the married women had ever used hormonal contraception, that be oral contraception or medroxyprogesteron injections, the same applied for 14 percent of the single women. Twelve months after the abortion, the proportion of women who stated they were using oral contraception or medroxyprogesteron injections had increased significantly among both married and single women. Hence, 77 percent of the married women and 84 percent of the single women stated they were using hormonal contraception when interviewed 12 months after the abortion</p>", "<title>Condom use</title>", "<p>In relation to the postabortion contraceptive service offered, it was stressed that condom use also was a means to avoid STIs/HIV infections. As indicated in table ##TAB##2##3##, single women were more likely to follow this advice. Hence among the single women, a significantly higher proportion stated they had used condoms more than half of the times they had had intercourse when interviewed 12 months after the abortion in comparison with before the abortion, whereas no significant difference in condom use was found among married women.</p>", "<title>The qualitative study</title>", "<p>To get more detailed information about the women's priorities and concerns in relation to their contraceptive use a number of in-depth interviews were performed among single and married women 6–12 months after their abortion. The findings from the in-depth interviews were in accordance with the survey results; hence the majority of the women had been using contraception after they had been discharged from the hospital. The in-depths interviews did, however, also reveal that many women for various reasons found it difficult to find the ideal method and found it difficult to use the method consistently.</p>", "<title>Barriers in relation to contraceptive use among married women</title>", "<p>Focusing on the married women, they were often concerned about their economical situation and how expensive it was to raise a child. Many felt they already had the number of children they were able to take care of and therefore used contraception to avoid a repeated unwanted pregnancy. However, they were often caught between a wish to limit their family size and fear of real or imagined side effects and were trying to navigate between these two conflicting concerns. As Theresa (46 years) explained:</p>", "<p>Life is very hard nowadays so it is very important to join the family planning program. I have previously tried other methods, once I used the pills and suffered from loss of appetite. Then I had the IUD inserted and suffered from severe abdominal pain so I had it removed ......Immediately after the abortion I was given the injection and I am now on my second injection.</p>", "<p>Another concern in relation to contraceptive use among married women was the problem of transportation fee; the women did, therefore, not always return in time for new supplies and were instead relying on less effective methods such as abstinence or withdrawal. This situation is illustrated by Rita (30) who had six children:</p>", "<p>I have come a bit late as we have transport problems, especially now with the rain. I am also living very fare from the hospital. I have to take more than one bus and the fare can come to 1000 TSH (0.86 USD). I am one month late but my husband and I have been very careful regarding this issue and we have been abstaining till now. Today when we got the fares we decided that I had to come.</p>", "<p>A number of married women were quite determined to limit their family size and opted for highly effective contraceptive methods such as e.g. sterilization. This was the situation for Grace, a 30 year old married woman, who had seven children. When Grace was discharged from the hospital, she was offered postabortion contraceptive service. She decided to use medroxyprogesteron injection and returned to the hospital after 3 months to have the injection renewed. She did, however, not show up for the six and nine months follow-up. A home based interview was performed 12 months after the abortion and Grace explained the following about her contraceptive use:</p>", "<p>I had financial problems [and did therefore not return to the hospital for new supplies] and right now we are not using any contraception instead my husband is practicing withdrawal. We have discussed that I latter will have to have a sterilization operation performed.</p>", "<title>Barriers in relation to contraceptive use among single women</title>", "<p>The single women had, not surprisingly, other priorities and concerns in relation to contraceptives than married women. Their main priority was to postpone childbirth until either they were married or until they had finished their education. Some of the girls were rather independent of their partner and felt they themselves had the decision making power both regarding the abortion and whether they should use a contraceptive method after the abortion. Crystle was a 16 year old single girl and her boyfriend was a couple of years older. She explained:</p>", "<p>When I experienced I was pregnant I told my boyfriend that I wanted to have the pregnancy terminated. He was against it but I decided to go ahead. My sister in law and my boyfriend's father gave me money. My boyfriend was annoyed and he blamed his father a lot.... I am not seeing him anymore. I want to work until I am 28 then I can get married.</p>", "<p>It was, however, not all single women who had the same knowledge and the same decision making power as Crystle. Some of the single women, who were sexually active, had no intention of becoming pregnant and did not use any contraception. These women were particularly at risk of experiencing a repeated unwanted pregnancy. For instance, Annie, who was 16 years and admitted to the hospital with complications from her second unsafe abortion, said about her contraceptive use:</p>", "<p>After having had the first abortion I was informed about different contraceptive methods ... I decided to use the pills .... When I came home from the hospital I talked with my sister and she said that it was not yet time for me to use the pills and she took them. Both my boyfriend and my sister advised me not to use contraception. Even myself I do not want to use pills because I might not be able to become pregnant later if I use the pills.</p>", "<title>Perceived HIV risk and condom use among married women</title>", "<p>Many women explained that they found it difficult to introduce the condom in their relationship. They were particularly afraid that raising the issue would create uncertainty about whether the couple was being faithful to each other. Hence, suggesting condom use would be interpreted by the partner as his wife suspecting that he was being unfaithful and perhaps infected either with an STI or HIV. As stressed by Claire (30) who had been married to her husband for five years:</p>", "<p>After coming back from the hospital I informed my husband about what the nurses had said and he accepted that it would be a good idea if I used the pills. I was also advised to use the condom, but I am sure my husband does not want to hear about it. If I tell him, he will ask whether I think he has a disease.</p>", "<p>Further, many of the married women trusted their partners and saw no reason to use condoms as they did not consider themselves at risk of becoming infected with HIV. Rita was 40 years old and had six children. She used injections to avoid experiencing another unwanted pregnancy, and she explained the following:</p>", "<p>I think the implant could be good to use. The pills are having side effects and are difficult to remember to take, whereas I have never used the condom. I see no use in using condoms as I am only sleeping with my husband. He too is not in favour of using the condoms since I am using injection. When asked about the risk of becoming infected with HIV, Rita said: I believe he has no other partner. It is good to trust each other and we do have this trust that is why we are not using condoms.</p>", "<title>Perceived HIV risk and condom use among single women</title>", "<p>The single women were generally more open-minded towards condom use, both at the time of enrolment in the study and at the 12 months follow-up. Especially the young girls seemed to be in favour of condoms; used either alone or in combination with hormonal contraception. Judith, for example, who was 20 years old, was using both condoms and oral contraceptives, but had only informed her partner about the condom:</p>", "<p>After the abortion I was told that the pills were for prevention of pregnancy and the condoms were for prevention of sexually transmitted diseases such as syphilis and HIV. After I left the hospital I told my lover that we had to use the condoms I had got at the hospital and he agreed. He is not aware that I am also using the pill. I think it is best not to tell him as it will be of no help to him. If I tell him, he might be against me using them.</p>", "<p>However, the majority of the single girls did not use condoms regularly and their motives for this differed. Some trusted their partners, others had introduced the condom but their partners were against using it and still others thought that the condom was mainly to be used at the beginning of a relationship, as for instance Maria (aged 20). Maria was single and had one daughter aged 4. Her relationship with the man, who was responsible for the pregnancy, had ended after the abortion. She now had a new partner, who she considered a fiancé. The importance of double protection in relation to inclusion in the study had been stressed to Maria and she had chosen to use Medroxyprogesteron injection and the condom. She was still using the injections but when asked about condom use Maria said:</p>", "<p>My relationship with the man, who had caused the pregnancy, stopped after the abortion. I am now having another boyfriend who is my fiancé; he went to my home town and introduced himself. I used the condom the first time we were having sex but after he has introduced himself to my family I have stopped using condoms with him.</p>" ]
[ "<title>Discussion</title>", "<p>The vast majority of the postabortion women accepted postabortion contraceptive services and stated they were using contraception 12 months after the abortion. Follow-up information, however, was only achieved among 59 percent of the included women and the long-term contraceptive adherence among the 41 percent of the women who were lost to follow-up is unknown. The fact that abortion is illegal in the studied setting is likely to have contributed to the high drop-out rate. Further, whether illegal or not, an induced abortion is often considered a traumatic or even stigmatising event, which women would like to avoid being confronted with at a later stage in their life – a fact that is likely also to have had a negative impact on the follow-up rate. Similar problems with high drop-out rates have been experienced in other studies focusing on the long-term impact of postabortion care [##REF##12132639##14##].</p>", "<p>The high drop-out rate experienced in the present study raises the question of the extent to which the women who contributed with follow-up data differ systematically from those who did not. Loss to follow-up was more pronounced among young, unmarried women who had not given birth previously. In Tanzania, as well as in many other low income societies, premarital sex is considered immoral [##REF##11352408##18##], and for fear of their abortion experience being disclosed, young or single women may thus have been more likely to provide wrong addresses to avoid a home visit, even though they accepted such a visit when they were enrolled in the study. In addition to the abortion related stigma, a high migration rate is likely also to have had a negative impact on the proportion of women who contributed with follow-up data. In Temeke Municipal, many settle in unplanned squatter areas with social problems and are likely to move when a better housing possibility becomes available, a situation which is reflected in a high yearly immigration rate of 25 percent [##REF##11914327##19##]. This situation that is likely to be more pronounced among young and single women, who have not yet established themselves with their own family, may also partly explain the low follow-up rate achieved.</p>", "<p>The findings form the present study, although hampered by a high drop-out rate, adds to the evidence that women having experienced unsafe abortions are likely to accept and use contraception. One explanation behind the high contraceptive prevalence rate found in this study may be that the service was offered as a ward based activity by well trained counsellors. An assumption which is supported by a Kenyan study where postabortion contraceptive service was accepted by 75 percent when it was on the ward, while only 41 percent obtained a method when asked to visit a separate site within the same hospital after discharge [##REF##10216893##7##]. In the present study, the women were further asked to return to the hospital every 3 month to discuss their contraceptive use and have experienced problems addressed. This approach may additionally have had a positive impact on the women's contraceptive adherence. Hence, women who have experienced an induced abortion may be reluctant to use contraception due to lack of detailed knowledge about contraception and fear of experienced or imagined side effects [##REF##17581034##20##,##REF##11424268##21##]. Frequent counselling that addresses the women's concerns may therefore increase the women's contraceptive adherence. Whether the approach of inviting the women to return to the hospital for ongoing counselling and service is the most appropriate way to strengthen the women's ongoing contraceptive use can be discussed. Hence, at a programmatic level, the cost-effectiveness of such an approach may be questioned and it may be more relevant to refer the women to a primary health facility.</p>", "<p>Postabortion contraceptive services are apparently well accepted by women who are admitted with complications after an unsafe abortion and should thus be considered an important means in addressing the problem of unsafe abortions. However, while hospitals incur only minor additional costs by introducing postabortion contraception, few have done so successfully. To begin with, contraceptive services and incomplete abortion treatment are typically delivered and administered separately – in different areas of one building, in separate buildings, or even within different institutions. In addition to separately administered services, lack of appropriate training, time, and institutional support can make physicians and nurses in the wards where patients receive postabortion care reluctant or unable to take on the duty of providing contraceptive services. To ensure postabortion contraceptive services are being offered in a comprehensive and sustainable way the following requirements should be addressed: qualified contraceptive counsellors should be present in the gynaecological ward; the most commonly used contraceptive methods (oral contraceptives, injections and condoms) should be made available in the ward and; the service should be monitored and evaluated through a simple routine supervision system.</p>", "<p>High HIV prevalence rates, ranging from 19 percent to 25 percent, have been found among Tanzanian single women who have experienced an unsafe abortion [##REF##16640622##22##]. These figures are considerable higher than sentinel surveillance results reporting HIV prevalence rates of 10–14 percent among pregnant women in Temeke Municipal. Therefore, in Tanzania as well as in other areas with high HIV prevalence rates, counselling about HIV and condom use should be considered an essential aspect of postabortion care. The present study documents that such counselling may lead to an increased number of single women practising safe sex. It has been documented that the vast majority of Tanzanian women are aware of risky behaviour and the protective role of abstinence, faithfulness to one uninfected partner and condom use [##UREF##9##23##]. However, as the present study shows, they face a number of obstacles in translating their knowledge into safer sex practices. Both women and men associate condoms with promiscuity, STIs and HIV. Suggesting condom use may, therefore, imply either that one has a sexually transmitted disease or that one mistrusts one's partner [##REF##16395947##24##]. This association of condom use with promiscuity undermines the use of condoms as an HIV-preventive measure, especially among the married women. These difficulties related to condom use should be acknowledged when counselling married women on contraceptive use. Especially, since marriage has been found to be a significant risk factor for HIV infection in Tanzania as well as in other sub-Saharan African countries [##REF##17445264##25##].</p>" ]
[ "<title>Conclusion</title>", "<p>Legal reforms ensuring women easy access to safe legal abortions is the primary option for addressing the tragic toll of unsafe abortion. However, this is not likely to take place in a foreseeable future in many low-income coutries; meanwhile there is a need for comprehensive postabortion care programs that ensure women prompt access to high-quality postabortion care. In that relation it should be acknowledged that postabortion women are likely to accept and use contraception when the service is offered as an integrated part of postabortion care.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Postabortion contraceptive service is considered an effective means in addressing the problem of unsafe abortion; in spite this fact this component remains one of the weakest parts of postabortion care. In this context, the paper aims to describe the impact of a postabortion contraceptive service intervention among women admitted with complications from unsafe abortions and to explore the women's long-term contraceptive adherence.</p>", "<title>Methods</title>", "<p>392 women having experienced unsafe abortion were identified by an empathetic approach and offered postabortion contraceptive service, which included counselling on HIV and condom use. Questionnaire interviews about contraceptive use were conducted at the time of inclusion and 12 months after the abortion. Additionally, in-depth interviews were performed 6–12 months after the abortion.</p>", "<title>Results</title>", "<p>Eighty-nine percent of the women accepted postabortion contraception. Follow-up information was obtained 12 months after the abortion among 59 percent of the women. Among these, 79 percent of the married women and 84 percent of the single women stated they were using contraception at 12 months. Condom use among the single women increased significantly during the 12 months follow up.</p>", "<title>Conclusion</title>", "<p>Postabortion contraceptive services appear to be well accepted by women who are admitted with complications after an unsafe abortion and should thus be recognized as an important means in addressing the problem of unsafe abortion. In addition, counselling about HIV and condom use should be considered an essential aspect of postabortion care.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>VR designed the study, performed the analyses, and led the writing. FY and SM assisted in designing the study and supervised the implementation of the study. All authors contributed to the interpretation of the findings and reviewed drafts of the paper.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2393/8/32/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>The study was supported by grants from RUF (Council for Development Research) within Danida (Danish International Development Agency) and from SAREC (Department for Development Research Cooperation) within Sida (Swedish International Development Cooperation Agency).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Study population, Women attending Temeke Municipal Hospital with unsafe abortions.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Demographic characteristics among women who had unsafe abortions and follow-up information achieved at 12 months</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\">Study cohort</td><td align=\"center\" colspan=\"3\">Follow-up information</td></tr><tr><td/><td colspan=\"5\"><hr/></td></tr><tr><td/><td align=\"center\"><break/><break/>N = 325</td><td align=\"center\"><break/><break/>%</td><td align=\"center\">Achieved <break/>N = 191<break/>%</td><td align=\"center\">Not achieved <break/>N = 134<break/>%</td><td align=\"center\">P value<break/><break/></td></tr></thead><tbody><tr><td align=\"left\">Age</td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> -19 years</td><td align=\"center\">70</td><td align=\"center\">22</td><td align=\"center\">18</td><td align=\"center\">27</td><td align=\"center\">0.09</td></tr><tr><td align=\"left\"> 20–24 years</td><td align=\"center\">108</td><td align=\"center\">33</td><td align=\"center\">32</td><td align=\"center\">35</td><td/></tr><tr><td align=\"left\"> 25–30 years</td><td align=\"center\">79</td><td align=\"center\">24</td><td align=\"center\">26</td><td align=\"center\">22</td><td/></tr><tr><td align=\"left\"> 30+ years</td><td align=\"center\">68</td><td align=\"center\">21</td><td align=\"center\">25</td><td align=\"center\">16</td><td/></tr><tr><td align=\"left\">Marital situation</td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Married</td><td align=\"center\">161</td><td align=\"center\">50</td><td align=\"center\">58</td><td align=\"center\">38</td><td align=\"center\">0.003</td></tr><tr><td align=\"left\"> Single</td><td align=\"center\">145</td><td align=\"center\">45</td><td align=\"center\">38</td><td align=\"center\">54</td><td/></tr><tr><td align=\"left\"> Divorced/wid.</td><td align=\"center\">18</td><td align=\"center\">6</td><td align=\"center\">4</td><td align=\"center\">8</td><td/></tr><tr><td align=\"left\">Education</td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> ≤ Std 4</td><td align=\"center\">46</td><td align=\"center\">14</td><td align=\"center\">12</td><td align=\"center\">17</td><td align=\"center\">0.305</td></tr><tr><td align=\"left\"> Std 5–7</td><td align=\"center\">239</td><td align=\"center\">74</td><td align=\"center\">77</td><td align=\"center\">69</td><td/></tr><tr><td align=\"left\"> ≥ Form 1</td><td align=\"center\">39</td><td align=\"center\">12</td><td align=\"center\">11</td><td align=\"center\">13</td><td/></tr><tr><td align=\"left\">Previous births</td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> 0</td><td align=\"center\">108</td><td align=\"center\">33</td><td align=\"center\">2</td><td align=\"center\">6</td><td align=\"center\">0.003</td></tr><tr><td align=\"left\"> 1</td><td align=\"center\">73</td><td align=\"center\">23</td><td align=\"center\">34</td><td align=\"center\">45</td><td/></tr><tr><td align=\"left\"> 2</td><td align=\"center\">61</td><td align=\"center\">19</td><td align=\"center\">27</td><td align=\"center\">27</td><td/></tr><tr><td align=\"left\"> 3+</td><td align=\"center\">82</td><td align=\"center\">25</td><td align=\"center\">36</td><td align=\"center\">22</td><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Contraceptive practice before and after the abortion among married and single women</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"5\">Married</td><td align=\"center\" colspan=\"5\">Single</td></tr><tr><td/><td colspan=\"10\"><hr/></td></tr><tr><td/><td align=\"center\" colspan=\"2\">Pre<break/>Abortion<break/>N = 110 %</td><td align=\"center\" colspan=\"2\">Post<break/>abortion*<break/>N = 110 %</td><td align=\"left\">P value<break/><break/></td><td align=\"center\" colspan=\"2\">Pre<break/>Abortion<break/>N = 81 %</td><td align=\"center\" colspan=\"2\">Post<break/>abortion*<break/>N = 81 %</td><td align=\"left\">P value<break/><break/></td></tr></thead><tbody><tr><td align=\"left\">Condom</td><td align=\"right\">12</td><td align=\"right\">11</td><td align=\"right\">11</td><td align=\"right\">10</td><td align=\"left\">NS</td><td align=\"right\">26</td><td align=\"right\">32</td><td align=\"right\">23</td><td align=\"right\">28</td><td align=\"left\">NS</td></tr><tr><td align=\"left\">Oral contracep</td><td align=\"right\">14</td><td align=\"right\">13</td><td align=\"right\">45</td><td align=\"right\">41</td><td align=\"left\">&lt; 0.000</td><td align=\"right\">9</td><td align=\"right\">11</td><td align=\"right\">38</td><td align=\"right\">48</td><td align=\"left\">&lt; 0.000</td></tr><tr><td align=\"left\">Medroxyprogesteron inj.</td><td align=\"right\">5</td><td align=\"right\">5</td><td align=\"right\">39</td><td align=\"right\">36</td><td align=\"left\">&lt; 0.000</td><td align=\"right\">3</td><td align=\"right\">3</td><td align=\"right\">29</td><td align=\"right\">36</td><td align=\"left\">&lt; 0.000</td></tr><tr><td align=\"left\">No use</td><td align=\"right\">79</td><td align=\"right\">72</td><td align=\"right\">23</td><td align=\"right\">21</td><td align=\"left\">&lt; 0.000</td><td align=\"right\">43</td><td align=\"right\">53</td><td align=\"right\">13</td><td align=\"right\">16</td><td align=\"left\">&lt; 0.000</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Condom use last month among married and single women</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"5\">Married</td><td align=\"center\" colspan=\"5\">Single</td></tr><tr><td/><td colspan=\"10\"><hr/></td></tr><tr><td/><td align=\"center\" colspan=\"2\">Pre<break/>Abortion<break/>N = 110 %</td><td align=\"center\" colspan=\"2\">Post <break/>abortion*<break/>N = 110 %</td><td align=\"left\">P value<break/><break/></td><td align=\"center\" colspan=\"2\">Pre<break/>Abortion<break/>N = 81 %</td><td align=\"center\" colspan=\"2\">Post<break/>abortion*<break/>N = 81 %</td><td align=\"left\">P value<break/><break/></td></tr></thead><tbody><tr><td align=\"left\">&gt; 1/2 the times</td><td align=\"right\">4</td><td align=\"right\">4</td><td align=\"right\">8</td><td align=\"right\">7</td><td align=\"left\">NS</td><td align=\"right\">8</td><td align=\"right\">10</td><td align=\"right\">18</td><td align=\"right\">23</td><td align=\"left\">&lt;0.05</td></tr><tr><td align=\"left\">1/2 the times</td><td align=\"right\">3</td><td align=\"right\">3</td><td align=\"right\">2</td><td align=\"right\">2</td><td align=\"left\">NS</td><td align=\"right\">12</td><td align=\"right\">15</td><td align=\"right\">3</td><td align=\"right\">4</td><td align=\"left\">&lt;0.05</td></tr><tr><td align=\"left\">&lt; 1/2 the times</td><td align=\"right\">103</td><td align=\"right\">94</td><td align=\"right\">99</td><td align=\"right\">91</td><td align=\"left\">NS</td><td align=\"right\">61</td><td align=\"right\">75</td><td align=\"right\">59</td><td align=\"right\">74</td><td align=\"left\">NS</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><p>* Adds more than 100% since some women used both condom and hormonal contraception</p></table-wrap-foot>", "<table-wrap-foot><p>*Information about condom use were missing among 1 married and 1 single women</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1471-2393-8-32-1\"/>" ]
[]
[{"surname": ["Cobb", "Putney", "Rochat", "Solo", "al."], "given-names": ["L", "P", "R", "J"], "source": ["Global Evaluation of USAID's Postabortion Care Program"], "year": ["2001"]}, {"surname": ["V", "D", "E.O."], "given-names": ["R", "H", "A"], "source": ["Report of the Postabortion Care Technical Advisory Panel"], "year": ["2007"], "publisher-name": ["USAID"]}, {"surname": ["Ministry of Health"], "given-names": ["BF"], "source": ["Introduction of Emergency Medical Treatment and Family Planning Services for Women with Complications from Abortion in Burkina Faso"], "year": ["1998"], "publisher-name": ["Nairobi, Population Council"]}, {"surname": ["Langer", "Heimburger", "Garc\u00eda-Barrios", "Winikoff", "Haberland N and Measham D"], "given-names": ["A", "A", "C", "B"], "article-title": ["Improving Postabortion Care in a Public Hospital in Mexico"], "source": ["Responding to Cairo: Case Studies of Changing Practice in Reproductive Health and Family Planning,"], "year": ["2002"], "publisher-name": ["New York, Population Council."], "fpage": ["236"], "lpage": ["256"]}, {"surname": ["Langer", "Garc\u00eda-Barrios", "Heimburger", "Campero", "Stein", "Winikoff", "V.", "Huntington D and Piet-Pelon N"], "given-names": ["A", "C", "A", "L", "K", "B", "B"], "article-title": ["Improving Postabortion Care with Limited Resources in a Public Hospital in Oaxaca, Mexico"], "source": ["Postabortion Care: Lessons from Operations Research"], "year": ["2005"], "publisher-name": ["New York, Population Council"], "fpage": ["80"], "lpage": ["105"]}, {"surname": ["Benson", "Huapaya"], "given-names": ["J", "V"], "source": ["Sustainability of Postabortion Care in Peru"], "year": ["2002"], "publisher-name": ["Washington, DC, Population Council."]}, {"surname": ["Savelieva", "Pile", "Sacci", "Loganathan"], "given-names": ["I", "JM", "I", "R"], "source": ["Postabortion Family Planning Operations Research Study in Perm, Russia (Full report)"], "year": ["2003"], "publisher-name": ["Washington DC, Frontiers"]}, {"surname": ["Dabash"], "given-names": ["R"], "source": ["Taking Postabortion Care Services Where They Are Needed: An Operations Research Project Testing PAC Expansion in Rural Senegal: Final Report\""], "year": ["2003"], "publisher-name": ["New York, NY, Engenderhealth."]}, {"article-title": ["The World's Abortion Laws"], "year": ["2008"]}, {"surname": ["National Bureau of Statistics"], "given-names": ["URT"], "source": ["Tanzania Demographic and Health Survey 2004-2005 Preliminary Report"], "year": ["2005"]}]
{ "acronym": [], "definition": [] }
25
CC BY
no
2022-01-12 14:47:25
BMC Pregnancy Childbirth. 2008 Jul 31; 8:32
oa_package/77/c1/PMC2529258.tar.gz
PMC2529259
18718007
[ "<title>Background</title>", "<p>Excessive bleeding after childbirth, the leading cause of maternal deaths worldwide, has received international attention among medical and research communities for decades. A crucial component in the treatment of PPH resulting from atonic uterus is the administration of injectable uterotonics. The most commonly used agents in hospital-based settings are oxytocin and/or ergometrine. Additional medical and surgical interventions, beyond the administration of conventional uterotonics, have also been investigated as alternative and adjuvant therapy options for postpartum bleeding. The off-label use of misoprostol, a prostaglandin E1 analogue, has entered into clinical practice for this indication because of its strong uterotonic properties, and its ease in oral administration, stability at ambient temperatures, wide availability, and low cost. A 600 mcg dose of oral misoprostol has been shown safe and effective in preventing PPH [##REF##17027730##1##]. Misoprostol is less effective than injectable oxytocin, and it has therefore been recommended only for prevention of PPH in settings where injectable conventional uterotonics are not available [##UREF##0##2##]. No misoprostol regimens have been proven for routine use as a stand alone or adjunct treatment for postpartum hemorrhage.</p>", "<p>To date, only three small randomized controlled trials (RCT) of misoprostol for the treatment of PPH have been conducted [##REF##11531635##3##, ####REF##15327620##4##, ##REF##15327620##5####15327620##5##]. The first RCT compared use of misoprostol (800 mcg rectally) as a first-line treatment with standard oxytocics; the latter two placebo-controlled trials assessed the additive effect of misoprostol when used in conjunction with standard oxytocics. The misoprostol regimens in these RCTs ranged from 600 to 1000 mcg and the routes of administration varied (oral, sublingual, and rectal). A trend in blood loss reduction in the misoprostol arms was shown, but the trials were not adequately powered to produce any statistically significant findings. A meta-analysis of the combined data from the two placebo-controlled trials testing misoprostol as an adjunct PPH treatment, however, does show a statistically significant reduction of blood loss of 500 ml or more after treatment [##REF##15842275##6##,##REF##17253486##7##]. Seven uncontrolled studies or case reports have demonstrated misoprostol's strong uterotonic effect in stopping bleeding when used as a last resort treatment in hospital-based settings [##REF##9699753##8##, ####REF##11214957##9##, ##UREF##1##10##, ##REF##15380748##11##, ##REF##11883024##12##, ##REF##11368143##13##, ##UREF##2##14####2##14##]. In community-based settings, findings from an intervention trial, where Tanzanian traditional birth attendants were trained to administer 1000 mcg of misoprostol rectally as first-line PPH treatment, provided insight on the drug's potential role in PPH care [##REF##15919088##15##]. Despite the promising results for misoprostol's therapeutic use as primary PPH treatment, as an additional treatment to standard oxytocics or as a last resort treatment option, the published RCTs have been small and the regimens studied have varied greatly, making it difficult to draw clear conclusions. A recent review on misoprostol for PPH noted that the current available evidence was insufficient to support the use of misoprostol for routine treatment of PPH [##UREF##3##16##]. In the absence of large randomized controlled trials with conclusive results, clinicians are left to decide on the best combination of medical interventions to control postpartum bleeding.</p>", "<p>In Pakistan, where PPH accounts for nearly 25% of maternal deaths [##UREF##4##17##], anecdotal evidence suggests that misoprostol is often used in hospital settings. However, in a country where statistics on maternal health outcomes are largely unavailable [##REF##17933645##18##], the benefits and/or risks of using misoprostol as an alternative or adjunct therapy for postpartum hemorrhage remain unknown. To establish the effectiveness of adding misoprostol to current treatment regimens, a randomized controlled trial was conducted to test a 600 mcg dose of sublingual misoprostol as an adjunct treatment at four hospitals in Karachi, Pakistan. The 600 mcg dose was selected for its demonstrated safety and efficacy in previous PPH trials of its prophylactic oral use, and the sublingual administration of misoprostol was preferred for its rapid onset of action, prolonged duration, and greater bioavailability [##REF##15842275##6##]. By testing the drug's efficacy as an adjunct PPH treatment, this trial provides insight on the potential of misoprostol to treat PPH more effectively.</p>" ]
[ "<title>Methods</title>", "<p>The purpose of this randomized controlled trial was to ascertain whether 600 mcg of misoprostol taken sublingually provides an additional benefit to a standard oxytocin regimen for treatment of PPH. The study had a pragmatic approach with minimal interference in the current routine management of delivery. Prior to trial commencement, a meeting was jointly conducted with representatives from the participating hospitals to document routine PPH prevention and treatment protocols at their facilities, and a management protocol was developed to guide PPH treatment practices over the course of the study. The trial was approved by the Ethical Review Committee at the Aga Khan University in Karachi.</p>", "<p>The study began in December 2005, with the last woman enrolled in April 2007 after 16 months of continuous recruitment. The study aimed to enroll 900 women over 18 months at four hospitals in Karachi, Pakistan: The Aga Khan University Hospital (AKUH) a large tertiary level hospital; and three secondary level facilities within the Aga Khan Network – Aga Khan Hospital for Women in Karimabad District, Aga Khan Hospital for Women in Garden District and Aga Khan Hospital for Women and Children in Kharadar District. Each of these hospitals has approximately 2,000 deliveries per year. Laboring women were given detailed information on the study protocol and invited to participate in the trial by trained hospital staff. A consent form (available in English and Urdu) was signed or thumb-printed by consenting women. Women with cesarean-section, gestational age less than 28 weeks at time of delivery, or not consenting were excluded from the study.</p>", "<p>All women underwent routine active management of the third stage of labor with standard uterotonics, controlled cord traction after delivery of baby, and gentle uterine massage after delivery of the placenta. At the delivery of the anterior shoulder of baby, one of two uterotonic regimens was administered: intravenous 10 IU of oxytocin or 5 IU of oxytocin plus 0.4 mg of ergometrine given either intramuscularly or intravenously. Immediately after delivery of the baby, blood loss was collected by placing a clean fracture bedpan directly under the woman's buttocks for a minimum of one hour. Markings were written onto the bedpan to show when 500 ml had been reached. Women losing less than 500 ml were not entered into the trial. Women losing 500 ml or more were enrolled in the trial, and a clean bedpan was placed underneath their buttocks to collect blood lost after PPH diagnosis. A fresh, large perineal pad with plastic backing was positioned just below the bedpan to capture any spattering blood. Once the delivery attendant considered active bleeding to have stopped, the blood was transferred to a calibrated jar for measurement.</p>", "<p>All women with diagnosed PPH thought to be due to inadequate uterine contraction, as per the provider's clinical judgment, were promptly given IV oxytocin as routine for PPH treatment. Women were reminded of their consent to participate in the trial and a member of study team gave each woman the pills in the next randomized study envelope and instructed her to place the tablets under her tongue, e.g. sublingually. Each study envelope contained three tablets of either misoprostol (200 mcg × 3) (Gymiso, HRA Pharma, France) or matching placebo. All women, providers, and investigators were blinded to the treatment assignments. Simultaneous to PPH treatment administration, blood collection was restarted with a clean bedpan and fresh perineal pad placed underneath the woman. Blood loss was then measured continuously until active bleeding ceased – or for a minimum of one hour. The additional blood loss after receiving PPH treatment was transferred to a calibrated jug and sealed, and all used gauzes and pads were counted and placed in a plastic bag. The plastic bag was then weighed; however, accurate use of the scales proved difficult, and these results could not be verified. Therefore, in this paper we share the validated results of total volume of blood collected.</p>", "<p>Based on previous studies, we estimated a 10% rate of additional blood loss ≥ 500 ml [##REF##15842275##6##] after receiving study treatment in the oxytocin-misoprostol arm and a rate of 16% in the oxytocin-placebo arm. To detect a difference of this size (one-sided test), we calculated that to achieve 80% power at p = 0.05, a sample size of 420 women in each arm was needed. The sample was randomized in blocks of ten, stratified by site, using a computer-generated random sequence provided by Gynuity Health Projects, New York, where the code was kept. Data were entered and cleaned in Epi Info (Epi Info, version 3.4.3). The randomization code was concealed until all data were entered and cleaned. Data analysis was conducted using the Statistical Package for the Social Sciences, version 13.0 (SPSS, Chicago, IL, USA). Using SPSS, categorical data were analyzed with chi-square tests and continuous data with t tests.</p>", "<p>The main study outcome was to determine if the addition of misoprostol to standard PPH care reduces postpartum bleeding. The primary endpoint was measured blood loss ≥ 500 mls after PPH treatment; secondary outcomes included change in hemoglobin, side effects, need for additional interventions including blood transfusion, additional uterotonics, balloon tamponade, hysterectomy, and mean blood loss. Hemoglobin levels were measured pre-delivery upon entry into labor ward and 12–24 hours post-delivery by taking a finger prick and using a Hemocue Hemoglobin machine (HemoCue AB, Ängleholm, Sweden). Side effects were recorded by the delivery attendant as they were observed or reported. Blood loss was documented for women who consented for the study but did not experience PPH. Regular monitoring and training of delivery ward staff continued throughout duration of the trial.</p>" ]
[ "<title>Results</title>", "<p>Blood loss following 5,171 vaginal deliveries was collected at the four participating hospitals. The mean blood loss and postpartum hemoglobin level were 150 ml and 11.2 g/dL, respectively. Sixty-one women (1.2%) were diagnosed with PPH and randomized to receive either oxytocin plus 600 mcg misoprostol sublingually (n = 29) or oxytocin plus matching placebo (n = 32). Deviations in study protocol occurred among two women randomized to the misoprostol arm. In one protocol deviation, the patient was given only 400 mcg of study medication because the delivery ward staff questioned whether her bleeding might be due to episiotomy. The second protocol deviation involved a woman whose blood mistakenly was not collected by hospital staff. Table ##TAB##0##1## details the baseline demographic and delivery characteristics of all study participants (n = 61). There were no significant differences between women in the two groups.</p>", "<p>All women had their third stage of labor actively managed as per standard hospital protocol. There were no statistical differences in the amount and route of prophylactic oxytocin administered between the two study groups. All women received oxytocin prophylaxis at the delivery of anterior shoulder of baby (IV administration 88.5%; IM administration 11.5%). The prophylactic use of ergometrine in addition to oxytocin was also standard practice at two of the four sites. In half of the deliveries (equally distributed across study arms), ergometrine was administered prophylactically (IV administration 60.0%; IM administration 40.0%) in conjunction with oxytocin.</p>", "<p>On average, PPH was diagnosed 35 minutes following delivery after losing an average of 655 ml (± 163 ml) of blood, at which time PPH treatment was administered. There were no differences in the dose, route or choice of standard uterotonics given for PPH treatment between the two study groups. Intravenous oxytocin (bolus 5–30 units and infusion 10–80 units in 500–1000 ml saline) and ergometrine up to 0.4 mg i.v. were initial treatment measures. Oxytocin infusion was given for all PPH cases; two-thirds were given intravenous oxytocin in bolus; and only one-third of cases received 0.2–0.4 mg ergometrine i.v. Six cases (four in the misoprostol group and two in the placebo group) were given an additional dose of 200–400 mcg misoprostol either rectally or sublingually. Prostaglandin alpha F-2 was administered to one woman in misoprostol group and three in the placebo group.</p>", "<p>There were no problems reported with sublingual administration of study medication. The study outcomes are summarized in Table ##TAB##1##2##. Due to the much lower than expected PPH rate, the intended sample size was not reached. Postpartum blood loss, drop in hemoglobin, and use of additional interventions (blood transfusion, uterine packing, and balloon tamponade) were lower in misoprostol group, but did not reach statistical significance. Fewer women in the misoprostol arm received i.v. fluids amounting to greater than 1000 ml (p = .056). Among those women receiving additional interventions (twelve in the misoprostol group; nineteen in the placebo group), total measured blood loss was significantly higher (940 ± 341 ml, p = .028) compared with women (n = 29) who did not receive any additional intervention beyond standard treatment (780 ± 176 ml). Similarly, drop in postpartum hemoglobin was significantly higher among women requiring additional interventions (2.5 g/dL (± 1.5) vs. 1.7 g/dL (± 0.9); p = .022). There were no hysterectomies or maternal deaths among study participants.</p>", "<p>Shivering and fever were more commonly reported in the misoprostol group. All other side effects were minimal (Table ##TAB##2##3##). Transient shivering was experienced by 51.7% of women receiving misoprostol compared with 6.2% in placebo group. The incidence of fever followed similar pattern to that of shivering. Eleven women (37.9%) in the misoprostol group experienced an elevated temperature of ≥ 37.5°C (≥ 99.5°F) compared with only three women (9.4%) in the placebo group. Three women receiving misoprostol characterized their fever as being severe. There was one case of high fever reported in the misoprostol group in which temperature at one hour post-treatment measured 40.1°C (104.2°F). There were no reports of severe side effects resulting in prolonged hospitalization or other adverse events in misoprostol group and all women made a full recovery.</p>" ]
[ "<title>Discussion</title>", "<p>The purpose of this trial was to explore whether 600 mcg sublingual misoprostol offers any additional benefit when added simultaneously to conventional methods for the treatment of primary postpartum bleeding. Although the study findings reported here are consistent with the two placebo-controlled trials on adjunct use of misoprostol [##REF##15327620##4##,##REF##15327620##5##], we were unable to measure statistical significance in our primary endpoints due to a much lower rate of PPH than expected. Our findings suggest a trend in reduced postpartum blood loss, smaller drop in postpartum hemoglobin, and fewer additional interventions among women treated with misoprostol in addition to standard oxytocics.</p>", "<p>The use of misoprostol was not associated with a significant reduction in any of the pre-specified primary outcome measures in this study. However, the relative risk reduction of 41% of blood loss ≥ 500 ml after treatment (RR 0.59 95% CI [0.12, 2.99]) is similar to the two hospital-based placebo-controlled trials conducted in South Africa (RR 0.56 95% CI [0.21, 1.46]) and the Gambia (RR 0.58, 95% CI [0.32, 1.06]) [##REF##15842275##6##]. Indeed, a combined analysis of results from this trial and the two placebo-controlled trials on adjunct use of misoprostol [##REF##15327620##4##,##REF##15327620##5##] confirms that misoprostol use is associated with a significant reduction of blood loss of ≥ 500 ml following treatment (RR 0.58, 95% CI [0.35, 0.95], p = .029). This confers with results from other recent meta-analyses [##UREF##0##2##,##REF##15842275##6##,##REF##17253486##7##].</p>", "<p>Our study findings on measured postpartum blood loss also highlight the clinical importance of a reduction of blood loss following PPH treatment. Women who bled less overall had a significantly smaller drop in hemoglobin and did not require additional interventions, such as blood transfusion, balloon tamponade, or uterine packing, to manage their postpartum bleeding. In contrast, women who had a significantly higher total blood loss were more likely to be given additional interventions and experience a larger drop in postpartum hemoglobin. A reduction in blood loss reduces need for more invasive procedures, results in a smaller change in postpartum hemoglobin, and as a result, may prevent more severe maternal morbidity experienced by recently delivered mothers. Based on the trends in blood loss we see among women in our study given misoprostol in addition to standard oxytocics for their PPH treatment, the adjunct use of misoprostol shows great potential in improving women's health outcomes after experiencing this obstetrical complication.</p>", "<p>As found in previous studies on misoprostol, use of the drug was significantly associated with fever and shivering. These effects, however, were described as transient and did not result in any additional complication to the women. Within the literature on misoprostol as adjunct treatment, a total of three cases of high fever ≥ 40.0°C (≥ 104.0°F) have been reported following a 1000 mcg dose of misoprostol (400 mcg sublingually + 400 mcg rectally + 200 mcg orally) [##REF##15327620##4##]. This study had one case of temperature over 40.0°C in the misoprostol group. Delivery ward staff should be trained to recognize and manage cases of severe shivering and high fever to ensure proper care of the patient.</p>", "<p>Great efforts were taken to standardize PPH care within and across the participating hospitals. Representatives from the hospitals reviewed their PPH management policies and developed a document to guide their treatment practices over the course of the study and thereafter. Despite efforts to standardize PPH care, there still existed variation in the pharmacological agents/regimens used, especially in the administration of second- or third-line therapies. For instance, six patients were given an additional dose of misoprostol beyond initial treatments. Although the hospitals in this study had policies cautioning against the use of prostaglandins due to lack of evidence, providers still relied on their use as a therapy options.</p>", "<p>Irrespective of the variability in PPH practices, a low rate of PPH &lt; 2% across the four participating hospitals was demonstrated in this study. This rate of PPH was validated using an objective measurement protocol with marked bedpan in over 5,000 normal vaginal deliveries. Studies on postpartum hemorrhage often find that the incidence of PPH is underreported in settings prior to objective measurement of postpartum blood loss [##REF##1995380##19##, ####REF##8798302##20##, ##REF##11044547##21##, ##REF##16626718##22####16626718##22##], however this study shows just the opposite with a measured PPH rate of 1.2%. According to the 2000 Cochrane Review, the rate of PPH ≥ 500 ml is roughly 5% for women receiving prophylactic uterotonics and 12% for those not receiving uterotonics prophylactically [##REF##10796082##23##]. Importantly, only two of the four studies included in the review specified the use of an objective blood measurement technique. In general, there is wide variability in reported PPH rates in the literature. As Soriano and colleagues note, PPH incidence ≥ 500 ml has been reported at 1.0%, 5.0%, 7.2%, and 14.5% in studies comparing oxytocin-ergometrine and oxytocin alone in the third stage of labor [##REF##8916990##24##]. This study, using objective measurement of postpartum blood loss, confirms that a very low rate of PPH can be achieved in hospital settings following the routine practice of active management of the third stage of labor. At the same time, the low rate of PPH in this study demonstrates the difficulty in conducting hospital-based studies on relatively rare, but clinically important obstetrical complications.</p>", "<p>The objective assessment of postpartum bleeding provided a valuable, initially unexpected, insight into the diagnosis and management of PPH in the four Karachi hospitals. Individual exit interviews with study staff revealed that they believed the use of the bedpan had corrected their \"over\"-estimation of postpartum blood loss. Both doctors and nurses explained that previously they had overestimated postpartum blood loss, resulting in misdiagnosis of PPH, unnecessary treatments, and prolonged hospitalization. During the study, staff was trained to diagnose PPH at 500 ml using the bedpan for objective assessment. However, PPH was diagnosed, on average, after losing nearly 650 ml, which suggests that the use of the bedpan did not lead to more prompt diagnosis at 500 ml. The timing of diagnosis, which commonly occurred around 30 minutes after delivery, also provided valuable insight on the duration of close-monitoring necessary during the postpartum period for all recently delivered mothers. Objective measures using the bedpan and Hemocue apparatus also verified that those women with greater postpartum bleeding and larger decline in postpartum hemoglobin were commonly provided with additional interventions beyond standard uterotonic treatments to control their bleeding. The use of the bedpan proved to be a valuable tool for educating delivery ward staff over the course of the study on the diagnosis and management of postpartum hemorrhage, but the value of its continued use outside a clinical trial is not yet apparent.</p>" ]
[ "<title>Conclusion</title>", "<p>Due to the logistical complexities of managing postpartum hemorrhage, this obstetrical complication continues to threaten women's lives, especially in facilities short-staffed or lacking uterotonics and protocols to manage PPH safely and effectively. It has been advocated that misoprostol should be available in community-based settings with limited access to conventional injectable uterotonics. This study suggests that misoprostol may also have an important role to play in hospital settings and its adjunct use should continue to be explored for its potential in quickly, safely, and effectively controlling postpartum bleeding, averting recourse to more invasive procedures, and preventing more severe maternal morbidity.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Postpartum hemorrhage (PPH) remains a major killer of women worldwide. Standard uterotonic treatments used to control postpartum bleeding do not always work and are not always available. Misoprostol's potential as a treatment option for PPH is increasingly known, but its use remains ad hoc and available evidence does not support the safety or efficacy of one particular regimen. This study aimed to determine the adjunct benefit of misoprostol when combined with standard oxytocics for PPH treatment.</p>", "<title>Methods</title>", "<p>A randomized controlled trial was conducted in four Karachi hospitals from December 2005 – April 2007 to assess the benefit of a 600 mcg dose of misoprostol given sublingually in addition to standard oxytocics for postpartum hemorrhage treatment. Consenting women had their blood loss measured after normal vaginal delivery and were enrolled in the study after losing more than 500 ml of blood. Women were randomly assigned to receive either 600 mcg sublingual misoprostol or matching placebo in addition to standard PPH treatment with injectable oxytocics. Both women and providers were blinded to the treatment assignment. Blood loss was collected until active bleeding stopped and for a minimum of one hour after PPH diagnosis. Total blood loss, hemoglobin measures, and treatment outcomes were recorded for all participants.</p>", "<title>Results</title>", "<p>Due to a much lower rate of PPH than expected (1.2%), only sixty-one patients were diagnosed and treated for their PPH in this study, and we were therefore unable to measure statistical significance in any of the primary endpoints. The addition of 600 mcg sublingual misoprostol to standard PPH treatments does, however, suggest a trend in reduced postpartum blood loss, a smaller drop in postpartum hemoglobin, and need for fewer additional interventions. Women who bled less overall had a significantly smaller drop in hemoglobin and received fewer additional interventions. There were no hysterectomies or maternal deaths among study participants. The rate of transient shivering and fever was significantly higher among women receiving misoprostol</p>", "<title>Conclusion</title>", "<p>A 600 mcg dose of misoprostol given sublingually shows promise as an adjunct treatment for PPH and its use should continue to be explored for its life-saving potential in the care of women experiencing PPH.</p>", "<title>Trial Registration</title>", "<p>Clinical trials.gov, Registry No. NCT00116480</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>NZ carried out the study, participated in its coordination and helped draft the manuscript. JD monitored the study, participated in its coordination, performed the statistical analysis and drafted the manuscript. RS carried out the study and reviewed the manuscript. NB carried out the study and reviewed the manuscript. JB contributed to design of the study, participated in its coordination, data interpretation and drafting the manuscript. GW designed the study, helped interpret data and draft the manuscript. All authors read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2393/8/40/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>The authors gratefully acknowledge the women who participated in this trial, the nurses and doctors who carefully collected the data, and Ms. Zia Sultana, who coordinated the trial from Karachi. We also would like to thank Dr. Charmine Gill at the Aga Khan Hospital for Women at Garden and Dr. Salva Nadeem at the Aga Khan Hospital for Women &amp; Children at Kharadar who oversaw the conduct of this trial at their respective hospitals. We are very grateful to the administration at the Aga Khan Health Services, Pakistan and at the Aga Khan University, Karachi for enabling this study to be carried out at the four participating study sites. The authors also want to thank Dr. Beverly Winikoff for reviewing this manuscript. This study was funded by the Bill and Melinda Gates Foundation through a grant to Gynuity Health Projects and Family Care International. The Foundation had no role in the actual planning, writing or submission of this paper.</p>" ]
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[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Baseline characteristics among women treated for PPH</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\"><bold>misoprostol</bold></td><td align=\"center\"><bold>placebo</bold></td><td/></tr></thead><tbody><tr><td/><td align=\"center\">n = 29</td><td align=\"center\">n = 32</td><td align=\"center\">RR [95% CI] or p-value</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Age (years) </bold>mean ± sd</td><td align=\"center\">25 ± 5</td><td align=\"center\">26 ± 4</td><td align=\"center\">p = .291</td></tr><tr><td align=\"left\"><bold>Parity </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> 0</td><td align=\"center\">62.1 (18)</td><td align=\"center\">40.6 (13)</td><td align=\"center\">1.53 [.92, 2.54]</td></tr><tr><td align=\"left\"> 1–3</td><td align=\"center\">31.0 (9)</td><td align=\"center\">50.0 (16)</td><td/></tr><tr><td align=\"left\"> 4–8</td><td align=\"center\">6.9 (2)</td><td align=\"center\">9.4 (3)</td><td/></tr><tr><td align=\"left\"><bold>Outcome of this delivery </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> Singleton, alive</td><td align=\"center\">93.1 (27)</td><td align=\"center\">96.9 (31)</td><td align=\"center\">.96 [.85, 1.08]</td></tr><tr><td align=\"left\"> Singleton, still birth</td><td align=\"center\">3.4 (1)</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"> Twins, both alive</td><td align=\"center\">3.4 (1)</td><td align=\"center\">3.7 (1)</td><td/></tr><tr><td align=\"left\"><bold>Episiotomy </bold>%(n)</td><td align=\"center\">75.9 (22)</td><td align=\"center\">68.8 (22)</td><td align=\"center\">1.10 [.81, 1.51]</td></tr><tr><td align=\"left\"><bold>Manual removal of placenta done </bold>%(n)</td><td align=\"center\">6.9 (2)</td><td align=\"center\">12.5 (4)</td><td align=\"center\">.55 [.11, 2.79]</td></tr><tr><td align=\"left\"><bold>Placental delivery within 5 minutes </bold>%(n)</td><td align=\"center\">62.1 (18)</td><td align=\"center\">78.1 (25)</td><td align=\"center\">.79 [.57,1.11]</td></tr><tr><td align=\"left\"><bold>Pre-delivery hemoglobin </bold>(g/dL)</td><td/><td/><td/></tr><tr><td align=\"left\"> mean ± sd</td><td align=\"center\">11.1 ± 1.0</td><td align=\"center\">10.9 ± 1.1</td><td align=\"center\">p = .502</td></tr><tr><td align=\"left\"> range</td><td align=\"center\">9.1 – 13.5</td><td align=\"center\">8.7 – 13.0</td><td/></tr><tr><td align=\"left\"><bold>Measured blood loss at diagnosis </bold>(ml)</td><td/><td/><td/></tr><tr><td align=\"left\"> mean ± sd</td><td align=\"center\">640 ± 139</td><td align=\"center\">669 ± 184^</td><td align=\"center\">p = .492</td></tr><tr><td align=\"left\"> range</td><td align=\"center\">400 – 1000</td><td align=\"center\">500 – 1000</td><td/></tr><tr><td align=\"left\"><bold>Time to diagnosis </bold>(minutes)</td><td/><td/><td/></tr><tr><td align=\"left\"> mean ± sd</td><td align=\"center\">37 ± 29</td><td align=\"center\">33 ± 24</td><td align=\"center\">p = .584</td></tr><tr><td align=\"left\"> range</td><td align=\"center\">5 – 122</td><td align=\"center\">5 – 100</td><td/></tr><tr><td align=\"left\"><bold>Use of oxytocics <italic>prior </italic>to study treatment </bold>%(n)</td><td align=\"center\">100 (29)</td><td align=\"center\">100 (32)</td><td align=\"center\">1.0</td></tr><tr><td align=\"left\"><bold>Use of oxytocics <italic>after </italic>study treatment </bold>%(n)</td><td align=\"center\">100 (29)</td><td align=\"center\">100 (32)</td><td align=\"center\">1.0</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Outcomes after receiving study treatment</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\"><bold>misoprostol</bold></td><td align=\"center\"><bold>placebo</bold></td><td/></tr></thead><tbody><tr><td/><td align=\"center\">n = 29</td><td align=\"center\">n = 32</td><td align=\"center\">RR [95% CI] or p-value</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Postpartum blood loss after study treatment</bold></td><td/><td/><td/></tr><tr><td/><td align=\"center\">(n = 27) ^</td><td align=\"center\">(n = 32)</td><td/></tr><tr><td align=\"left\"><bold>Total blood loss post-treatment </bold>(ml)</td><td/><td/><td/></tr><tr><td align=\"left\"> mean ± sd</td><td align=\"center\">175 ± 168</td><td align=\"center\">187 ± 207</td><td align=\"center\">p = .809</td></tr><tr><td align=\"left\"> Range</td><td align=\"center\">10–700</td><td align=\"center\">10–900</td><td/></tr><tr><td align=\"left\"><bold>Blood loss ≥ 500 ml post-treatment </bold>%(n)</td><td align=\"center\">7.4 (2)</td><td align=\"center\">12.5 (4)</td><td align=\"center\">.59 [.12, 2.99]</td></tr><tr><td align=\"left\"><bold>Postpartum hemoglobin measures</bold></td><td/><td/><td/></tr><tr><td align=\"left\"><bold>Post-delivery Hb</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> mean ± sd</td><td align=\"center\">9.0 ± 1.4</td><td align=\"center\">8.7 ± 1.2</td><td align=\"center\">p = .291</td></tr><tr><td align=\"left\"> range</td><td align=\"center\">5.9 – 11.3</td><td align=\"center\">5.9 – 10.2</td><td/></tr><tr><td align=\"left\"><bold>Drop in Hb</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> mean ± sd</td><td align=\"center\">2.0 ± 1.1</td><td align=\"center\">2.2 ± 1.4</td><td align=\"center\">p = .614</td></tr><tr><td align=\"left\"> range</td><td align=\"center\">0.4 – 4.2</td><td align=\"center\">0.1 – 5.1</td><td/></tr><tr><td align=\"left\"><bold>Postpartum Hb ≥ 2 g/dL lower than pre-delivery Hb </bold>%(n)</td><td align=\"center\">41.4 (12)</td><td align=\"center\">56.3 (18)</td><td align=\"center\">.74 [.43, 1.25]</td></tr><tr><td align=\"left\"><bold>Additional interventions</bold></td><td/><td/><td/></tr><tr><td align=\"left\"><bold>Amount of IV fluids given</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> 500–1000 ml</td><td align=\"center\">75.9 (22)</td><td align=\"center\">53.1 (17)</td><td/></tr><tr><td align=\"left\"> &gt; 1000 ml</td><td align=\"center\">24.1 (7)</td><td align=\"center\">46.9 (15)</td><td align=\"center\">.51 [.24, 1.08]</td></tr><tr><td align=\"left\"><bold>Blood transfusion </bold>%(n)</td><td align=\"center\">17.2 (5)</td><td align=\"center\">18.8 (6)</td><td align=\"center\">.92 [.31, 2.69]</td></tr><tr><td align=\"left\"><bold>Uterine packing </bold>%(n)</td><td align=\"center\">6.9 (2)</td><td align=\"center\">18.8 (6)</td><td align=\"center\">.37 [.08, 1.68]</td></tr><tr><td align=\"left\"><bold>Balloon tamponade </bold>%(n)</td><td align=\"center\">0.0 (0)</td><td align=\"center\">3.1 (1)</td><td align=\"center\">.00 [.00, 43.0]</td></tr><tr><td align=\"left\"><bold>Referrals for additional PPH care </bold>%(n)</td><td align=\"center\">3.4 (1)</td><td align=\"center\">3.2 (1)</td><td align=\"center\">1.1 [.07, 16.9]</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Side effects after receiving study treatment</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\"><bold>misoprostol</bold></td><td align=\"center\"><bold>placebo</bold></td><td/></tr></thead><tbody><tr><td/><td align=\"center\">n = 29</td><td align=\"center\">n = 32</td><td align=\"center\">RR [95% CI] or p-value</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Nausea </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> none</td><td align=\"center\">93.1 (27)</td><td align=\"center\">93.8 (30)</td><td align=\"center\">.99 [.87, 1.13]</td></tr><tr><td align=\"left\"> mild or moderate</td><td align=\"center\">6.9 (2)</td><td align=\"center\">6.3 (2)</td><td/></tr><tr><td align=\"left\"> severe</td><td align=\"center\">--</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"><bold>Vomiting </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> none</td><td align=\"center\">93.1 (27)</td><td align=\"center\">96.9 (31)</td><td align=\"center\">.96 [.85, 1.08]</td></tr><tr><td align=\"left\"> mild or moderate</td><td align=\"center\">6.9 (2)</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"> severe</td><td align=\"center\">--</td><td align=\"center\">3.1 (1)</td><td/></tr><tr><td align=\"left\"><bold>Diarrhea </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> none</td><td align=\"center\">100 (29)</td><td align=\"center\">100 (32)</td><td align=\"center\">1.0</td></tr><tr><td align=\"left\"> mild or moderate</td><td align=\"center\">--</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"> severe</td><td align=\"center\">--</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"><bold>Fainting </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> none</td><td align=\"center\">100 (29)</td><td align=\"center\">96.9 (31)</td><td align=\"center\">1.03 [.97, 1.10]</td></tr><tr><td align=\"left\"> mild or moderate</td><td align=\"center\">--</td><td align=\"center\">3.1 (1)</td><td/></tr><tr><td align=\"left\"> severe</td><td align=\"center\">--</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"><bold>Fatigue </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> none</td><td align=\"center\">100 (29)</td><td align=\"center\">100 (32)</td><td align=\"center\">1.0</td></tr><tr><td align=\"left\"> mild or moderate</td><td align=\"center\">--</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"> severe</td><td align=\"center\">--</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"><bold>Headache </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> none</td><td align=\"center\">93.1 (27)</td><td align=\"center\">100 (32)</td><td align=\"center\">.93 [.84, 1.03]</td></tr><tr><td align=\"left\"> mild or moderate</td><td align=\"center\">6.9 (2)</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"> severe</td><td align=\"center\">--</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"><bold>Shivering </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> none</td><td align=\"center\">48.3 (14)</td><td align=\"center\">93.8 (30)</td><td/></tr><tr><td align=\"left\"> mild or moderate</td><td align=\"center\">37.9 (11)</td><td align=\"center\">6.2 (2)</td><td align=\"center\">8.28 [2.1. 33.1]</td></tr><tr><td align=\"left\"> severe</td><td align=\"center\">13.8 (4)</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"><bold>Fever </bold>%(n)</td><td/><td/><td/></tr><tr><td align=\"left\"> none</td><td align=\"center\">48.3 (14)</td><td align=\"center\">90.6 (29)</td><td/></tr><tr><td align=\"left\"> mild or moderate</td><td align=\"center\">41.4 (12)</td><td align=\"center\">9.4 (3)</td><td align=\"center\">5.52 [1.8, 17.1]</td></tr><tr><td align=\"left\"> severe</td><td align=\"center\">10.3 (3)</td><td align=\"center\">--</td><td/></tr><tr><td align=\"left\"><bold>Temperature at 1 hour </bold>(Celsius)</td><td/><td/><td/></tr><tr><td align=\"left\"> mean ± sd</td><td align=\"center\">37.4 ± .94</td><td align=\"center\">37.0 ± .35</td><td align=\"center\">p = .022</td></tr><tr><td align=\"left\"> range</td><td align=\"center\">36.2 – 40.1</td><td align=\"center\">36.3 – 37.8</td><td/></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>^ There is one outlier excluded in which the woman was diagnosed after losing 1,750 ml.</p></table-wrap-foot>", "<table-wrap-foot><p>^ Two cases in the misoprostol arm have incomplete blood loss measurements and were excluded from analysis of measured postpartum blood loss.</p></table-wrap-foot>" ]
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[{"surname": ["Alfirevic", "Blum", "Walraven", "Weeks", "Winikoff"], "given-names": ["A", "J", "G", "A", "B"], "article-title": ["Prevention of postpartum hemorrhage with misoprostol"], "source": ["Int J Gynecol Obstet"], "year": ["2007"], "volume": ["99"], "fpage": ["198"], "lpage": ["201"], "pub-id": ["10.1016/j.ijgo.2007.09.012"]}, {"surname": ["Abdel-aleem", "El-Nashar", "Abdel-Aleem"], "given-names": ["H", "I", "A"], "article-title": ["Management of severe postpartum hemorrhage with misoprostol"], "source": ["Int J Gynecol Obstet"], "year": ["2001"], "volume": ["72"], "fpage": ["75"], "lpage": ["76"], "pub-id": ["10.1016/S0020-7292(00)00321-0"]}, {"surname": ["Oboro", "Tabowei", "Bosah"], "given-names": ["VO", "TO", "JO"], "article-title": ["Intrauterine misoprostol for refractory postpartum hemorrhage"], "source": ["Int J Gynecol Obstet"], "year": ["2003"], "volume": ["80"], "fpage": ["67"], "lpage": ["68"], "pub-id": ["10.1016/S0020-7292(02)00301-6"]}, {"surname": ["Blum", "Alfirevic", "Walraven", "Weeks", "Winikoff"], "given-names": ["J", "Z", "G", "A", "B"], "article-title": ["Treatment of postpartum hemorrhage with misoprostol"], "source": ["Int J Gynaecol Obstet"], "year": ["2007"], "fpage": ["202"], "lpage": ["205"], "pub-id": ["10.1016/j.ijgo.2007.09.013"]}, {"surname": ["Bhutta", "Jafarey", "Midhet", "Bhutta ZA"], "given-names": ["S", "SN", "F"], "article-title": ["Safe motherhood. A situation analysis and recommendations for evidence-based approaches"], "source": ["Maternal and child health in Pakistan Challenges and opportunities"], "year": ["2004"], "publisher-name": ["Karachi: Oxford University Press"], "fpage": ["1"], "lpage": ["18"]}]
{ "acronym": [], "definition": [] }
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CC BY
no
2022-01-12 14:47:25
BMC Pregnancy Childbirth. 2008 Aug 21; 8:40
oa_package/0e/ab/PMC2529259.tar.gz
PMC2529260
18721462
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[ "<title>Discussion</title>", "<p>Primary lung cancer is the most frequently diagnosed cancer in the USA and the most common cause of cancer mortality worldwide. It occurs most often between the ages of 40 and 70 years, with a peak incidence in the fifties and sixties. Only 2% of all cases appear before the age of 40. Primary lung cancer in childhood is a rare entity and primary bronchogenic squamous cell carcinoma is extremely rare. To our knowledge, only eight cases of primary bronchogenic squamous cell carcinoma in childhood have been reported in English literature.</p>", "<p>Pulmonary squamous cell carcinoma is most commonly found in men and is closely correlated with a smoking history. Histologically, the tumor is characterized by the presence of keratinization and intercellular bridges. It is graded according to the degree of keratinization, squamous pearl formation, or intercellular bridges. These features are obvious in the well-differentiated tumors but only focally demonstrated in the poorly-differentiated tumors.</p>", "<p>Cayler et al [##REF##14846416##1##] in 1951 reported 16 cases of primary carcinoma of the lung in children less than 15 years of age Primary bronchogenic squamous cell carcinoma is extremely rare in childhood and adolescence. Eight histologically confirmed cases reported in the English literature [##REF##4809785##2##, ####REF##7968793##3##, ##REF##894451##4##, ##REF##10661854##5##, ##REF##5438571##6##, ##REF##1267282##7##, ##UREF##0##8##, ##REF##8609692##9####8609692##9##] are summarized along with the present case [Table 1].</p>", "<p>In 1974, Niitu et al [##REF##4809785##2##] reported one case of squamous cell carcinoma in a boy and reviewed the world literature and found 39 cases of primary lung cancer in children less than 16 years of age. These cases included two cases of bronchogenic squamous cell carcinoma [##REF##5438571##6##,##UREF##0##8##]. Since then only five additional cases of primary brochogenic SCC have been reported, including one case with substantial family history of cancer [[##REF##7968793##3##, ####REF##894451##4##, ##REF##10661854##5####10661854##5##,##REF##1267282##7##], and [##REF##8609692##9##]]. Most of the patients (eight out of nine) are boys. The clinical presentation of these bronchial cancers varies with the extent of the primary tumor. In our case, the patient presented with bone pain due to metastasis. Four of the reported cases presented with recurrent pneumopathies and hemoptysis. Three of the reported cases were incidentally found by chest x-ray. One case was discovered by routine chest radiograph. There have been no clearly identified risk factors. There is no standard treatment and management essentially depends on the initial findings of the extent of tumor and the presence of metastases. Generally, the prognosis is poor due to metastatic disease.</p>", "<p>The high frequency of p53 mutations have been seen in all histological types of lung carcinoma. Loss of tumor suppressor gene RB, inactivation of CDK-inhibitor, and overexpression of epidermal growth-factor receptor might contribute to the development of neoplasm. There are no p53 mutations or loss of tumor suppressor gene in our case. However, NUT1, a gene homologous to the major nitrogen regulatory gene [##REF##11383511##10##], is positive for gene rearrangement. It is unknown whether this rearrangement plays any role in the pathogenesis of primay lung bronchogenic squamous cell carcinoma.</p>" ]
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[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>We are reporting a very rare case of primary bronchogenic squamous cell carcinoma (SCC) with bone metastasis in a 13-year-old boy. A brief review of the English literature on this rare neoplasm in childhood is presented.</p>" ]
[ "<title>Case presentation</title>", "<p>A 13-year-old boy presented with a two-month history of left shoulder pain. Radiographs followed by MRI demonstrated a destructive lesion of the metaphysis of the proximal third of the left humerus [Fig ##FIG##0##1##]. The metaphysis of the humerus was replaced by a 5-cm tumor. There was some erosion of the cortex with minimal soft tissue extension by the tumor. The initial impression of the lesion was an osteosarcoma, however, an open biopsy revealed metastatic squamous cell carcinoma. The tumor showed islands and nests of squamous cells with a basaloid appearance at the periphery with maturation and squamous pearl formation in the center [Fig ##FIG##1##2##]. The cells appeared anaplastic with prominent nucleoli, numerous mitoses and focal necrosis [Fig ##FIG##2##3##].</p>", "<p>Molecular cytogenetic studies on the tumor were positive for rearrangement of the NUT1 region in 54% of the interphase cells. Additional interphase FISH studies were negative for the NUT/BDR4 fusion. The patient underwent a PET-CT that showed multiple foci of increased uptake in the right upper lobe of the lung. Additional abnormal uptake was also noted at T1, T8, T10, and T11, left proximal humerus, right hilar nodes and right acetabulum. A subsequent chest CT confirmed. the malignant lung neoplasm [Fig. ##FIG##3##4##] Since lung cancer is an extremely rare childhood neoplasm, radon tests are performed in the family home and the results were negative. Upon further questioning, a history of lung cancer in both grandmothers was uncovered. One grandfather also had a history of non-Hodgkin lymphoma and pancreatic cancer. The patient was treated with various chemotherapeutic agents. There was no significant clinical improvement and he died one year later.</p>", "<title>Consent</title>", "<p>Written consent was obtained from the patient's parents for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this Journal.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>JFW conceived and drafted the manuscript, BW helped with the photomicrographs, EES and JAJ revised and proof-read the manuscript. DPS reviewed the references, made the final corrections and submitted the manuscript. All authors have read and approved the final manuscript.</p>" ]
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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>X-ray of left shoulder shows destructive lesion of humerus.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Invasive islands and nests of squamous cells with basaloid appearance (Hematoxylin and eosin stain, 10×).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Squamous cells with prominent nucleoli, numerous mitoses and necrosis (Hematoxylin and eosin stain, 20×).</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>Chest CT confirms the neoplasm in the right lung.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"1757-1626-1-123-1\"/>", "<graphic xlink:href=\"1757-1626-1-123-2\"/>", "<graphic xlink:href=\"1757-1626-1-123-3\"/>", "<graphic xlink:href=\"1757-1626-1-123-4\"/>" ]
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[{"surname": ["Ikeda"], "given-names": ["S"], "article-title": ["A case of lung cancer in a 2-year-old infant"], "source": ["Therapeutics"], "year": ["1968"], "volume": ["21"], "fpage": ["270"], "lpage": ["271"]}]
{ "acronym": [], "definition": [] }
10
CC BY
no
2022-01-12 14:47:25
Cases J. 2008 Aug 22; 1:123
oa_package/c0/ae/PMC2529260.tar.gz
PMC2529261
18718014
[ "<title>Introduction</title>", "<p>Transient osteoporosis of pregnancy (TOP) is a rare, idiopathic self-limiting condition typically associated with the third trimester of pregnancy. It almost always affects a single hip although bilateral presentation and involvement of the knee have been reported [##REF##16642889##1##, ####REF##17828410##2##, ##REF##17345549##3####17345549##3##].</p>", "<p>TOP usually presents with a sudden, quite severe onset of unilateral groin pain with no history of trauma. The patient may be unable to walk, or may have an antalgic gait. Pain is elicited by hip rotation, although a full range of motion is common. Radiographs are avoided in pregnancy where possible, and are a poor investigation for demonstrating early osteopaenia. Magnetic Resonance Imaging (MRI) reveals low signal intensity of bone marrow on T1 weighted images, and high signal on T2 weighted images suggestive of bone marrow oedema[##REF##1914294##4##]. The natural history is of resolution of symptoms over the course of 3 to 6 months</p>", "<p>Hip fracture secondary to TOP is very rare with only 12 reported patients in the literature to date; in two cases the hip fractures were bilateral[##REF##17828410##2##,##REF##17345549##3##,##REF##2777856##5##, ####REF##17495737##6##, ##REF##9266303##7##, ##REF##14504581##8####14504581##8##]. The majority of these fractures were caused by a traumatic event. Atraumatic hip fractures secondary to TOP are even more unusual and are easily overlooked and hence may present to the orthopaedic surgeon at a late stage, making management more challenging.</p>", "<p>We report of a case of bilateral TOP leading bilateral atraumatic femoral neck fractures that were diagnosed post partum. Despite the delay in presentation internal fixation was successfully carried out. We highlight the importance of adequate investigation of hip pain during pregnancy and discuss the role of MRI.</p>" ]
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[ "<title>Discussion</title>", "<p>Musculoskeletal complaints are very common in pregnancy. The position and weight of the gravid uterus alters the centre of gravity and loading patterns of the axial and appendicular skeleton, whilst hormonal changes lead to joint laxity, and fluid retention may cause neural compression[##REF##18199383##9##]. The majority of musculoskeletal complaints are not serious, and are managed conservatively without a specific diagnosis.</p>", "<p>Pregnant women frequently complain of hip or pelvic pain. The differential diagnosis includes some serious problems that need to be excluded, namely transient osteoporosis, osteonecrosis and pubic symphysiolysis.</p>", "<p>Conventionally ionising radiation is avoided during pregnancy although Brodell <italic>et al. </italic>suggested that in the third trimester of pregnancy the benefits of adequate investigation of hip pain may outweigh the minimal risks[##REF##2777856##5##]. There is no conclusive evidence that MRI has deleterious effects, however the safety of MRI has yet to be definitively proven[##REF##12006687##10##]. It is in common use in the third trimester of pregnancy where clinically indicated[##REF##15556670##11##] and is generally considered to be safe[##REF##16572573##12##]. MRI has a high sensitivity for diagnosis of occult hip fracture[##REF##15910835##13##] and can reliably distinguish between osteonecrosis transient osteoporosis[##REF##1914294##4##], making it the investigation of choice for hip pain in the third trimester of pregnancy.</p>", "<p>Displaced intracapsular fractures have a high incidence of non-union and avascular necrosis[##REF##8288658##14##]. It has however been shown that the risk of non-union is independent of bone quality[##REF##15773648##15##] therefore in young patients with high value hips internal fixation should be the goal.</p>" ]
[ "<title>Conclusion</title>", "<p>This case report highlights the need for vigilance in the assessment of musculoskeletal complaints in pregnancy, and demonstrates that the more conservative approach of internal fixation is viable. We suggest MRI should be considered for women presenting with significant hip pain in the third trimester of pregnancy.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>We describe a case of bilateral femoral neck fractures secondary to transient osteoporosis of pregnancy, which were diagnosed after delivery due to the desire to avoid ionising radiation. These fractures were presumed to be secondary to transient osteoporosis of pregnancy and were treated successfully with internal fixation despite delayed presentation. We discuss the role of MRI in the evaluation of hip pain in pregnancy.</p>" ]
[ "<title>Case presentation</title>", "<p>A 34 year old Persian woman, gravida 1, para 0, presented at 22 weeks of pregnancy with a two week history of left hip pain with no apparent precipitating event. Her past medical history included mild Multiple Sclerosis from which she was asymptomatic. She did not smoke or drink alcohol, had no history of corticosteroid, anticonvulsant or anticoagulant use and was not on any other medication. Clinical examination was unremarkable and no investigations were deemed appropriate. The working diagnosis at this stage was non-specific hip pain related to pregnancy and supportive measures were instituted.</p>", "<p>Over the following 12 weeks her hip pain worsened, and she started to experience pain in the contra lateral hip. Again there was no history of a traumatic event. Because of her pregnancy imaging of her hips was avoided. By 36 weeks of pregnancy she was unable to weight bear and became wheelchair bound. Pain in her hips and limitation of motion meant that a normal vaginal delivery was impossible; hence she underwent a caesarean delivery of a healthy baby at full term.</p>", "<p>She was brought to the attention of the orthopaedic team when plain radiographs (see figure ##FIG##0##1##) following delivery revealed a displaced intracapsular femoral neck fracture on the left and a valgus impacted right intracapsular femoral neck fracture on the right. The radiographs also revealed considerable osteopaenia. MRI (see figure ##FIG##1##2##) revealed these fractures, with reduced signal on T1 and increased signal on T2 in the femoral necks in keeping with TOP.</p>", "<p>She underwent closed reduction and internal fixation of the left hip. The right hip was internally fixed in situ. Two hole 135 degree dynamic hip screws were used in order to provide sufficient stability to allow immediate mobilisation despite bilateral fractures. Difficulty was encountered in ensuring that the threads of the dynamic hip screw had crossed the fracture site in the left hip as the level of the fracture was high in the femoral neck, consequently the tip of the implant had to be implanted close to the subchondral plate (see figure ##FIG##2##3##).</p>", "<p>The post-operative course was uncomplicated and the hip pain significantly improved immediately. Full weight bearing on the right, and partial weight bearing on the left was initiated on the first postoperative day, and maintained for the first 12 weeks. Check radiographs at 3 months showed no loss of fixation and the fractures appeared to be uniting in an adequate position. At six months she was pain free with no evidence of avascular necrosis or implant failure.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>AC assessed the patient at first presentation, JD and AL carried out the surgery, CWO was involved throughout admission and follow-up and was the major contributor in writing the manuscript. All authors contributed to, read and approved the final manuscript.</p>" ]
[ "<title/>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Antero-posterior radiograph of the pelvis post partum.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>T1 weighted coronal MRI scan of the pelvis post partum.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Antero-posterior radiograph of the pelvis post fixation with dynamic hip screws.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"1757-1626-1-120-1\"/>", "<graphic xlink:href=\"1757-1626-1-120-2\"/>", "<graphic xlink:href=\"1757-1626-1-120-3\"/>" ]
[]
[]
{ "acronym": [], "definition": [] }
15
CC BY
no
2022-01-12 14:47:25
Cases J. 2008 Aug 21; 1:120
oa_package/3d/64/PMC2529261.tar.gz
PMC2529262
18687129
[ "<title>Background</title>", "<p>Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by a defect of DNA-repair occurring during UV-induced damage. The disease is quite complex and different subsets of abnormalities in the DNA-repair system may be present during the course of the disease. Thus, patients with XP have a decreased cutaneous immune surveillance which results in an increased risk of UV-induced skin tumours at an early age. Basal cell carcinoma (BCC), squamous cell carcinoma (SCC), actinic keratoses, atypical moles and malignant melanoma, all associated with severe photoaging are commonly seen in such patients. The prognosis of XP is based on early diagnosis as to permit strict UV avoidance and early detection and treatment of skin tumours, especially in photo-exposed areas.</p>" ]
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[ "<title>Discussion</title>", "<p>To our knowledge, the association between psoriasis and XP has not been previously reported and may be regarded as unlikely when considering the pathogenesis of both diseases.</p>", "<p>XP patients exhibit Langerhans cell depletion, intensified impairment of natural killer (NK) cell function and enhanced UV-immunosuppression, possibly mediated through increased prostaglandin E2 production [##REF##1739289##1##, ####REF##11764287##2##, ##REF##14632192##3##, ##REF##16323244##4####16323244##4##]. By opposition, psoriatic plaques are characterized by immune activation, with NK cell activation and decreased cyclo-oxygenase activity [##REF##15577845##5##,##REF##3458681##6##].</p>", "<p>In addition, in XP, unrestricted cellular proliferation is associated with inactivation of members of the iNK4a/Arf locus, such as p14 and p16 [##REF##11078762##7##]. By opposition, in psoriasis, members of the iNK4a/Arf locus are overexpressed, which may contribute to the senescent switch and resistance of psoriatic plaques to cellular transformation despite altered differentiation, angiogenesis, increased telomerase activity, proliferative changes and apoptosis resistance characterising psoriatic skin [##REF##12507899##8##,##REF##11168753##9##].</p>", "<p>Further data are required to determine why and how those apparently antinomic diseases can coexist.</p>" ]
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[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>We present the case of an eighteen-year-old Caucasian white boy who was diagnosed with xeroderma pigmentosum type A at age 5 and who experienced over the past year disseminated small plaque psoriasis confirmed with skin punch biopsy. The psoriatic lesions were successfully treated with multipotent topical corticosteroids and systemic retinoids. To our knowledge, the association between psoriasis and xeroderma pigmentosum has not been previously reported and may be regarded as unlikely when considering the pathogenesis of both diseases.</p>" ]
[ "<title>Case presentation</title>", "<p>We present the case of an eighteen-year-old white Caucasian boy who was diagnosed with XP type A at age 5. Other clinical signs include photophobia, keratitis and loss of eyelashes. Despite rigorous monitoring and photoprotection, the patient developed over 100 facial skin cancers (mainly BCC and SCC) treated with cryotherapy and surgery. Surprisingly, the patient experienced over the past year disseminated small plaque psoriasis (Fig. ##FIG##0##1##) confirmed with skin punch biopsy. S100 staining showed reduced density of Langerhans cells in healthy skin (Fig. ##FIG##1##2##), as previously reported in XP group A [##REF##1739289##1##]. The psoriatic lesions were successfully treated with multipotent topical corticosteroids and systemic retinoids.</p>", "<title>Abbreviations</title>", "<p>BCC: Basal cell carcinoma; DNA: desoxy ribonucleic acid; iNK: invariant natural killer; NK: natural killer; SCC: squamous cell carcinoma; UV: ultraviolet; XP: xeroderma pigmentosum.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>KE, TS, MH examined the patient. KE and TS were the major contributors in writing the manuscript. DM first seen the patient and helped to the redaction of the manuscript. MH and MC treated the patient and MH performed the histological examination for the skin biopsy. All authors read and approved the final manuscript.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>" ]
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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Association of small erythematous scaly plaques with multiple areas of hyperpigmentation resembling freckles on the patient's back.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>S100 staining showing reduced density of Langerhans cells in healthy skin.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"1757-1626-1-74-1\"/>", "<graphic xlink:href=\"1757-1626-1-74-2\"/>" ]
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[]
{ "acronym": [], "definition": [] }
9
CC BY
no
2022-01-12 14:47:25
Cases J. 2008 Aug 7; 1:74
oa_package/12/0f/PMC2529262.tar.gz
PMC2529263
18671865
[ "<title>Background</title>", "<p>Juvenile dermatomyositis, a systemic vasculopathy, is the most common of the inflammatory myopathies in children and is characterized by symmetrical proximal muscle weakness, and a pathognomonic rash, which includes a heliotrope discoloration about the eyes, dilated capillaries at the nailbeds and eyelid margins, and thickened erythema over the knuckles (Gottron's papules). The three other diagnostic criteria are: serum elevation of muscle derived enzymes, a muscle biopsy with specific histological features that include mononuclear cell infiltrates as well as perifasicular atrophy with evidence of an occlusive vasculopathy, and a positive electromyogram documenting inflammation [##REF##1090839##1##].</p>", "<p>There is little information describing the critical variables that influence the development and course of this often devastating illness. Over 3.2 cases/million children/year are diagnosed, with a 2.1 girl to 1 boy ratio [##REF##12794783##2##]. At the time of their first symptom, rash or weakness, the mean age of the patient population is 6.7 years, while 25% of the children are age 4 or younger. It usually takes 4 months or more for the children to be diagnosed with JDM, when the muscle biopsy is obtained [##REF##9632086##3##]. At diagnosis, the extent and severity of the skin and muscle inflammatory response can be assessed using validated disease activity scores (DAS) for skin and muscle involvement [##REF##12794783##2##,##REF##12579588##4##,##REF##15818654##5##].</p>", "<p>Children with JDM often have a family history of autoimmune disease. The frequencies of the HLA antigens DQA1*0501, DQA1*0301, and DRB3 have been reported to be higher in the JDM population than control populations suggesting a genetic contribution to this disorder, which may be additionally influenced by the TNF-α-308 allelic polymorphisms [##REF##1783570##6##,##REF##8666549##7##].</p>", "<p>The precise stimulus initiating the inflammatory process is not known. However, there is evidence that newly diagnosed children with JDM have a history of infection, often respiratory or gastrointestinal in nature, within three months prior to the appearance of rash or muscle weakness [##REF##15818654##5##]. In addition, gene expression profile data from MRI directed diagnostic biopsies of muscle from untreated children with active symptoms of JDM identified up-regulation of many type I interferon- (IFNα/β) inducible genes [##REF##11937576##8##]. These findings support the interpretation that the inflammatory milieu in JDM is similar to that seen in anti-microbial responses. In that investigation we also found a marked down-regulation of genes associated with protein synthesis; both observations were subsequently confirmed in studies of muscle from adults with DM [##REF##15852401##9##].</p>", "<p>Untreated chronic inflammation in children with JDM is associated with the development of pathological calcifications [##REF##16492437##10##]. The phenotype of the children who present in clinic early in their disease course differs from those diagnosed later, both with respect to height and weight, and specific JDM symptoms. Children diagnosed early in the disease course are much weaker than those who have a longer time to diagnosis [##REF##16492437##10##]. However, the extent and severity of skin involvement appears to be stable over time [##REF##16492437##10##].</p>", "<p>With respect to diagnostic laboratory testing, serum levels of muscle enzymes, generally used to evaluate muscle inflammation, are more likely to be in the normal range when blood is collected 2–4.7 months after the child's first symptom (rash or weakness), making it more difficult to establish a diagnosis of definite JDM [##REF##16492437##10##]. The purpose of the present study was to examine the impact of the duration of untreated chronic inflammation on gene expression in diagnostic muscle biopsies obtained from a large group of untreated girls with clinical symptoms of active JDM in order to identify genes and biological processes associated with disease progression in JDM.</p>" ]
[ "<title>Methods</title>", "<title>Patient population</title>", "<p>Age appropriate informed consent was obtained from a total of 31 girls with definite/probable JDM (IRB# 2002-11762) and the 4 healthy age and sex-matched controls (IRB# 2001-11715) who were enrolled in this study. Ethical approval for this study was obtained from the Children's Memorial Hospital Institutional Review Board, and all procedures were carried out in accordance with the Helsinki Declaration.</p>", "<p>All the girls with JDM were negative for myositis specific or associated antibodies or for antibodies indicating overlap syndromes at the time of biopsy, and, as part of their diagnostic evaluation, had an MRI directed muscle biopsy. In the first step, 23 muscle biopsies from partially treated (n = 4) patients and untreated (n = 19) JDM patients and 4 control biopsies were expression profiled and used for gene filtering. In the second step, only untreated JDM patients at the time of muscle biopsy (n = 19, 3 short and 16 long duration) were analyzed statistically. In the third step, specific genes identified by the expression profiles were confirmed by testing additional samples from 8 untreated children with JDM. There was enough muscle biopsy material from 2 of the rare group of children with disease duration less than 2 months to be used for qRT-PCR enabling comparison of 5 patients each with long and short disease duration, as well as immunohistochemical studies.</p>", "<p>The date of recognition of the first symptom (rash or weakness) was defined as the \"disease onset date\". The \"duration of disease\" at the time of the muscle biopsy was defined as the time from disease onset to the date of muscle biopsy. We have reported the results from profile analyses performed using only samples from untreated girls with JDM; their demographics are presented in Table ##TAB##0##1##.</p>", "<p>Confirmation of the gene profiles by q-RT PCR utilized a separate set of 5 muscle biopsies from girls with a short duration of disease (1.4 ± 0.4 SD months), matched with 5 muscle biopsies' from girls with a long duration of untreated symptoms (6.8 ± 3.5 SD months); 2 children tested were also part of the gene profile long duration group. The girls were matched for age (8.2 ± 2.2 years compared with 9.5 ± 3.5 years), as well as disease activity, with respect to DAS skin (5.4 ± 0.9, 5.8 ± 1.5 respectively) and DAS muscle (8.2 ± 0.8, 8.0 ± 1.2 respectively). Details are presented in table ##TAB##1##2##.</p>", "<p>The four control muscle from non-inflammatory female donors were biopsies, obtained with informed consent, from thoracic muscle from girls undergoing plastic surgery for repair of cleft palate, which do not appear to have evidence of the genetic dysregulation observed in JDM [##REF##16415175##43##]. Their age range was 8–10.3; their mean age was 9.5 years.</p>", "<title>Disease Activity Scores</title>", "<p>Overall measure of severity of the JDM disease activity was assessed at the time of the diagnostic muscle biopsy using the total disease activity score (DAS), a 20 point scale which has two sub-scales, which reflect skin involvement (ranging from 0–9) and muscle inflammation (ranging from 0–11) [##REF##12579588##4##]. The skin component (DAS skin) is based on extent and severity of rash, the presence of telangiectasia (nailfold, palate, eyelids) and Gottron's papules [##REF##12579588##4##] The muscle component (DAS muscle) includes measures of muscle function and the extent of weakness in eight manoeuvres as evaluated by a single physician (LMP) on routine diagnostic physical examination. Both sub scores have been validated for inter-rater reliability.</p>", "<title>Muscle biopsy samples</title>", "<p>A diagnostic muscle biopsy, frequently the vastus lateralis, was obtained from the area of inflammation as defined by an MRI, using a T-2 weighted image with fat suppression. The sample was divided so that one portion was saved for immunohistochemical studies, while the other portion of the sample was used for gene expression profile studies and gene confirmation. Both parts of the muscle biopsy samples were snap frozen and stored in liquid nitrogen (-180°C).</p>", "<title>Determination of DQA1*0501 and DQA1*0301</title>", "<p>At the diagnostic visit, immediately prior to the muscle biopsy, peripheral blood mononuclear cells were obtained by Ficoll-Hypaque separation, and frozen in liquid nitrogen at -180°C.</p>", "<p>Genomic DNA was extracted from whole blood or frozen lymphocytes using the Puregene DNA Purification Kit (Gentra Systems, Minneapolis, MN). DNA from JDM patients was genotyped for HLA-DQA1*0301 and HLA-DQA1*0501 alleles using PCR amplification with sequence-specific primers as previously reported [##REF##8668926##44##,##REF##9096369##45##]. Aliquots of DNA (2 μl) were amplified using <italic>Taq</italic>Bead™ Hot Start Polymerase wax beads (1.25 U/bead; Promega Corporation, Madison, WI) in a reaction volume of 50 μl that contained 5× Green GoTaq™ Reaction Buffer (Promega Corporation, Madison, WI), dNTPs (0.2 mM each), and 0.4 μM allele specific primers for HLA-DQA1*0301 or HLA-DQA*0501.</p>", "<p>The primers for HLA-DQA1*0301 were: 5'-TTCACTCGTCAGCTGACCAT-3' (Forward) and 5'-CAAATTGCGGGTCAAATCTTCT-3' (Reverse), which amplify a 183 bp product. The primers for HLA-DQA*0501 were: 5'-ACGGTCCCTCTGGCCAGTA-3' (Forward) and 5'-AGTTGGAGCGTTTAATCAGAC-3' (Reverse), which amplify a 186 bp product.</p>", "<p>Denaturation was performed in each PCR at 94°C for 45 seconds, annealing at 62°C for 45, and extension at 72°C for 2 min (for the final step, extension was for 7 min). Absence or presence of PCR products was visualized by agarose gel electrophoresis.</p>", "<title>Determination of the TNF-α-308 polymorphism</title>", "<p>The TNF-α-308 polymorphism consists of a single base pair G to A substitution. PCR was used to amplify a 107 bp fragment that incorporated the polymorphic site into an NcoI restriction site as previously described [##REF##11037898##23##,##REF##9096369##45##] distinguishes AA from AG from GG.</p>", "<title>Expression profiles of JDM patient muscle biopsies</title>", "<p>Total RNA was isolated from each biopsy, processed for production of biotinylated cRNA and hybridization to microarrays, as we have previously described [##REF##11121445##28##]. Each sample was then hybridized to Affymetrix U133A microarrays containing approximately 14,500 well-characterized transcripts. Standard operating procedure and quality control was done as previously described [##REF##14970825##46##]. Muscle samples from 23 female JDM patients and 4 healthy age- and sex-matched controls were initially profiled.</p>", "<p>Generation of hybridization signals (probe set algorithms) of the microarrays was done using Affymetrix MAS (Version 5.0) (Affymetrix, CA), and dCHIP [##REF##11532216##47##]. After the absolute analysis, the gene expression levels were imported into GeneSpring software. The JDM samples were normalized to the mean of the profiles of age- and sex-matched control samples. Data filtering was done by retaining only those probe sets that showed at least two MAS5.0 \"present calls\" across all profiles. This resulted in retention of 67% of probe sets for the U133A microarrays.</p>", "<p>Welch t-test was used to calculate the probabilities of significant gene expression changes between samples with shorter (&lt; 2 months) and longer (≥ 2 months) disease duration. To reduce false positives, correction for multiple testing was done using Benjamini and Hochberg false discovery rate (5%) [##UREF##1##48##,##UREF##2##49##]. In addition, we used all treated and untreated patients to first generate a gene list with genes that showed statistically significant changes to reduce number of genes for multiple testing. We then used only the profiles of untreated patients and performed t-tests on the filtered genes with multiple testing corrections to minimize the false positives. To visualize transcripts showing coordinate regulation as a function of active disease duration, genes sharing temporal patterns were identified by hierarchical clustering using GeneSpring software. Clustering algorithm was based on standard correlation (r = 0.95). For hierarchical clustering, we included all genes with p &lt; 0.05 after multiple testing correction. In order to determine the presence of significant linear relationships between gene expression and duration of untreated disease, linear regression was performed between the gene expression levels of each of the 79 genes and duration of untreated disease. All profiles are publicly accessible via NCBI GEO <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ncbi.nlm.nih.gov/geo/\"/> (GSE11971).</p>", "<title>Quantitative Real Time-PCR verification (qRT-PCR)</title>", "<p>Total cellular RNA was extracted using Trizol Reagent (Invitrogen Corp.) and subsequently DNase treated using DNA-free (Ambion, Austin, TX). Reverse transcription reactions were performed using Superscript III Reverse Transcriptase (Invitrogen Corp., Carlsbad, CA) and random hexamer primers. Relative cDNA quantification of smooth muscle myosin heavy chain (SMMHC), clusterin and an internal reference gene, β-actin, were done using a TaqMan PCR Core Reagent Kit (Applied Biosystems; Roche Molecular Systems, Inc., New Jersey) using fluorescence-based detection method (Applied Biosystems 7500 Fast Real-Time PCR System; Applied Biosystems, Foster City, CA). The PCR reaction was performed using standard methodology as previously described for each gene of interest and the β-actin reference gene was used to normalize input cDNA.</p>", "<p>The primers and probe for SMMHC are as follows: 5'-CTGGGCAACGTAGTAAAACC-3' (Forward), 5'-TATAGCTCATTGCAGCCTCG-3' (Reverse), and 6FAM-ATAAGCTGGGCGTGGTGGTACACACCT-TAMRA (Probe) [##REF##14567992##50##].</p>", "<p>The primers and probe for Clusterin are as follows: 5'-GAGCAGCTGAACGAGCAGTTT-3' (Forward) 5'-CTTCGCCTTGCGTGAGGT-3' (Reverse) and 6FAM-ACTGGGTGTCCCGGCTGGCA-TAMRA (Probe) [##REF##15026542##51##]</p>", "<p>The primers and probe for β-actin are as follows: 5'-TGAGCGCGGCTACAGCTT-3' (Forward) 5'-TCCTTAATGTCACGCACGATTT-3' (Reverse) and 6FAM-ACCACCACGGCCGAGCGG-TAMRA (Probe) [##REF##15014008##52##]</p>", "<p>For relative cDNA quantification of class II antigen, HLA-DQA1 (#QT00060130), plexin D1 (#QT00036134), tenomodulin (#QT01024590), and an internal reference gene, β-actin (#QT00095431), QuantiTect Primer Assays and QuantiFast Syber Green PCR kits (Qiagen Inc., Valencia, CA) were used (Applied Biosystems 7500 Fast Real-Time PCR System; Applied Biosystems, Foster City, CA). The PCR reaction was performed according to manufacturer's protocol for these genes using β-actin reference gene to normalize input cDNA.</p>", "<title>Immunohistochemistry assay for mature dendritic cells</title>", "<p>Three untreated girls with long duration of active JDM, were age-race matched with three girls with duration of untreated symptoms of less than 2 months and 3 age-matched healthy female muscle donors and studied for presence of mature dendritic cells. Serial 6 um-thick frozen muscle sections were fixed in cold anhydrous acetone. Sections were then blocked for 30 minutes in 10% normal goat or donkey sera and incubated with primary antibody overnight at 4°C. Monoclonal antibodies against DC-LAMP (Beckman coulter, CA), raised in mice, were used at a dilution of 1:10. Polyclonal antibody against BDCA2 (Santa Cruz Biotechnology, CA), raised in goats, were used at a dilution of 1:50. After 3 washes with 1× PBS, slides were incubated for 1 hour at room temperature with biotin conjugated secondary antibodies against mouse and goat respectively (Jackson Immunoresearch, PA). Subsequently, slides were stained with Vectastain Elite (Vector Laboratories, CA), followed by a BioGenex Liquid DAB Substrate Kit (yielding brown coloration at site of positive antibody binding). The slides were then counterstained with haematoxylin and Scott's bluing solution (Ricca Chemical, Texas), dehydrated, and prepared for viewing.</p>" ]
[ "<title>Results</title>", "<title>Demographics</title>", "<p>All diagnostic biopsies studied were from untreated girls who had not been given any nonsteroidal or immunosuppressive therapy, and for whom a Disease Activity Score (DAS) was obtained at the time of biopsy (Table ##TAB##0##1##). The short disease duration group (mean 0.9 ± 0.3 months) was compared with the long duration group (mean 18 ± 27.9 months). Their ages at disease onset date were similar: 5.6 ± 1.0 years for the short duration group compared with 5.2 ± 2.7 years for long duration group. They were similar in disease activity scores. Total DAS for the short duration group was 11.7 ± 4.0 (mean DAS skin 5.0 ± 1.7, mean DAS muscle 6.7 ± 2.5) compared with the Total DAS for the long disease duration group of 12.0 ± 3.9 (mean DAS skin, 6.2 ± 1.4, mean DAS muscle 5.8 ± 3.1). Microarray analysis did not identify any gene significantly differentially expressed among different genotypes (TNF-α-308, DQA1*0301 and DQA1*0501). The second group of girls tested for confirmation of the gene expression changes by immunohistochemistry or RT-PCR, 5 patients each of long and short duration of untreated disease were also similar with respect to age at onset, race, and DAS scores (Table ##TAB##1##2##).</p>", "<title>Comparison of muscle biopsy gene profiles based on disease duration</title>", "<p>As noted above, duration of disease, measured as more than 2 months of symptoms is a critical variable, influencing both clinical and laboratory data [##REF##16492437##10##]. Since changes at the molecular level occur prior to phenotypic changes, we used 2 months (≥ 2 months compared with &lt; 2 months) as the grouping cut-off in the present study. In order to control for gender effects, this study was limited to girls. We identified 79 genes represented by 85 Affymetrix probe sets differentially expressed in the muscle samples. All genes reached statistical significance (p &lt; 0.05) after multiple testing correction with the false positive rate at 5%. In addition, in order to \"bracket\" the optimal cut-off time we compared data from patients with disease duration of 3 months or less with greater than 3 months, and also compared 4 months or less with greater than 4 months. There was no significant difference in expressed genes when the 4 month cut-off was used, and only 3 genes were differentially expressed when the 3 month cut-off was used.</p>", "<p>For an overview of the changes in relation to the baseline generated using the control samples from age-matched healthy girls undergoing cleft palate repair, we used hierarchical clustering analysis to visualize changes of gene expression patterns associated with the three groups (control, short and long active disease duration) (Figure ##FIG##0##1##). Six clusters were identified by visualizing the gene tree in Figure ##FIG##0##1##. Among the 79 genes, 44 genes were expressed at a lower level in the group with active disease greater than 2 months vs. shorter than 2 months (Figure ##FIG##0##1##, cluster A, B, and E), while 35 genes were expressed at a higher level in the group (Figure ##FIG##0##1##, cluster C, D, and F).</p>", "<p>Cluster A represents genes up-regulated only in JDM patients with short duration of active disease. Cluster B represents genes significantly down-regulated only in JDM patients with long duration of active JDM. Many genes in these two clusters are involved in protein turn over and stress responses [see Additional file ##SUPPL##0##1##]. Most genes in cluster E are either encoding enzymes or proteins with unknown functions. These genes were highly up-regulated in patients with short duration of active disease but not in the other groups suggesting that they are activated at the acute phase of the disease.</p>", "<p>For genes up-regulated in patients with longer duration of active disease (cluster C, D, and F), genes in cluster C and D were up-regulated in both groups when compared to healthy controls. However, the changes were significantly higher in the patients with long duration of active disease. Interestingly, most of the immune response genes identified in this study were grouped into these two clusters (Table ##TAB##2##3##). While genes in cluster F were expressed at higher level in the group with long duration, most of the genes in this cluster were down-regulated compared to the baseline (Table ##TAB##3##4##). In cluster F, most genes are associated with functions of vasculature remodelling. In this report, we will focus on genes in cluster C, D and F (Table ##TAB##2##3## and ##TAB##3##4##), while the complete gene list of each cluster is reported in Additional file ##SUPPL##0##1##.</p>", "<p>In a previous profiling study, we identified a list of immune response genes differentially expressed in JDM patients, in which many of the genes were induced by type I interferon (IFNα/β) [##REF##11937576##8##]. Although many probe sets on the U133A microarry are different from those on the Affymetrix FL microarray, the changes identified previously were verified using the new microarray. However we found no differential expression of those IFN inducible genes in the short and long disease duration group, suggesting the expression of these inflammatory response genes is independent of disease chronicity.</p>", "<title>Infiltration of mature dendritic cells in the patients with longer duration of active symptoms</title>", "<p>To interpret the changes of genes involved in immune responses in cluster C and D (Table ##TAB##2##3##), we first searched for genes that were highly expressed in specific immune cell types. Although most of the genes were not specifically expressed by a single cell type, HLA-DR and glycoprotein nmb have been shown to be highly expressed in dendritic cells compared to B cells, monocytes and T cells [##REF##12176896##11##]. This pattern of up-regulation of the genes suggested that there might be a higher number of dendritic cells in the muscle of children with a longer duration of active disease.</p>", "<p>Using qRT-PCR, we first verified the expression changes HLA-DQA1 which could be either directly involved in antigen presentation or a surrogate marker of an active site. Because supply of diagnostic muscle biopsy tissue was limited, we tested a different group of 5 girls with short duration of disease, less than 2 months and a different group of 3 girls with disease duration greater or equal to 2 months, plus 2 girls from the original long disease duration group, which had been gene profiled in this study (Table ##TAB##1##2##).</p>", "<p>The expression level of HLA-DQA1 was 4.28 fold (p &lt; 0.05) up-regulated in patients with longer duration of active disease, which verified our array data (5.6 fold, p &lt; 0.005) (Figure ##FIG##1##2##). We then performed immunohistochemistry, using an antibody directed against a dendritic cell maturation marker, DC-LAMP to localize the cells in the muscle tissues (Figure ##FIG##2##3##). Long disease duration JDM patients displayed a greater overall presence of mature dendritic cells (Figure ##FIG##2##3A##) compared to short disease duration JDM patients (3C). Greater concentrations of mature dendritic cells were found in perivascular regions compared with the endomesium, In addition, many mature dendritic cells were BDCA2 positive suggesting a plasmacytoid origin. No significant differences were found in the distribution of the BDCA2 positive plasmacytoid dendritic cells in JDM of either long or short disease duration (B, D). Muscle from healthy children displayed an absence of mature dendritic cells (E), but a similar display of BDCA2 positive cells (F) compared with JDM.</p>", "<title>Vasculature remodelling in JDM patients</title>", "<p>Based on the array data, genes involved in vasculature remodelling, including the structural gene, SMMHC (two probe sets, 7.9 and 6.4 folds, p &lt; 0.005), and regulatory factors, tenomodulin (13.1 fold, p &lt; 0.001), prostaglandin E receptor 4 (1.9 fold, p &lt; 0.005), plexin D1 (1.6 fold, p &lt; 0.001), Akt-3 (1.3 fold, p &lt; 0.001), clusterin (2.9 fold, p &lt; 0.001), and LPGDS (two probe sets, 2.4 and 1.9 folds, p &lt; 0.005) [##REF##15967161##12##, ####REF##10330402##13##, ##REF##11169458##14##, ##REF##14695456##15##, ##REF##9887984##16##, ##REF##15240344##17##, ##REF##15950187##18##, ##REF##15239959##19####15239959##19##], were significantly differentially expressed between patients with long and short duration of active disease, suggesting the involvement of vasculature remodelling during this period of time (Table ##TAB##2##3## and ##TAB##3##4##). In addition, matrix metalloproteinase 2 (2.0 fold, p &lt; 0.005) and collagen VIα2 (1.6 fold, p &lt; 0.005) were also up-regulated indicating extracellular matrix remodelling, which is part of angiogenesis [##REF##12726863##20##,##REF##11134507##21##]. Interestingly, most of these genes belonged to cluster F, in which genes were down-regulated more in patients with short duration of active disease when comparing to the baseline (Figure ##FIG##0##1##, cluster F), suggesting that the expression changes of these specific genes were preferentially misregulated in the earlier stage of the inflammatory disease process.</p>", "<p>Using qRT-PCR, we confirmed the higher expression of 4 genes related to vascular remodelling in muscle biopsies of girls with longer disease duration compared with disease of short duration. The expression level of smooth muscle heavy chain myosin was elevated 5.9 fold (p &lt; 0.05), clusterin was 4.74 fold increased (p &lt; 0.05); the level of plexin D1 was increased in the long duration group by 2.35 fold (p &lt; 0.005), and tenomodulin was increased by 4.6 fold, (p &lt; 0.05) (Figure ##FIG##1##2##).</p>", "<p>A regression analysis was performed of 79 genes and 9 genes (10 probe sets) were identified that had a statistically significant linear relationship between the level of gene expression and duration of active disease (Table ##TAB##4##5##). Both probe sets representing SMMHC showed linear relationships, suggesting that the gene expression changes are dependent on the duration of the chronic inflammation of active disease.</p>" ]
[ "<title>Discussion</title>", "<title>Impact of duration of untreated inflammation on clinical and laboratory findings in children with JDM</title>", "<p>Previous studies have confirmed the observation that prolonged duration of untreated symptoms in children with JDM are highly associated with the development of pathologic calcifications, one of the most debilitating complications of JDM [##REF##3871616##22##,##REF##11037898##23##]. Untreated children with a longer duration of chronic symptoms had a higher frequency of loss of nailfold capillary end row loops [##REF##15290747##24##], which persisted after 36 months of therapy [##REF##18240225##25##]. The chronic inflammation associated with nailfold capillary loss was accompanied by impaired absorption of orally administered corticosteroids [##REF##18240180##26##]. In addition, chronic untreated inflammation at diagnosis of JDM was associated with severe bone loss and an abnormal osteoprotegerin: RANKL ratio [##REF##16492437##10##]. A study of 166 previously untreated children with JDM enrolled in a large national NIAMS JDM Research Registry showed that the clinical presentation of the children with shorter duration of active JDM was quite different from those who came to diagnosis later in their disease course. Children diagnosed early in the disease course were more likely to be weaker than later on, and muscle enzymes were more likely to be in the normal range 2–4.7 months after the child's first symptom (rash or weakness) [##REF##16492437##10##] which may be associated with differential utilization of the TRAIL apoptotic pathway [##REF##16492437##10##,##REF##17704000##27##]. In this study we used a 2 month cut-off and identified 79 genes differentially expressed in patients with short (less than 2 months) and long (greater than 2 months or equal) duration of active disease by expression profiling. Identifying these changes provide insight concerning the observed differences in clinical phenotypes at the molecular level over time.</p>", "<title>Down regulation of genes involved in protein turnover and metabolism</title>", "<p>Down-regulation of protein turnover and stress response genes in skeletal muscles has been associated with muscular dystrophies and conditions associated with muscle atrophy [##REF##11121445##28##,##REF##11259386##29##]. In this study, genes involved in both protein turnover and stress responses were down-regulated significantly in patients with longer duration of active disease [see Additional file ##SUPPL##0##1##]. The genes encoding heat shock proteins (eg. heat shock 70 kDa protein 9B and heat shock 40) and genes involved in proteasome functions (eg. proteasome regulatory particle subunit p44S10, ubiquitination factor E4B, located in cluster A) showed slight upregulation in the short duration group compared to controls, but approached the baseline in the long duration group, reflecting the acute phase of the disease. In contrast, genes involved in protein synthesis (eg. eukaryotic translation initiation factor 2, mitochondrial ribosomal protein S10 and L15) were mildly downregulated in the short duration group and achieve significant down-regulation in the long duration group, suggesting continuous suppression of protein synthesis commonly associated with muscle atrophy. The changes at the molecular level support the observation of ongoing muscle loss in untreated patients with JDM. Gene involved in ER stress response have been previously reported to be differentially expressed in adults with inflammatory myopathies [##REF##15934115##30##]. In our study, we did not observe significant differences between the groups, based on the duration of untreated disease.</p>", "<title>Dendritic cell infiltration</title>", "<p>The presence of dendritic cells in the muscle of patients with dermatomyositis was previously reported [##REF##17469160##31##,##REF##16504012##32##]. Recent investigation presented data showing that the localization and maturation of resident plasmacytoid dendritic cells <italic>in situ </italic>in the perivascular areas was essential to the initiation and perpetuation of muscle inflammation in juvenile DM [##REF##17469160##31##]. The present study confirms that the typical pattern of dendritic cell infiltration (with heavy infiltration surrounding the blood vessels) in JDM is a progressive process associated with persistent inflammation without focal infiltration in muscle of patients with short duration of active disease. Many DC-LAMP positive dendritic cells were positive for BDCA2, again confirming that they were of plasmacytoid dendritic cell lineage as recently reported [##REF##17469160##31##]. Dendritic cells are well characterized for efficient antigen presentation to T cells and are speculated to affect both tolerance and T cell activation [##REF##16202211##33##]. Models of autoimmunity through activated dendritic cells in rheumatoid arthritis and in systemic lupus erythematosus (SLE) associated with a Type 1 IFN-α/β initiated response have been proposed by other investigators [##REF##16224814##34##, ####REF##15569606##35##, ##REF##16126005##36####16126005##36##]. In addition, increased expression of IFNα/β induced genes have been identified in peripheral blood of children with SLE, systemic onset juvenile rheumatoid arthritis, and in JDM [##REF##16859997##37##, ####REF##15851489##38##, ##REF##12960479##39####12960479##39##]. In this study, we found large number of dendritic cells in perivascular areas with dense mononuclear infiltrates in biopsies from children with longer duration of active disease, which suggested that dendritic cells might be actively involved in the progression of the disease by modulating T cells function in the muscle vasculature.</p>", "<title>Vasculature remodelling</title>", "<p>Vascular smooth muscle cells (SMCs) are highly plastic capable of profound alterations in phenotype in response to changes in local environmental cues [##REF##15269336##40##]. Vascular injury initiates a transition in the phenotype of vascular SMCs whereby \"contractile\" (differentiated) SMCs are capable of undergoing transient modification to a highly \"synthetic\" (dedifferentiated) state. These synthetic vascular SMCs are migratory, highly proliferative, and play a critical role in repair of the vascular injury. Upon resolution of the injury, SMCs reacquire their contractile phenotype and associated markers, which include smooth muscle isoforms of contractile apparatus proteins such as SMMHC [##REF##15269336##40##]. In this study, we found clusterin, tenomodulin and prostaglandin E receptor 4 were down-regulated in JDM patients with short disease duration. In contrast, for children with longer duration of active disease, expression of these genes approached the baseline, and additional genes involved in vasculature remodelling were up-regulated, (plexin D1, Akt-3, LPGDS). These genes are associated with contractile (differentiated) vascular SMCs. These data suggest that early in the course of JDM, a relatively high proportion of vascular SMCs at the site of inflammation are in a synthetic (dedifferentiated or undifferentiated) state; conversely, as time passes and the inflammation becomes chronic, vascular SMCs further differentiate. The linear regression relationship between the duration of active disease and the SMMHC expression suggests that the vasculature remodelling in the muscles is a progressive process associated with disease chronicity and the duration of active disease. This is consistent with the documentation of evidence of cardiovascular compromise in older patients who had JDM in childhood [##UREF##0##41##]. Among the genes involved in vasculature remodelling, plexin D1 and prostaglandin E receptor 4, are two potent angiogenic factors [##REF##15967161##12##,##REF##15239959##19##]. Plexin D1 is expressed in vascular endothelial cells of developing blood vessels. Signalling between semaphorin 3E and its receptor plexin D1 controls endothelial cell positioning and the patterning of the vasculature during embryonic development, and can negatively regulate angiogenesis as well as activate T cells [##REF##17318185##42##]. Because the hallmark of JDM is occlusion and obliteration of capillaries and arterioles, it is not surprising that expression of tenomodulin is increased, for it is overexpressed in hypovascular connective tissue. The up-regulation of these genes might underlie the molecular mechanisms of the vasculature loss and remodelling we observed in the patients' muscles.</p>", "<p>In summary, the findings from this study support the clinical observation that identification of the duration of the inflammatory process is a critical component in children with JDM, influencing both the gene expression and the pathophysiology of the immune responses in children with active symptoms. Recognition of this variable, which identifies the chronicity of the process, is important in dissecting out factors involved in immune progression and response.</p>" ]
[ "<title>Conclusion</title>", "<p>We conclude that the duration of the chronic inflammatory process in untreated JDM alters mRNA expression patterns, including both dendritic cell maturation and vascular remodelling with increased expression of anti-angiogenic factors. We propose that the duration of the inflammatory process must be considered when interpreting gene profiling data as well as clinical and laboratory findings in children with JDM in order to gain insight into potential modes of intervention. We speculate that interventions that diminish the antiangiogenic remodelling present in children with JDM who have a longer duration of untreated disease may be of benefit.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>To evaluate the impact of the duration of chronic inflammation on gene expression in skeletal muscle biopsies (MBx) from untreated children with juvenile dermatomyositis (JDM) and identify genes and biological processes associated with the disease progression, expression profiling data from 16 girls with active symptoms of JDM greater than or equal to 2 months were compared with 3 girls with active symptoms less than 2 months.</p>", "<title>Results</title>", "<p>Seventy-nine genes were differentially expressed between the groups with long or short duration of untreated disease. Genes involved in immune responses and vasculature remodelling were expressed at a higher level in muscle biopsies from children with greater or equal to 2 months of symptoms, while genes involved in stress responses and protein turnover were expressed at a lower level. Among the 79 genes, expression of 9 genes showed a significant linear regression relationship with the duration of untreated disease. Five differentially expressed genes – HLA-DQA1, smooth muscle myosin heavy chain, clusterin, plexin D1 and tenomodulin – were verified by quantitative RT-PCR. The chronic inflammation of longer disease duration was also associated with increased DC-LAMP<sup>+ </sup>and BDCA2<sup>+ </sup>mature dendritic cells, identified by immunohistochemistry.</p>", "<title>Conclusion</title>", "<p>We conclude that chronic inflammation alters the gene expression patterns in muscle of untreated children with JDM. Symptoms lasting greater or equal to 2 months were associated with dendritic cell maturation and anti-angiogenic vascular remodelling, directly contributing to disease pathophysiology.</p>" ]
[ "<title>Authors' contributions</title>", "<p>Y-WC oversaw the expression profiling, qRT-PCR and IHC experiments, determined data analysis strategies and performed the analysis, interpreted the profiling, qRT-PCR and IHC data, and prepared the manuscript. RS performed expression profiling and immunohistochemistry. HG-D performed statistical analyses and interpreted results. SS performed the qRT-PCR, supervised the muscle biopsy selection for study and wrote some of the experimental methods. NG performed the immunohistochemistry. LMP created the IRB protocol, oversaw the study, recruited the patients and supervised the biopsy collection, qRT-PCR and immunohistochemistry assays and participated in the literature review and manuscript preparation. All authors have read and approved the final manuscript.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>The study is supported by NIH RO-1AR48289 (LMP and Y-WC), and grant from The Myositis Association and Cure JM Program of Excellence in Myositis Research (LMP). Y-WC and RS are partially supported by NIH 1R24HD050846 and 1U54HD053177. We thank Dr. Paveen Patel for the contribution of normal controls with their consent.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Gene tree generated by hierarchical clustering based on gene expression patterns. </bold>Genes up-regulated in girls with long (≥ 2 m) vs. short (&lt; 2 m) duration of active disease were clustered into cluster C, D and F, while down-regulated genes were clustered into clusters A, B and E. The color codes represent the ratio between each of the JDM group compared with the age- and sex-matched control samples.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Five differentially expressed genes, HLA-DQA1, smooth muscle myosin heavy chain (SMMHC), clusterin, plexin D1, and tenomodulin were verified by quantitative RT-PCR, and their level of expression compared in diagnostic muscle biopsies from 5 girls with untreated symptoms of JDM for a short disease duration &lt; 2 months (open bars) and 5 girls with untreated symptoms of JDM of ≥ 2 months duration (black bars). </bold>* p &lt; 0.05, **p &lt; 0.005.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Comparisons of dendritic cells in skeletal muscle biopsies from JDM, long and short disease duration, and normal patients.</bold> DC-LAMP is a membrane bound protein produced by mature activated dendritic cells, while BDCA2 is an antigen produced by immature plasmacytoid dendritic cells (both markers are stained dark brown). Long disease duration JDM patients displayed a greater overall presence of mature dendritic cells (A) compared to short disease duration JDM patients (C). Greater concentrations of mature dendritic cells were found in perivascular and perifasicular regions compared with the endomysium. Many mature dendritic cells co-expressed plasmacytoid markers (A, B). No substantiated differences were found in the distribution of the BDCA2 positive plasmacytoid dendritic cells in JDM of either long or short disease duration (B, D). Normal pediatric muscle displayed an absence of mature dendritic cells with presence of BDCA2 positive cells (E). Images were taken at 10× on a Leica Upright Light Microscope. Scale bars represent 10 μm.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Demographics of children with JDM in expression profiling study.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Patient No.</bold></td><td align=\"center\"><bold>Age at Onset</bold></td><td align=\"center\"><bold>DAS Skin</bold></td><td align=\"center\"><bold>DAS Weak</bold></td><td align=\"center\"><bold>DAS Total</bold></td><td align=\"center\"><bold>Age at MBx</bold></td><td align=\"center\"><bold>Duration of Untreated Disease (Months)</bold></td><td align=\"center\"><bold>DQA 0301</bold></td><td align=\"center\"><bold>DQA 0501</bold></td><td align=\"center\"><bold>TNF Type</bold></td></tr></thead><tbody><tr><td align=\"left\" colspan=\"10\"><bold>Short Disease Duration</bold></td></tr><tr><td align=\"left\">1</td><td align=\"center\">4.7</td><td align=\"center\">4.0</td><td align=\"center\">7.0</td><td align=\"center\">11.0</td><td align=\"center\">4.8</td><td align=\"center\">1.1</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GA</td></tr><tr><td align=\"left\">2</td><td align=\"center\">6.8</td><td align=\"center\">7.0</td><td align=\"center\">9.0</td><td align=\"center\">16.0</td><td align=\"center\">6.8</td><td align=\"center\">1.1</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">3</td><td align=\"center\">5.5</td><td align=\"center\">4.0</td><td align=\"center\">4.0</td><td align=\"center\">8.0</td><td align=\"center\">5.50</td><td align=\"center\">0.6</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GA</td></tr><tr><td colspan=\"10\"><hr/></td></tr><tr><td align=\"left\">Mean</td><td align=\"center\">5.6</td><td align=\"center\">5.0</td><td align=\"center\">6.7</td><td align=\"center\">11.7</td><td align=\"center\">5.7</td><td align=\"center\">0.9</td><td/><td/><td/></tr><tr><td align=\"left\">ST. Dev.</td><td align=\"center\">1.0</td><td align=\"center\">1.7</td><td align=\"center\">2.5</td><td align=\"center\">4.0</td><td align=\"center\">1.1</td><td align=\"center\">0.3</td><td/><td/><td/></tr><tr><td colspan=\"10\"><hr/></td></tr><tr><td align=\"left\" colspan=\"10\"><bold>Long Disease Duration</bold></td></tr><tr><td align=\"left\">4</td><td align=\"center\">1.0</td><td align=\"center\">6.0</td><td align=\"center\">5.5</td><td align=\"center\">11.5</td><td align=\"center\">3.5</td><td align=\"center\">30.6</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GA</td></tr><tr><td align=\"left\">5</td><td align=\"center\">1.8</td><td align=\"center\">6.0</td><td align=\"center\">3.0</td><td align=\"center\">9.0</td><td align=\"center\">2.3</td><td align=\"center\">6.1</td><td align=\"center\">pos</td><td align=\"center\">neg</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">6</td><td align=\"center\">8.3</td><td align=\"center\">9.0</td><td align=\"center\">10.0</td><td align=\"center\">19.0</td><td align=\"center\">8.5</td><td align=\"center\">2.1</td><td align=\"center\">neg</td><td align=\"center\">neg</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">7</td><td align=\"center\">8.8</td><td align=\"center\">5.0</td><td align=\"center\">4.0</td><td align=\"center\">9.0</td><td align=\"center\">9.0</td><td align=\"center\">2.6</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GA</td></tr><tr><td align=\"left\">8</td><td align=\"center\">3.9</td><td align=\"center\">6.0</td><td align=\"center\">9.0</td><td align=\"center\">15.0</td><td align=\"center\">5.2</td><td align=\"center\">16.1</td><td align=\"center\">neg</td><td align=\"center\">neg</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">9</td><td align=\"center\">1.6</td><td align=\"center\">7.0</td><td align=\"center\">9.0</td><td align=\"center\">16.0</td><td align=\"center\">2.2</td><td align=\"center\">7.7</td><td align=\"center\">pos</td><td align=\"center\">neg</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">10</td><td align=\"center\">7.2</td><td align=\"center\">8.0</td><td align=\"center\">6.0</td><td align=\"center\">14.0</td><td align=\"center\">7.4</td><td align=\"center\">2.2</td><td align=\"center\">pos</td><td align=\"center\">neg</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">11</td><td align=\"center\">8.3</td><td align=\"center\">4.0</td><td align=\"center\">8.0</td><td align=\"center\">12.0</td><td align=\"center\">8.7</td><td align=\"center\">4.9</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">AA</td></tr><tr><td align=\"left\">12</td><td align=\"center\">6.0</td><td align=\"center\">7.0</td><td align=\"center\">3.0</td><td align=\"center\">10.0</td><td align=\"center\">7.7</td><td align=\"center\">20.6</td><td align=\"center\">pos</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">13</td><td align=\"center\">7.7</td><td align=\"center\">6.0</td><td align=\"center\">7.0</td><td align=\"center\">13.0</td><td align=\"center\">8.2</td><td align=\"center\">6.4</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">14</td><td align=\"center\">2.5</td><td align=\"center\">3.0</td><td align=\"center\">1.0</td><td align=\"center\">4.0</td><td align=\"center\">3.6</td><td align=\"center\">13.6</td><td align=\"center\">neg</td><td align=\"center\">neg</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">15</td><td align=\"center\">2.6</td><td align=\"center\">7.0</td><td align=\"center\">3.0</td><td align=\"center\">10.0</td><td align=\"center\">11.3</td><td align=\"center\">105.6</td><td align=\"center\">neg</td><td align=\"center\">neg</td><td align=\"center\">GA</td></tr><tr><td align=\"left\">16</td><td align=\"center\">6.1</td><td align=\"center\">7.0</td><td align=\"center\">10.0</td><td align=\"center\">17.0</td><td align=\"center\">6.6</td><td align=\"center\">6.6</td><td align=\"center\">neg</td><td align=\"center\">neg</td><td align=\"center\">GA</td></tr><tr><td align=\"left\">17</td><td align=\"center\">4.5</td><td align=\"center\">6.0</td><td align=\"center\">6.0</td><td align=\"center\">12.0</td><td align=\"center\">5.2</td><td align=\"center\">8.5</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">18</td><td align=\"center\">5.1</td><td align=\"center\">6.0</td><td align=\"center\">8.0</td><td align=\"center\">14.0</td><td align=\"center\">10.3</td><td align=\"center\">63.5</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">AA</td></tr><tr><td align=\"left\">19</td><td align=\"center\">8.4</td><td align=\"center\">6.0</td><td align=\"center\">1.0</td><td align=\"center\">7.0</td><td align=\"center\">8.6</td><td align=\"center\">3.2</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td colspan=\"10\"><hr/></td></tr><tr><td align=\"left\">Mean</td><td align=\"center\">5.1</td><td align=\"center\">6.2</td><td align=\"center\">5.8</td><td align=\"center\">12.0</td><td align=\"center\">6.7</td><td align=\"center\">18.8</td><td/><td/><td/></tr><tr><td align=\"left\">ST. Dev.</td><td align=\"center\">2.8</td><td align=\"center\">1.4</td><td align=\"center\">3.1</td><td align=\"center\">3.9</td><td align=\"center\">2.9</td><td align=\"center\">27.9</td><td/><td/><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Demographics of children with JDM in qRT-PCR validation.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Patient No.</bold></td><td align=\"center\"><bold>Age at Onset</bold></td><td align=\"center\"><bold>DAS Skin</bold></td><td align=\"center\"><bold>DAS Weak</bold></td><td align=\"center\"><bold>DAS Total</bold></td><td align=\"center\"><bold>Age at MBx</bold></td><td align=\"center\"><bold>Duration of Untreated Disease (Months)</bold></td><td align=\"center\"><bold>DQA 0301</bold></td><td align=\"center\"><bold>DQA 0501</bold></td><td align=\"center\"><bold>TNF Type</bold></td></tr></thead><tbody><tr><td align=\"left\" colspan=\"10\"><bold>Short Disease Duration</bold></td></tr><tr><td align=\"left\">1</td><td align=\"center\">6.8</td><td align=\"center\">5.0</td><td align=\"center\">9.0</td><td align=\"center\">14.0</td><td align=\"center\">6.9</td><td align=\"center\">1.2</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GA</td></tr><tr><td align=\"left\">2</td><td align=\"center\">6.9</td><td align=\"center\">5.0</td><td align=\"center\">8.0</td><td align=\"center\">13.0</td><td align=\"center\">7.0</td><td align=\"center\">1.7</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">3</td><td align=\"center\">6.1</td><td align=\"center\">7.0</td><td align=\"center\">9.0</td><td align=\"center\">16.0</td><td align=\"center\">6.3</td><td align=\"center\">1.8</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">4</td><td align=\"center\">11.6</td><td align=\"center\">5.0</td><td align=\"center\">8.0</td><td align=\"center\">13.0</td><td align=\"center\">11.7</td><td align=\"center\">1.0</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">5</td><td align=\"center\">8.9</td><td align=\"center\">5.0</td><td align=\"center\">7.0</td><td align=\"center\">12.0</td><td align=\"center\">9.0</td><td align=\"center\">1.5</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td colspan=\"10\"><hr/></td></tr><tr><td align=\"left\">Mean</td><td align=\"center\">8.1</td><td align=\"center\">5.4</td><td align=\"center\">8.2</td><td align=\"center\">13.6</td><td align=\"center\">8.2</td><td align=\"center\">1.4</td><td/><td/><td/></tr><tr><td align=\"left\">ST. Dev.</td><td align=\"center\">2.2</td><td align=\"center\">0.9</td><td align=\"center\">0.8</td><td align=\"center\">1.5</td><td align=\"center\">2.2</td><td align=\"center\">0.4</td><td/><td/><td/></tr><tr><td colspan=\"10\"><hr/></td></tr><tr><td align=\"left\" colspan=\"10\"><bold>Long Disease Duration</bold></td></tr><tr><td align=\"left\">6</td><td align=\"center\">8.5</td><td align=\"center\">6.0</td><td align=\"center\">7.0</td><td align=\"center\">13.0</td><td align=\"center\">9.6</td><td align=\"center\">12.9</td><td align=\"center\">neg</td><td align=\"center\">neg</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">7</td><td align=\"center\">5.6</td><td align=\"center\">8.0</td><td align=\"center\">8.0</td><td align=\"center\">16.0</td><td align=\"center\">5.9</td><td align=\"center\">4.0</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GA</td></tr><tr><td align=\"left\">8</td><td align=\"center\">8.3</td><td align=\"center\">4.0</td><td align=\"center\">8.0</td><td align=\"center\">12.0</td><td align=\"center\">8.7</td><td align=\"center\">4.9</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">AA</td></tr><tr><td align=\"left\">9</td><td align=\"center\">7.7</td><td align=\"center\">6.0</td><td align=\"center\">7.0</td><td align=\"center\">13.0</td><td align=\"center\">8.2</td><td align=\"center\">6.4</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td align=\"left\">10</td><td align=\"center\">14.7</td><td align=\"center\">5.0</td><td align=\"center\">10.0</td><td align=\"center\">15.0</td><td align=\"center\">15.2</td><td align=\"center\">5.9</td><td align=\"center\">neg</td><td align=\"center\">pos</td><td align=\"center\">GG</td></tr><tr><td colspan=\"10\"><hr/></td></tr><tr><td align=\"left\">Mean</td><td align=\"center\">9.0</td><td align=\"center\">5.8</td><td align=\"center\">8.0</td><td align=\"center\">13.8</td><td align=\"center\">9.5</td><td align=\"center\">6.8</td><td/><td/><td/></tr><tr><td align=\"left\">ST. Dev.</td><td align=\"center\">3.4</td><td align=\"center\">1.5</td><td align=\"center\">1.2</td><td align=\"center\">1.6</td><td align=\"center\">3.5</td><td align=\"center\">3.5</td><td/><td/><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Up-regulation of immune response genes in skeletal muscles of patients with active JDM longer than 2 months (Cluster C and D; genes are in order shown in figure 1)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Affymetrix accession</bold></td><td align=\"left\"><bold>p-value</bold></td><td align=\"left\"><bold>Fold change (long/short)</bold></td><td align=\"left\"><bold>Gene description</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>Cluster C</bold></td><td/><td/><td/></tr><tr><td align=\"left\">203981_s_at</td><td align=\"left\">6.2E-03</td><td align=\"left\">1.5</td><td align=\"left\">ATP-binding cassette, sub-family D (ALD), member 4</td></tr><tr><td align=\"left\">210105_s_at</td><td align=\"left\">1.5E-03</td><td align=\"left\">1.6</td><td align=\"left\">FYN oncogene related to SRC, FGR, YES</td></tr><tr><td align=\"left\">214430_at</td><td align=\"left\">1.4E-03</td><td align=\"left\">1.6</td><td align=\"left\">galactosidase, alpha</td></tr><tr><td align=\"left\">200602_at</td><td align=\"left\">6.5E-04</td><td align=\"left\">2.0</td><td align=\"left\">amyloid beta (A4) precursor protein</td></tr><tr><td align=\"left\">201103_x_at</td><td align=\"left\">5.7E-03</td><td align=\"left\">1.4</td><td align=\"left\">hypothetical protein LOC200030</td></tr><tr><td align=\"left\">209765_at</td><td align=\"left\">2.3E-03</td><td align=\"left\">1.6</td><td align=\"left\">a disintegrin and metalloproteinase domain 19</td></tr><tr><td align=\"left\">216510_x_at</td><td align=\"left\">9.6E-04</td><td align=\"left\">5.6</td><td align=\"left\">immunoglobulin heavy constant gamma 1</td></tr><tr><td align=\"left\">201069_at</td><td align=\"left\">3.9E-03</td><td align=\"left\">2.0</td><td align=\"left\">matrix metalloproteinase 2</td></tr><tr><td align=\"left\">203473_at</td><td align=\"left\">1.1E-04</td><td align=\"left\">2.2</td><td align=\"left\">solute carrier organic anion transporter family, member 2B1</td></tr><tr><td align=\"left\">203742_s_at</td><td align=\"left\">1.9E-03</td><td align=\"left\">1.5</td><td align=\"left\">thymine-DNA glycosylase</td></tr><tr><td align=\"left\">205917_at</td><td align=\"left\">6.5E-04</td><td align=\"left\">1.6</td><td align=\"left\">ZNF264</td></tr><tr><td align=\"left\">212671_s_at</td><td align=\"left\">2.5E-04</td><td align=\"left\">5.6</td><td align=\"left\">major histocompatibility complex, class II, DQ alpha 1/2</td></tr><tr><td/><td/><td/><td/></tr><tr><td align=\"left\"><bold>Cluster D</bold></td><td/><td/><td/></tr><tr><td align=\"left\">218376_s_at</td><td align=\"left\">4.1E-03</td><td align=\"left\">2.5</td><td align=\"left\">NEDD9 interacting protein with calponin homology and LIM domains</td></tr><tr><td align=\"left\">209079_x_at</td><td align=\"left\">9.1E-04</td><td align=\"left\">1.6</td><td align=\"left\">protocadherin gamma subfamily</td></tr><tr><td align=\"left\">38671_at</td><td align=\"left\">5.9E-04</td><td align=\"left\">1.6</td><td align=\"left\">plexin D1</td></tr><tr><td align=\"left\">201279_s_at</td><td align=\"left\">2.0E-03</td><td align=\"left\">1.6</td><td align=\"left\">disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila)</td></tr><tr><td align=\"left\">211066_x_at</td><td align=\"left\">6.5E-04</td><td align=\"left\">1.6</td><td align=\"left\">protocadherin gamma subfamily</td></tr><tr><td align=\"left\">215599_at</td><td align=\"left\">3.8E-04</td><td align=\"left\">2.1</td><td align=\"left\">SMA4</td></tr><tr><td align=\"left\">205717_x_at</td><td align=\"left\">1.8E-03</td><td align=\"left\">1.4</td><td align=\"left\">protocadherin gamma subfamily</td></tr><tr><td align=\"left\">217659_at</td><td align=\"left\">1.3E-03</td><td align=\"left\">1.6</td><td align=\"left\">KIAA0261</td></tr><tr><td align=\"left\">212607_at</td><td align=\"left\">1.1E-04</td><td align=\"left\">1.3</td><td align=\"left\">AKT3 (protein kinase B, gamma)</td></tr><tr><td align=\"left\">211748_x_at</td><td align=\"left\">4.0E-03</td><td align=\"left\">2.4</td><td align=\"left\">prostaglandin D2 synthase 21 kDa (brain)</td></tr><tr><td align=\"left\">215376_at</td><td align=\"left\">2.4E-04</td><td align=\"left\">1.4</td><td align=\"left\">CDNA FLJ12295 fis, clone MAMMA1001818</td></tr><tr><td align=\"left\">202259_s_at</td><td align=\"left\">1.1E-04</td><td align=\"left\">1.6</td><td align=\"left\">phosphonoformate immuno-associated protein 5</td></tr><tr><td align=\"left\">212187_x_at</td><td align=\"left\">2.1E-03</td><td align=\"left\">1.9</td><td align=\"left\">prostaglandin D2 synthase 21 kDa (brain)</td></tr><tr><td align=\"left\">206666_at</td><td align=\"left\">3.7E-04</td><td align=\"left\">2.5</td><td align=\"left\">granzyme K (serine protease, granzyme 3; tryptase II)</td></tr><tr><td align=\"left\">217947_at</td><td align=\"left\">5.9E-04</td><td align=\"left\">1.9</td><td align=\"left\">chemokine-like factor super family 6</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Genes involved in vasculature remodelling were down-regulated in patients with active JDM shorter than 2 months (Cluster F; genes are in order shown in figure 1).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Affymetrix accession</bold><break/></td><td align=\"left\"><bold>p-value</bold><break/></td><td align=\"left\"><bold>Fold change </bold><break/><bold>(long/short)</bold></td><td align=\"left\"><bold>Gene description</bold><break/></td></tr></thead><tbody><tr><td align=\"left\">213290_at</td><td align=\"left\">4.2E-03</td><td align=\"left\">1.6</td><td align=\"left\">collagen, type VI, alpha 2</td></tr><tr><td align=\"left\">201141_at</td><td align=\"left\">2.1E-06</td><td align=\"left\">3.2</td><td align=\"left\">glycoprotein (transmembrane) nmb</td></tr><tr><td align=\"left\">221796_at</td><td align=\"left\">1.9E-03</td><td align=\"left\">2.0</td><td align=\"left\">cDNA clone IMAGE:452016</td></tr><tr><td align=\"left\">207695_s_at</td><td align=\"left\">1.9E-03</td><td align=\"left\">5.8</td><td align=\"left\">immunoglobulin superfamily, member 1</td></tr><tr><td align=\"left\">201369_s_at</td><td align=\"left\">6.7E-04</td><td align=\"left\">1.6</td><td align=\"left\">zinc finger protein 36, C3H type-like 2</td></tr><tr><td align=\"left\">222043_at</td><td align=\"left\">1.1E-04</td><td align=\"left\">2.9</td><td align=\"left\">Clusterin</td></tr><tr><td align=\"left\">201497_x_at</td><td align=\"left\">1.3E-03</td><td align=\"left\">7.9</td><td align=\"left\">myosin, heavy polypeptide 11, smooth muscle</td></tr><tr><td align=\"left\">207961_x_at</td><td align=\"left\">4.9E-04</td><td align=\"left\">6.4</td><td align=\"left\">myosin, heavy polypeptide 11, smooth muscle</td></tr><tr><td align=\"left\">204897_at</td><td align=\"left\">1.2E-03</td><td align=\"left\">1.9</td><td align=\"left\">prostaglandin E receptor 4</td></tr><tr><td align=\"left\">220065_at</td><td align=\"left\">6.5E-04</td><td align=\"left\">13.1</td><td align=\"left\">Tenomodulin</td></tr><tr><td align=\"left\">205573_s_at</td><td align=\"left\">5.0E-04</td><td align=\"left\">1.9</td><td align=\"left\">sorting nexin 7</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Nine genes (10 probe sets, SMMCH are represented by two probe sets) for which the expression level correlated with the duration of untreated duration of JDM</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Affymetrix accession</bold></td><td align=\"left\"><bold>Gene description</bold></td><td align=\"left\"><bold>r square</bold></td><td align=\"left\"><bold>p-value</bold></td><td align=\"left\"><bold>Cluster</bold></td></tr></thead><tbody><tr><td align=\"left\">207961_x_at</td><td align=\"left\">myosin, heavy polypeptide 11, smooth muscle</td><td align=\"left\">0.47</td><td align=\"left\">0.002</td><td align=\"left\">F</td></tr><tr><td align=\"left\">201369_s_at</td><td align=\"left\">zinc finger protein 36, C3H type-like 2</td><td align=\"left\">0.40</td><td align=\"left\">0.005</td><td align=\"left\">F</td></tr><tr><td align=\"left\">203742_s_at</td><td align=\"left\">thymine-DNA glycosylase</td><td align=\"left\">0.39</td><td align=\"left\">0.006</td><td align=\"left\">C</td></tr><tr><td align=\"left\">214430_at</td><td align=\"left\">galactosidase, alpha</td><td align=\"left\">0.34</td><td align=\"left\">0.011</td><td align=\"left\">C</td></tr><tr><td align=\"left\">201497_x_at</td><td align=\"left\">myosin, heavy polypeptide 11, smooth muscle</td><td align=\"left\">0.33</td><td align=\"left\">0.013</td><td align=\"left\">F</td></tr><tr><td align=\"left\">204807_at</td><td align=\"left\">transmembrane protein 5</td><td align=\"left\">0.28</td><td align=\"left\">0.025</td><td align=\"left\">A</td></tr><tr><td align=\"left\">214437_s_at</td><td align=\"left\">serine hydroxymethyltransferase 2 (mitochondrial)</td><td align=\"left\">0.27</td><td align=\"left\">0.027</td><td align=\"left\">E</td></tr><tr><td align=\"left\">215376_at</td><td align=\"left\">CDNA FLJ12295 fis, clone MAMMA1001818</td><td align=\"left\">0.27</td><td align=\"left\">0.027</td><td align=\"left\">D</td></tr><tr><td align=\"left\">202854_at</td><td align=\"left\">hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan syndrome)</td><td align=\"left\">0.25</td><td align=\"left\">0.034</td><td align=\"left\">E</td></tr><tr><td align=\"left\">221931_s_at</td><td align=\"left\">SEH1-like (S. cerevisiae)</td><td align=\"left\">0.23</td><td align=\"left\">0.042</td><td align=\"left\">A</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p><bold>Supplemental table 1</bold>. Complete gene list of genes in cluster A-F. All genes are in order shown in figure ##FIG##0##1##.</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><p>DAS: disease activity score; MBx: muscle biopsy; DQA: Major histocompatibility complex, class II, DQ alpha-1; TNF type: Tumor necrosis factor -308 allele.</p></table-wrap-foot>", "<table-wrap-foot><p>DAS: disease activity score; MBx: muscle biopsy; DQA: Major histocompatibility complex, class II, DQ alpha-1; TNF type: Tumor necrosis factor -308 allele.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1471-2172-9-43-1\"/>", "<graphic xlink:href=\"1471-2172-9-43-2\"/>", "<graphic xlink:href=\"1471-2172-9-43-3\"/>" ]
[ "<media xlink:href=\"1471-2172-9-43-S1.xls\" mimetype=\"application\" mime-subtype=\"vnd.ms-excel\"><caption><p>Click here for file</p></caption></media>" ]
[{"surname": ["Eimer", "Young", "Abbott", "Seshadri", "Gursahney", "Smulevitz", "Rhew", "Ramsey-Goldman", "McPherson"], "given-names": ["MJ", "L", "K", "R", "A", "B", "E", "R", "D"], "article-title": ["Abnormal cardiovascular risk profiles in adult patients with juvenile dermatomyositis."], "source": ["Arthritis Rheum"], "year": ["2006"], "volume": ["54:S519"]}, {"surname": ["Benjamini", "Hochberg"], "given-names": ["Y", "Y"], "article-title": ["On the adaptive control of the false discovery rate: A practical and powerful approach to multiple testing."], "source": ["Journal of Educational and Behavioral Statistics"], "year": ["2000"], "volume": ["25"], "fpage": ["60"], "lpage": ["83"]}, {"surname": ["Benjamini", "Hochberg"], "given-names": ["Y", "Y"], "article-title": ["Controlling the false discovery rate: A practical and powerful approach to multiple testing."], "source": ["Journal of the Royal Statistical Society"], "year": ["1995"], "volume": ["Series B 57"], "fpage": ["289"], "lpage": ["300"]}]
{ "acronym": [], "definition": [] }
52
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2022-01-12 14:47:25
BMC Immunol. 2008 Jul 31; 9:43
oa_package/4f/65/PMC2529263.tar.gz
PMC2529264
18710581
[ "<title>Background</title>", "<p>The cytokine interleukin 1 beta (IL-1β) is a potent mediator in response to infection and injury [##REF##9646173##1##]. It is produced mainly by blood monocytes, but also by macrophages, dendritic cells and a variety of other cells in the body [##REF##8630372##2##,##REF##11994473##3##]. A minute amount of IL-1β <italic>in vivo </italic>can evoke fever, hypotension, release of adrenocorticotrophic hormone and production of cytokines which in turn induce various inflammatory and immune responses.</p>", "<p>Increased IL-1β production has been reported in patients with various infections, inflammation, trauma (surgery), ischemic diseases, tumors, intravascular coagulation, autoimmune disorders, UV radiation, graft-versus-host disease, transplant rejection, and in healthy subjects after strenuous exercise [##REF##15362934##4##,##REF##3493347##5##]. An increasing IL-1β production was observed in patients with Alzheimer's disease and a possible role for IL-1β in the release of the amyloid precursor protein was proposed [##REF##9772027##6##]. Significant elevations of plasma IL-1β have been detected in healthy humans injected with lipopolysaccharide (LPS) and in patients with septic shock and burns [##REF##1395662##7##]. Correlations have been found between plasma IL-1β levels and severity of acute attacks of rheumatoid arthritis, thermal burns, and mortality in septic shock [##REF##8379598##8##]. Agents that reduce the production and activity of IL-1β are likely to have an impact on clinical applications. In fact, IL-1Ra, a blocker of IL-1β transduction, has been administered to patients with septic shock, rheumatoid arthritis, steroid resistant graft-versus-host disease, AML, CML and so on [##REF##8379598##8##, ####REF##8124953##9##, ##REF##8196140##10##, ##REF##8049450##11####8049450##11##].</p>", "<p>Development of a method to monitor IL-1β gene promoter activity <italic>in vivo </italic>will facilitate its use in the study of related diseases and preclinical evaluation of anti-inflammatory drugs. For this purpose, in this paper, we have established a transgenic mouse model using the human IL-1β gene promoter [##REF##8021507##12##] to direct the expression of luciferase reporter gene. When combining with the approach of \"biophotonic\" imaging using a highly light-sensitive camera system [##REF##11549548##13##, ####REF##9461201##14##, ##REF##11801687##15####11801687##15##], this model allows us to non-invasively study the transcriptional activity of IL-1β gene promoter in real time. Our data indicate that human IL-1β gene promoter functions in transgenic mice and this model can be used to study transcriptional regulation of the IL-1β gene expression in the inflammatory process and evaluate the effects of anti-inflammatory agents on IL-1β gene induction <italic>in vivo</italic>.</p>" ]
[ "<title>Methods</title>", "<title>Reagents</title>", "<p>Bacterial LPS (from Escherichia Coli Serotype 0127:B8), zymosan A (a cell wall preparation from Saccharomyces cerevisiae), dexamethasone and oxazolone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Luciferin (Biosyth, Basel, Switzerland) was dissolved in PBS at 15 mg/ml and stored at -20°C.</p>", "<title>Generation of cHS4I-hIL-1βP-Luc transgenic mice</title>", "<p>A 4.5-kb <italic>Kpn </italic>I-<italic>Bgl </italic>II fragment of human IL-1β gene promoter (hIL-1βP) was isolated from human genomic DNA by PCR amplification according to the literature [##REF##8021507##12##]. The promoter fragment was verified by DNA sequencing and cloned into the polylinker sites <italic>Kpn </italic>I and <italic>Bgl </italic>II of pGL3-Basic (Promega) to direct the expression of luciferase reporter gene (hIL-1βP-Luc). The resulting plasmid was named as pGL3-hIL-1βP-Luc. In order to eliminate the transgenic chromosomal insertion site effect on transgene expression[##REF##12893244##31##], the <italic>Not </italic>I-<italic>Kpn </italic>I fragment of the chicken HS4 insulator (cHS4I) was cloned into the <italic>Not </italic>I and <italic>Kpn </italic>I sites upstream to hIL-1βP. Then, the constructed cHS4I-hIL-1βP-Luc cassette was cut and subcloned into the <italic>Apa I </italic>and <italic>Sal </italic>I sites of plasmid pGFP-1. The cHS4I-hIL-1βP-Luc transgenic cassette (Fig. ##FIG##0##1A##) was cut out by <italic>Apa </italic>I and <italic>Mlu </italic>I, and used to generate transgenic mice in the C57XCBA background by standard microinjection techniques. The transgenic mice were bred to BALB/c for 5 generations before testing. All animals were housed in a specific pathogen-free environment in accordance with institutional guidelines for animal care. Animal handling was performed in accordance with institutional guidelines and approved by the local institutional animal care and use committee.</p>", "<title>Genotyping of cHS4I-hIL-1βP-Luc transgene in mice</title>", "<p>Transgenic founders and their offspring were identified by PCR using the forward-luc (5' TTCCGCCCTTCTTGGCCTTTATGA 3') and reverse-luc (5' CAGCTATTCTGATTACACCCGAGG 3') primers specific for luciferase gene.</p>", "<title><italic>In vivo </italic>imaging</title>", "<p><italic>In vivo </italic>bioluminescent imaging was performed using an IVIS imaging system (Xenogen, Alameda, CA) as previously described. 150 ul sodium salt luciferin (dissolved in PBS) was injected into the intraperitoneal cavity at a dose of 150 mg/ml. Mice were anesthetized with isoflurane/oxygen and placed on the imaging stage. 12 min after luciferin injection, mice were imaged for 1 to 5 min. Photons emitted from specific regions were quantified using a LivingImage software (Xenogen). <italic>In vivo </italic>luciferase activity was presented in photons emitted per second.</p>", "<title>Functional screening of cHS4I-hIL-1βP-Luc transgenic lines</title>", "<p>For primary functional screening of transgenic lines, the luciferase gene PCR positive mice were imaged for expression of the luciferase transgene without any treatment or at 5 h after LPS injection at the does of 3.0 mg/kg. The criteria used for screening available transgenic lines were: (1) lower basal luciferase expression in normal status and (2) higher induced luciferase expression in whole body after LPS injection.</p>", "<title>Acute septic shock model by intraperitoneal LPS injection</title>", "<p>The acute septic shock model was produced by i.p. injection of LPS (3.0 mg/kg) into cHS4I-hIL-1βP-Luc transgenic mice at the age of 2–3 mo. Control mice were injected with saline. At selected time points after the treatment, mice were i.p. injected with luciferin and imaged 12 min later with the IVIS imaging system described above. To test the effect of dexamethasone on LPS-triggered luciferase expression, male mice were cotreated with dexamethasone and LPS, and the control mice were injected with saline and LPS. The luciferase signal was monitored through imaging.</p>", "<title>Acute arthritis model by intra-articular administration of zymosan</title>", "<p>Zymosan A was suspended in sterile saline containing 5% glucose at a concentration of 30 mg/ml. Female cHS4I-hIL-1βP-Luc mice at the age of 2–3 mo were anesthetized with isoflurane. Then the hind legs of mice were shaved and the skin was sterilized with 70% ethanol. The right knee tendon was exposed and 10 μl of zymosan A suspension was intra-articularly injected through the tendon with a 25-gauge needle. The left rear knees of the same mice were intra-articularly injected with 5% glucose in saline (sham control). Mice were imaged at selected time points after the injection. Separately, mice were pretreated with dexamethasone (50 μg/mouse i.p.) half hour before the zymosan administration, and the luciferase signal was monitored through imaging.</p>", "<title>Induction of contact hypersensitivity (CHS) reaction</title>", "<p>Female cHS4I-hIL-1βP-Luc mice, 3–6 mo of age, were shaved in the thorax area and treated topically with 50 μl of 2% oxazolone solution (in acetone/olive oil (4/1, vol/vol)). Six days after sensitization, the right ears were challenged by topical application of 10 μl of 1% oxazolone solution, and the left ears were treated with vehicle (acetone/olive oil, 4:1 vol/vol) alone. The extent of inflammation was assessed by measuring ear thickness with a micrometer. Bioluminescent images were collected on the day of sensitization (day -6), the day of challenge (day 0), and at day 1 and 30 after treatment.</p>", "<title>Luciferase activity assay <italic>in vitro</italic></title>", "<p>Luciferase activity was assayed using the Luciferase Assay System (Promega) on a Luminometer (Lumat LB9507, EG&amp;G, Berthold, Germany).</p>", "<title>Total RNA isolation</title>", "<p>Female cHS4I-hIL-1βP-Luc mice, 3–6 mo of age, were treated with i.p. injection of LPS (3.0 mg/kg). Control mice were injected with saline. After 5 h, total RNA was isolated from selected mouse tissues using TRIZOL (Invitrogen, Paisley, Scotland, UK) according to the manufacturer's instruction and the extracted RNAs were kept at -80°C prior to use.</p>", "<title>Real-time reverse transcription-polymerase chain reaction (real time RT-PCR)</title>", "<p>Two μg of the RNA samples isolated from the LPS-treated female cHS4I-hIL-1βP-Luc mice was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Promega) in a final reaction volume of 25 μl. For real time PCR amplification, 0.5 μl of cDNA per reaction and 0.2 μM primer sets were used. Reaction conditions were as follows: 94°C/4 min; 51 cycles of 94°C/30 s, 60°C/30 s, 72°C/30 s; and 72°C/10 min. Evagreen used for detecting PCR products was purchased from Molecular Probes (Gentaurer, Brussels, Belgium). Amplification and detection of Evagreen were performed with RG-3000 Real Time Thermal Cycler (Corbett. Research, Sydney, Australia). The expression of the mRNA for the murine β-actin gene was used as a reference to normalize expression levels. All data were expressed relative to β-actin to compensate for any difference in sample loading, and all experiments were performed in triplicates. Data were obtained as threshold cycle (Ct) values (PCR cycle numbers at which fluorescence was threshold) using Rotor-Gene Real-Time Analysis Software 6.0 and used to calculate ΔCT values (ΔCT is the Ct of the target gene subtracted from the Ct of the housekeeping gene). Fold changes relative to saline controls were determined by the 2-ΔΔCT method.</p>", "<p>Primers for real time PCR were synthesized as the sequences listed below (Sangon, Shanghai, China). The forward murine IL-1β primer used was 5' AAGGAGAACCAAGCAACGACAAAA 3', the reverse murine IL-1β primer used was 5' TGGGGAACTCTGCAGACTCAAACT 3'. The forward murine β-actin primer used was 5' CCTGTATGCCTCTGGTCGTA 3', the reverse murine β-actin primer used was 5' CCATCTCCTGCTCGAAGTCT 3'.</p>", "<title>Western blot analysis of mIL-1β and luciferase activity assay in tissues</title>", "<p>Mouse tissues were removed and homogenized in 3 volumes of phosphate buffered saline (PBS) containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) and lysed with passive lysis buffer (Promega). After centrifugation at 14,000 rpm for 10 min at 4°C, the supernatant was collected. Luciferase activity was measured using a luminometer and the Luciferase Assay System (Promega). Protein concentration in lysate was estimated by Bradford reagent (Sangon, Shanghai, China). For Western blot analysis, equal amounts of proteins (120 μg) were loaded onto SDS-PAGE gels. After transfer, the membrane was probed with a specific anti-mIL-1β rabbit polyclonal Ab (Chemicon International, Temecula, CA, USA). The blots were developed using the ECL Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ, USA).</p>", "<title>Statistics</title>", "<p>All data are expressed as means ± SE. The data were analyzed by one-way ANOVA. A P value of less than 0.05 was considered significant.</p>" ]
[ "<title>Results</title>", "<title>Founder screening and molecular characterization</title>", "<p>The plasmid used for construction of transgenic mice was illustrated in Fig. ##FIG##0##1A##. Transgenic founders is identified by PCR detection of luciferase gene (using the primer pair marked in Fig. ##FIG##0##1A##) in tail-clip DNA (Fig. ##FIG##0##1B##). Four founder mice were obtained and crossed to BALB/c mice for five generations to generate progeny for further experiments.</p>", "<title>Induction of luciferase expression in cHS4I-hIL-1βP-Luc transgenic mice by LPS</title>", "<p>The progenies of cHS4I-hIL-1βP-Luc transgenic founders were screened for luciferase expression in response to LPS as described in materials and methods. All transgenic lines showed robust inducible luciferase activity in the whole body after LPS treatment while injection with saline did not induce luciferase expression. One line named BALB/cTg(cHS4I-hIL-1βP-Luc)Xen had the highest LPS-induced luciferase expression (Fig. ##FIG##1##2##) and was selected for further studies. In these mice, luciferase activity was detectable 1 h (<italic>n </italic>= 3/group, <italic>p </italic>&gt; 0.05 in males compared with the baseline level, <italic>p </italic>&lt; 0.01 in females compared with the baseline level) after LPS treatment all over the body and relatively higher expression levels were seen at the position of liver, intestine and lungs. The expression signal peaked at 3 h (<italic>n </italic>= 3/group, <italic>p </italic>&lt; 0.001 in males and females compared with the baseline level, respectively) after treatment and then gradually declined; by 168 h (<italic>n </italic>= 3/group, <italic>p </italic>&gt; 0.05 in males compared with the baseline level, <italic>p </italic>&lt; 0.01 in females compared with the baseline level) the signal had returned to the baseline level (Fig. ##FIG##1##2A, B##). As quantified by the LivingImage software, both male and female mice showed significant LPS-induced luciferase signal above the baseline level from 1 to 72 h after treatment, and the kinetics and magnitude of the signals were similar in the two sexes (Fig. ##FIG##1##2C##). At the peak, the luciferase signal was induced by 7–8 fold in both sexes. In another experiment, no significant difference in LPS-induced luciferase expression was found among mice aged from 2 to 7 months (data not shown).</p>", "<title>Expression of luciferase was induced in parallel with mouse endogenous IL-1β in multiple tissues after LPS treatment</title>", "<p>To confirm the <italic>in vivo </italic>activity data observed by live imaging, in another set of experiments, luciferase activity was detected <italic>ex vivo </italic>in dissected organs of cHS4I-hIL-1βP-Luc female mice at 5 h after LPS injection (Fig. ##FIG##2##3A##). The luciferase activity was significantly higher in the liver, lungs, duodenum, kidneys, brain, and heart. As compared with the saline treated mice, LPS treatment induced the luciferase activity by 11-fold in the liver, 19.6-fold in the lungs, 21-fold in the duodenum, 10-fold in the kidneys, 5-fold in the brain, 4.5-fold in the heart, 5.5-fold in the stomach, 2.7-fold in the thymus, and 1.5-fold in the spleen (Fig. ##FIG##2##3B##), which was consistent with the <italic>in vivo </italic>results at 5 h in Fig. ##FIG##1##2B##.</p>", "<p>The LPS-induced expression of mouse endogenous IL-1β mRNA was evaluated by real-time RT-PCR (Fig. ##FIG##2##3C##). In a large degree in parallel with that of the induced luciferase expression, LPS treatment increased endogenous IL-1β mRNA expression by 15.6-fold in the liver, 9.5-fold in the lungs, 4.7-fold in the kidneys, 9-fold in the brain, 5-fold in the heart, 2.5-fold in the stomach, 12.7-fold in the thymus, 4.2-fold in the spleen, and 1.3-fold in the duodenum. Western Blot analysis showed that LPS significantly induced the expression of pro-IL-1β protein in lung, spleen, and thymus of mice (Fig. ##FIG##2##3D##).</p>", "<title>Luciferase expression in cHS4I-hIL-1βP-Luc transgenic mice was inducible during acute arthritis</title>", "<p>The inducibility of IL-1β gene expression during acute inflammatory arthritis was studied using the cHS4I-hIL-1βP-Luc transgenic mouse model. Intra-articular injection of zymosan into the right knee joint of cHS4I-hIL-1βP-Luc mice caused a local induction of luciferase signal which was detectable at 2 h, peaked at 8 h, started to decline at 24 h and was still detectable at 96 h after the local treatment (Fig. ##FIG##3##4A, B##). The luciferase signal at the zymosan-injected right knee joints were 3.1-, 7.6-, 6.9-, and 2.8-fold of the base level at 3, 8, 24, and 96 h, respectively (Fig. ##FIG##3##4B##). The saline injected left knees showed slight increase in luciferase signal, possibly caused by a systemic response to zymosan administration and/or the lesion at the injection site. The induction of luciferase signal correlated with an increase of knee joint volume, as measured across lateral/medial axis and the anterior/posterior axis of the knee joints (results not shown).</p>", "<title>Luciferase expression was inducible during CHS reaction</title>", "<p>In a third model of inflammatory disease, cHS4I-hIL-1βP-Luc female mice were sensitized with oxazolone and topically challenged at the right ear with oxazolone 6 days later. Oxazolone challenge induced luciferase expression in the right ears at the level of 2.6 fold of the base level at day 1 after challenge (Fig. ##FIG##4##5B##). A weak induction of luciferase expression was also observed in the vehicle injected left ear. Induction of luciferase expression correlated with increase in ear thickness (Fig. ##FIG##4##5C##).</p>", "<title>Dexamethasone inhibited the induction of luciferase expression</title>", "<p>Dexamethasone, a synthetic glucocorticoid, has been well characterized for its ability to inhibit IL-1β production. A major mechanism is that dexamethasone inhibits IκB degradation and therefore suppresses IL-1β gene transcription promoted by NF-κB signaling pathway [##REF##10936515##16##,##REF##9143378##17##]. Male cHS4I-hIL-1βP-Luc mice were cotreated with LPS and dexamethasone (3 mg/kg i.p.) or LPS and saline. The dexamethasone cotreated mice had 51.5% lower induced luciferase expression as compared with the saline cotreated mice (Fig. ##FIG##5##6A, B##). Similarly, dexamethasone inhibited the induced luciferase expression in the zymosan induced acute arthritis model (Fig. ##FIG##5##6C, D##).</p>" ]
[ "<title>Discussion</title>", "<p>We have generated a cHS4I-hIL-1βP-Luc transgenic mouse model for monitoring hIL-1β gene transcriptional activity during inflammation. Using an <italic>in vivo </italic>bioluminescence imaging system, we demonstrated that luciferase signals in the cHS4I-hIL-1βP-Luc transgenic mice were dramatically induced in the whole body (especially in liver, duodenum, lung, kidneys, stomach, brain, and thymus) following treatment with LPS. Direct quantification of luciferase signals using the LivingImage software yielded data comparable to those obtained by the conventional luminometer assay performed on excised and homogenized tissues. In an arthritis and a CHS reaction model, luciferase signals were induced locally in the treated area. Dexamethasone, a well known blocker of IL-1β production [##REF##10936515##16##, ####REF##9143378##17##, ##REF##11778222##18####11778222##18##], suppressed the induced expression of luciferase in transgenic mice in these inflammation models. These data indicate that cHS4I-hIL-1βP-Luc mice are useful as a sensitive and convenient model for monitoring IL-1β gene expression during the disease process in a broad range of inflammatory conditions and for evaluating the effects of anti-inflammatory drugs.</p>", "<p>Conventional methods for monitoring IL-1β gene expression rely mostly on either measuring circulating levels of IL-1β in the serum or mRNA expression in tissues. Compared with these methods, the approach reported in this study is convenient and sensitive, and more importantly, with this approach the kinetics as well as the anatomical location of IL-1β gene expression can be conveniently studied and use a minimal number of animals. This is extraordinarily useful in monitoring both systematic and local inflammation, such as sepsis, arthritis, and chemical-induced skin toxicity.</p>", "<p>The pattern of LPS-induced luciferase expression in transgenic mice was generally in agreement with the endogenous murine IL-1β mRNA expression. However, the fold of increase did not match exactly between luciferase activity and endogenous IL-1β mRNA. This might be explained by the facts that the promoter for the luciferase is from human and IL-1β mRNA level and protein level are often not in linear correlation. In addition, translational regulation of IL-1β mRNA and luciferase mRNA, as well as posttranslational regulation of IL-1β protein and luciferase protein, may also be different. So it should be note that the signal of luciferase in the reporter mice may be not consist with the IL-1β protein level since the regulation of IL-1β protein translational and posttranslational process is rather complex in vivo[##REF##17803913##19##]. However, these differences do not affect the application of our transgenic model in inflammation research to monitor the mRNA level of IL-1β. In the cHS4I-hIL-1βP-Luc transgenic mice described here, there were no clear gender differences in both baseline of luciferase signals and response to LPS where nearly identical kinetics and signal intensity were observed. This result is in agreement with the results reported previously [##REF##15265539##20##].</p>", "<p>IL-1β have been found in the synovial fluid recovered from inflamed joints of rheumatoid arthritis patients [##REF##2545778##21##]. We clearly demonstrated that local administration of zymosan into knee joints triggered induction of luciferase expression in the joints, which correlated with the increases in both IL-1β mRNA and protein in zymosan-treated knee joint tissue reported earlier [##REF##2901567##22##]. It is believed that locally synthesized IL-1β by synovial cells in inflamed joints plays a key role in the pathogenesis of rheumatoid arthritis, and IL-1β can be used as a marker for evaluating the efficacy of therapeutics for rheumatoid arthritis.</p>", "<p>Luciferase expression was also clearly induced in the ear of the transgenic mice in a CHS model. Contact hypersensitivity is a two-phase process. The induction phase begins on initial epicutaneous application of a hapten (low-molecular-weight chemical), resulting in activation and rapid lymphocyte proliferation. The challenge or elicitation phase occurs when sensitized individuals are re-exposed to the sensitizing antigen. This local CHS reaction is characterized by infiltrating T lymphocytes, macrophages, and neutrophils. It has been reported that mice with oxazolone treatment in the ear showed ear swelling and increased expression levels of IL-1β, IL-4, IL-18 and GM-CSF [##REF##15649273##23##,##REF##17660848##24##]. In line with these reports, we showed that luciferase expression was induced in the ear of the transgenic mice at day 1 after oxazolone challenge and the expression level correlated with the increase in ear thickness.</p>", "<p>The regulatory region for human IL-1β gene expression was found to be distributed over several thousand basepairs upstream and a few basepairs downstream from the transcriptional start site [##REF##7727690##25##]. There are two independent enhancer regions, -2782 to -2729 and -2896 to -2846 that appear to act cooperatively [##REF##8441379##26##]. The latter contains a cAMP response element, whereas the former is a composite cAMP response element (NFIL-6) that is responsive to LPS. The 80-bp fragment (-2800 to -2720) is required for transcription and contains, in addition to a cAMP response element, an NF-κB-like binding site. Activating protein-l (AP-l) sites also participate in endotoxin-induced IL-1β gene expression. Proximal promoter elements have also been identified between -131 and +14 [##REF##8441379##26##]. Sequences in this region contain binding sites for the novel nuclear factor NFβA [##REF##8157962##27##], which appears to be similar to nuclear factors termed NFβl and NFβ2 [##REF##8441379##26##]. This proximal promoter is required for maximal IL-1β gene expression. The nucleotide binding sequences of NFβA are found to be identical to those of the transcription factor Spi-1/PU.1 [##REF##7609733##28##,##REF##7799967##29##], a well-established NF in cells of myeloid and monocyte lineage [##REF##8474451##30##]. The requirement for Spi-1/PU.1 for IL-1β gene expression imparts its tissue specificity. Human blood monocytes, which constitutively express Spi-l/PU.1, are exquisitely sensitive to LPS inducible IL-1β gene expression. In our case, the promoter region used for driving luciferase expression in the transgenic mice is 4.5-kb long which contains all the cis-elements discussed above. The chicken HS4 insulator (cHS4I) upstream of the hIL-1βP may eliminate the transgenic chromosomal insertion site effect on transgene expression[##REF##12893244##31##]. Therefore, the expression of the luciferase gene in the transgenic mice was expected to reflect the natural pattern of IL-1β gene expression.</p>", "<p>Bioluminescence imaging techniques have been used to study the expression of several inflammatory factors in reporter mice, such as NF-κB-binding cis elements[##REF##11549548##13##,##REF##11801687##15##], the inducible NO synthase gene(iNOS)[##REF##12794164##32##], the vascular endothelial growth factor-2 (VEGFR2)[##REF##14512298##33##]. These reporter systems can be successfully used to monitor and quantify different inflammatory processes in vivo. Together with our model in this paper, we believe these reporter mice systems will facilitate us a better understanding of the molecular mechanism of different diseases.</p>" ]
[ "<title>Conclusion</title>", "<p>The cHS4I-hIL-1βP-Luc transgenic mouse, with its IL-1β promoter driven luciferase expression, is able to provide information about anatomical sites of IL-1β expression in an inflammatory process. The induction was observed in the whole body (especial in liver, lungs, and duodenum) in a LPS-sepsis model, and was restricted to the treated joints and ears in a zymosan-arthritis model and in an oxazolone-CHS model, respectively. Treatment with dexamethasone, a proven IL-1β expression blocker, significantly suppressed LPS- or zymosan-induced IL-1β promoter-driven luciferase expression. As IL-1β induction is featured in the pathogenesis of a number of acute and chronic inflammatory diseases, the cHS4I-hIL-1βP-Luc mouse is a useful tool not only for tracking various inflammatory processes <italic>in vivo</italic>, but also for testing the efficacies of therapeutic compounds that are targeted to inflammatory diseases, especially those that involve induction of IL-1β expression.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Interleukin 1 beta (IL-1β) plays an important role in a number of chronic and acute inflammatory diseases. To understand the role of IL-1β in disease processes and develop an <italic>in vivo </italic>screening system for anti-inflammatory drugs, a transgenic mouse line was generated which incorporated the transgene firefly luciferase gene driven by a 4.5-kb fragment of the human IL-1β gene promoter. Luciferase gene expression was monitored in live mice under anesthesia using bioluminescence imaging in a number of inflammatory disease models.</p>", "<title>Results</title>", "<p>In a LPS-induced sepsis model, dramatic increase in luciferase activity was observed in the mice. This transgene induction was time dependent and correlated with an increase of endogenous IL-1β mRNA and pro-IL-1β protein levels in the mice. In a zymosan-induced arthritis model and an oxazolone-induced skin hypersensitivity reaction model, luciferase expression was locally induced in the zymosan injected knee joint and in the ear with oxazolone application, respectively. Dexamethasone suppressed the expression of luciferase gene both in the acute sepsis model and in the acute arthritis model.</p>", "<title>Conclusion</title>", "<p>Our data suggest that the transgenic mice model could be used to study transcriptional regulation of the IL-1β gene expression in the inflammatory process and evaluation the effect of anti-inflammatory drug <italic>in vivo</italic>.</p>" ]
[ "<title>Authors' contributions</title>", "<p>LL and JF designed and analyzed all the experiments, and wrote the manuscript. LL carried out the molecular genetic studies and the immunoassays, participated in the sequence alignment and the design of the study, and performed the statistical analysis. LW and XS participated in transgenic research. ZF, JR, RS, ZL, ZS, JY and ZW participated in its design and coordination. All authors read and approved the final manuscript.</p>" ]
[ "<title>Acknowledgements</title>", "<p>We thank Mrs. Xixia ZHOU (Institute of Biochemistry and Cell biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) for the care of the animals used in this study, Mrs. Jiajuan SHEN (School of Life Science and Technology, Tongji University, Shanghai) for her lab administration affairs, Mr. Zhen Yu (Shanghai Genomics, Inc) for his guidance of the IVIS imaging system, as well as for Mr. Boliang LI (Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) for valuable advice. This study was supported by grants from the Science and Technology Commission of Shanghai Municipality (06DZ19004; 06XD14014; 06DZ05907) and E-Institutes of Shanghai Municipal Education Commission (Project Number: E03003).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Schematic diagram of IL-1β-luc reporter system used for microinjection</bold>. (A) The cHS4I-hIL-1βP-Luc transgene was constructed by inserting a 4.5-kb 5' flanking promoter region of the human IL-1β gene in front of firefly luciferase cDNA. (B) Genotyping by PCR yielded a 600-bp fragment. PCR products were run on a 1% agarose gel. Lane 1 was a DL 2,000 DNA ladder, lane 2 was the product from a wild-type CBA control, lane 3 was the product from a wild-type C57BL/6 control, lane 4 was the buffer for dissolving the genomic DNA, and lanes 5–8 were samples from cHS4I-hIL-1βP-Luc heterozygous transgenic mice. P1: forward-luc primer, P2: reverse-luc primer.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Induction of hIL-1βP-driven luciferase expression in IL-1β-luc transgenic mice by LPS</bold>. Dorsal and ventral views of a representative mouse are depicted for male (A) and female (B) mice. The color overlay on the images represents the photons/sec/cm<sup>2</sup>/steradian (p/s/cm<sup>2</sup>/sr), as indicated by the color scale next to the images. (C) Quantification of luciferase activity (photons/sec) of the whole body with the LivingImage software. Data presented are group means of 3 mice ± standard errors.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>hIL-1βP-driven luciferase expression is induced in multiple tissues of IL-1β-luc transgenic mice after LPS treatment</bold>. (A) Female cHS4I-hIL-1βP-Luc mice (n = 3) were treated with LPS (3 mg/kg). At 5 h after LPS injection, mice were sacrificed and selected organs were rapidly harvested and homogenized for luciferase activity measurement with a luminometer. Saline-treated mice were used as controls. (B) Fold induction of the luciferase expression in the selected organs. Degree of change in gene expression is based on the expression level of the saline-treated mice. All values were expressed as mean ± SE. (C) Quantification-RT-PCR analysis for IL-1β gene expression in female mice. The mRNA levels of IL-1β gene expression in the selected organs of LPS-treated mice were quantified using real-time RT-PCR with Evagreen detection at 5 h after treatment. Degree of change in gene expression is based on the expression level of the saline-treated mice. All values were expressed as mean ± SE (n = 3/group). (D) Western blot for the presence of pro-IL-1β protein in the spleen, thymus and lung at 5 h following treatment with saline or LPS.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Monitoring hIL-1βP-driven luciferase expression in IL-1β-luc transgenic mice during zymosan-induced acute arthritis</bold>. (A) Female cHS4I-hIL-1βP-Luc mice (n = 3/group). were injected with zymosan (300 μg/knee) into the right rear knee joint, and vehicle (5% glucose in PBS) into the left rear knee joint. The mice were imaged at 0, 1, 3, 4, 6, 8, 24, 30, 48, 72, and 96 h following injection. The color overlay on the images represents the photons/sec/cm<sup>2</sup>/steradian (p/s/cm<sup>2</sup>/sr), as indicated by the color scale next to the images. (B) Quantification of luciferase activity (photons/sec) of the knee region by the LivingImage software. Data presented were mean ± SE.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>hIL-1βP-driven luciferase expression in IL-1β-luc transgenic mice in an oxazolone-CHS model</bold>. (A) Female cHS4I-hIL-1βP-Luc mice (n = 3) are sensitized with oxazolone on day -6. On day 0, the right ears were challenged with oxazolone and the left ears were treated with vehicle. Imaging analysis was performed on day – 6, 0, 1, and 30. (B) Quantification of luciferase expression (photons/sec) in all the ears with LivingImage software. Data were presented as mean ± SE. (C) Ear thickness was measured on day – 6, 0, 1, and 30 with a micrometer. Data represent mean ± SE. *** indicate significant differences at p &lt; 0.001. p/s/cm<sup>2</sup>/sr: photons/sec/cm<sup>2</sup>/steradian.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Dexamethasone inhibits hIL-1βP-driven luciferase expression in IL-1β-luc transgenic mice in the LPS and zymosan induced inflammation models</bold>. (A) Dexamethasone inhibits LPS-induced hIL-1βP-driven luciferase expression. Male cHS4I-hIL-1βP-Luc mice (n = 3/group) were cotreated with vehicle or dexamethasone (3 mg/kg) during the LPS (3 mg/kg) injection. All the mice were imaged at 0 and 5 h after LPS injection. (B) Quantification of luciferase activity (photons/sec) with the LivingImage software. Data presented were mean ± SE. (C) Dexamethasone inhibits hIL-1βP-driven luciferase expression during zymosan induced acute arthritis. Female cHS4I-hIL-1βP-Luc mice (n = 3/group) were cotreated with vehicle or dexamethasone (50 μg/mouse) during the administration of zymosan (300 μg/mouse). All the mice were imaged at 0 and 4 h after zymosan injection. (D) Quantification of luciferase activity (photons/sec) of the articular region of the acute arthritis mice with the LivingImage software. Data presented were mean ± SE. *** indicate significant differences at p &lt; 0.001. p/s/cm<sup>2</sup>/sr: photons/sec/cm<sup>2</sup>/steradian.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"1471-2172-9-49-1\"/>", "<graphic xlink:href=\"1471-2172-9-49-2\"/>", "<graphic xlink:href=\"1471-2172-9-49-3\"/>", "<graphic xlink:href=\"1471-2172-9-49-4\"/>", "<graphic xlink:href=\"1471-2172-9-49-5\"/>", "<graphic xlink:href=\"1471-2172-9-49-6\"/>" ]
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{ "acronym": [], "definition": [] }
33
CC BY
no
2022-01-12 14:47:25
BMC Immunol. 2008 Aug 19; 9:49
oa_package/8b/53/PMC2529264.tar.gz
PMC2529265
18673556
[ "<title>Background</title>", "<p>After antigen encounter populations of naive lymphocytes cells differentiate through an effector state into memory cells that confer protective immunity to the host [##REF##16824118##1##,##REF##15023416##2##]. Memory lymphocytes acquire functions during this differentiation process that are necessary for their immunologic efficacy, including a lowered threshold for proliferation and acquisition of effector functions, and the ability to self-renew for the life-time of the host.</p>", "<p>Historically, vaccines have been used to induce antigen-specific T and B cell memory and are highly effective against many pathogens [##REF##12001996##3##]. However, diseases like HIV and cancer that are characterized by defective T cell function are refractory to current vaccination approaches [##REF##16551249##4##,##REF##15340416##5##]. The ability to manipulate directly the differentiation of memory lymphocytes would therefore have great importance in improving immunotherapies for cancer and chronic viral diseases. However, the regulatory mechanisms that govern the differentiation of naive lymphocytes into memory cells are not completely understood. This has precluded the development of targeted therapies that could influence the cellular differentiation program underlying memory development [##REF##15023416##2##].</p>", "<p>High-throughput small-molecule screens are being increasingly used as a tool to identify compounds that can direct cellular differentiation from a precursor cell into a more differentiated cell-type [##REF##18295572##6##]. This raises the possibility that similar approaches could be used to identify small molecules that induce the full or partial differentiation of naive cells into those that have functional characteristics of memory cells. Small molecules capable of promoting memory differentiation could 1) serve as tool compounds to understand better memory differentiation: and 2) have therapeutic applications in the <italic>ex-vivo</italic> expansion of antigen-specific T cells or as molecularly targeted vaccine adjuvants. Applying this approach to memory differentiation, however, is limited by significant obstacles. Differentiation screens rely on high-throughput assays that can detect the emergence of a more differentiated state. This is often achieved by the use of a reporter gene assay that monitors the expression level of a single differentiation marker gene [##REF##12465946##7##,##REF##14871063##8##]. Reporter gene screens have been used to identify compounds that partially induce either neural or cardiac differentiation in an undifferentiated cell line. However, the memory lymphocyte pool is highly heterogeneous, and no single gene can reliably distinguish naive and memory lymphocytes [##REF##16824118##1##]. Other screening approaches have assayed for a functional read-out such as proliferation to detect compounds that promote a more differentiated state in neural precursor cells [##REF##17884634##9##], but the cardinal functional characteristics of memory cells such as longevity and the ability to self-renew are not easily assayed in vitro.</p>", "<p>Genome-wide transcriptional profiling of naive, effector and memory T cells has demonstrated that extensive reprogramming of gene expression occurs during memory differentiation [##REF##15548615##10##,##REF##12526810##11##]. We have previously used cross-species genomic analysis to identify the transcriptional programs that occur during memory differentiation in humans and mice [##REF##18641323##12##]. We demonstrated that these coordinately regulated transcriptional programs, or differentiation signatures, are highly evolutionarily conserved, and shared by multiple lymphoid lineages [##REF##18641323##12##]. This suggests that although no single gene can serve as a marker for memory differentiation, a complex signature of genes may be used to identify populations of cells that have adopted a differentiation state of interest. We therefore sought to develop a robust assay that could discriminate between naive and memory states in primary human lymphocytes based not on a single reporter gene, but on a complex gene expression profile. Previous work in cancer cell lines has demonstrated the utility of using a complex gene expression signature to detect cellular differentiation [##REF##14770183##13##,##REF##17425403##14##]. We therefore tested whether a gene expression-based assay could be used as a high-throughput assay to distinguish primary human naive and memory-phenotype T cells.</p>" ]
[ "<title>Methods</title>", "<title>Research Subjects and cell purification</title>", "<p>Peripheral blood samples were obtained from healthy volunteers who gave informed consent for research and were enrolled on a protocol approved by the DFCI IRB. Written informed consent was obtained from the subjects for publication of data in this report. A copy of the written consent is available for review by Editor-in-Chief of this journal. Peripheral blood mononuclear cells were purified by density centrifugation. Naive or effector/memory phenotype CD4 T cells were obtained either by negative selection over a magnetic column (Naive or Memory CD4+ T cell Isolation Kit, Miltenyi Biotec) or by flow sorting. For sorting, cells were stained with a cocktail of antibodies designed to exclude irrelevant lineages, Annexin V to exclude dead/dying cells, and CD4, CD45RA and CD62L to identify memory-phenotype and naive CD4 T cells as shown in Figure ##FIG##1##2##. Purity of MACS isolated naive cells was routinely &gt; 95%, and sorted cells was &gt; 99%.</p>", "<title>Ligation-mediated amplification</title>", "<p>Purified populations of naive or memory-phenotype cells were plated in 384-well tissue-culture plates at 25,000 cells per well in 50 uL of RPMI supplemented with 10% human AB-serum (Valley Biomedical, Inc.) and antibiotics, and incubated at 37°C with 5% CO<sub>2 </sub>for ~18 hours. After incubation, 40 uL of medium was removed from the wells with a Multimek robot (Beckman Coulter). Cells were lysed with the addition of 10 ul of TCL lysis buffer (Qiagen) for 20 minutes at room temperature. The lysate was transferred to a 384-well oligo-dT coated plated (TurboCapture 384 mRNA kit, Qiagen) and incubated for 1 hour at room temperature. Excess lysate was removed by briefly centrifuging inverted uncovered plates onto absorbent paper towel. Reverse transcription was carried out in a 5 uL reaction volume using MMLV reverse transcriptase at 37°C (Promega). After a 90 minute incubation, liquid was removed from the wells by inverted centrifugation of the plates. Gene-specific oligonucleotide probes (Additional file ##SUPPL##0##1##) were hybridized to the plate-bound cDNA using 2 nM of each probes (118 probes in total). Upstream probes were designed to contain a T7 primer sequence, Luminex-designed FlexMap barcode tag (24 nucleotides in length) and the gene-specific sequence (20 nt). Downstream probes were phosphorylated at the 5' end to allow subsequent DNA ligation, and contained gene-specific sequence followed by a T3 primer sequence. Gene-specific probes were designed such that the upstream and downstream probes were of similar G-C composition, minimal repeats, and abutted at C and G or G and C nucleotides. Probe hybridization was performed at 95°C for 2 minutes followed by 50°C for 1 hour. Excess probe was removed by inverted centrifugation of the plate. DNA ligation step was then performed using Taq ligase (New England Biolabs) in a 5 uL volume at 45°C for 1 h followed by incubation at 65°C for 10 minutes. Excess ligation mix was spun out by inverted centrifugation of the plate. The ligated products were amplified with a universal, biotinylated T3 primer (5'-ATT AAC CCT CAC TAA AGG GA-3') and a universal T7 primer (5'-TAA TAC GAC TCA CTA TAG GG-3') using HotStarTaq DNA polymerase (Qiagen) in a 5 uL reaction volume.</p>", "<p>Amplicon detection was carried out using xMAP Multi-Analyze COOH microspheres (Luminex, 2.5 × 10<sup>6</sup>) coupled to FlexMap barcode sequences that were complementary to barcode sequences contained in the gene-specific probe pairs. Coupling reactions were performed as previously described [##REF##17425403##14##]. A 5 uL aliquot of the ligation-mediated amplification mix was hybridized to a mixture of microspheres containing ~2500 fluorescently distinguishable microspheres per gene in 18 uL of 1.5× TMAC (4.5 M tetramethylammonium chloride, 0.15% N-lauryl sarcosine, 75 mM Tris-HCl [pH 8], and 6 mM EDTA [pH 8]) and 5 uL of TE [pH8] at 95°C for 2 min and then 45°C for 1 h. To detect the amplicon bound to each bead, the sample was incubated with 20 ul of streptavidin-PE (Invitrogen) in 1× TMAC for 5 min at 45°C, washed twice, and resuspended in 1× TMAC. Dual-color fluorescence was detected with a Luminex 200, or high-throughput detection instruments. A minimum of 30 events was recorded per microsphere, and the median intensity on the PE channel recorded per bead.</p>", "<title>Data analysis</title>", "<p>The median fluorescence intensity of PE corresponding to a gene-specific bead was used as a measure of the gene's expression level. Expression levels of each signature gene in the well were indexed to a mean of 4 control genes (<italic>ACTB, TUBG, TUBB </italic>and <italic>HNRAPB</italic>) in the corresponding well to minimize well-to-well variability. Filtering was performed to eliminate wells with aberrant control gene expression from further analysis, using the mean plus or minus two times the standard deviation of the control genes as a filtering threshold. Aggregate scores of all genes in the panel created a summed score of the indexed expression values of the memory and naive genes with a sign determined by the expected direction of expression in the two differentiation states. For some visualizations, normalization of the gene expression values were performed using a Z-score derived by subtracting the mean expression level of the gene in that class from a the raw gene expression value in a given well and then dividing the difference by the standard deviation of the gene's expression in that class.</p>" ]
[ "<title>Results</title>", "<title>Detection of differentiation signatures by ligation-mediated amplification</title>", "<p>Ligation-mediated amplification with bead-based detection has been described in detail previously [##REF##17425403##14##, ####REF##17010675##15##, ##REF##16859521##16####16859521##16##]. In brief, mRNAs are captured on oligo-dT-coated 384-well plates and reverse transcribed to generate first strand cDNA covalently linked to the plate (Fig. ##FIG##0##1##). Gene-specific 20-mer oligonucleotides corresponding to the signature genes are then annealed to the cDNA template. The oligos are designed in such a way that primer pairs anneal to adjacent stretches of the gene, allowing the ends of the primer pairs to abut. This is necessary for the ligation step, which will only occur when the abutting ends of the primer pairs are held adjacent to each other by binding the corresponding cDNA. Both gene-specific oligos incorporate a common flanking sequence that allows PCR amplification using a single set of PCR primers that will amplify all gene-specific amplicons. This amplification technique enables the simultaneous amplification of the signature transcripts in a highly reproducible manner that is faithful to the relative abundance of the starting mRNAs. To resolve the relative amounts of each gene-specific amplicon, one partner of each of the oligo pairs incorporates a unique barcode sequence that binds to a fluorescent bead linked to an oligo with a corresponding antisense sequence. Following PCR amplification, individual gene-specific amplicons are distinguished by the fluorescence emission spectra of the barcode-oligo-tagged beads to which they bind. To identify how much amplicon is bound to each bead, one of the PCR primers is biotinylated to allow detection of the relative abundance of the amplicon bound to each bead using streptavidin-phycoerythrin. Up to 100 fluorescently distinct beads are used allowing the quantitation of transcripts from up to 100 genes. The emission spectrum of each bead denotes the identity of the gene; the phycoerythrin intensity the transcript's abundance.</p>", "<title>Selection of Marker Genes to Distinguish Naive and Memory-Phenotype CD4 T cells</title>", "<p>As proof of principle, we tested whether we could use this approach to distinguish between naive human CD4 cells and memory-phenotype cells on the basis of a well-defined differentiation signature. To identify a differentiation signature that was characteristic of the memory state, we analyzed our previously-published gene expression profiles of human and mouse memory lymphocytes [##REF##18641323##12##]. In a two-step analytic process, we first identified a set of genes differentially expressed by human memory-phenotype CD4 T cells compared to their naive precursors (Fig ##FIG##1##2##.). Then, we refined this list using gene-set enrichment analysis to identify only those genes present in the human signature that were also upregulated during the differentiation of mouse memory CD8 T cells as we have done previously [##REF##18641323##12##]. This \"filtering\" step through a gold-standard model of functional memory lymphocytes allowed us to focus on a robust signature of genes characteristic of the memory, rather than memory-phenotype, state. From this list we selected genes that showed the highest signal-to-noise ratio in the microarray analysis of naive and memory-phenotype CD4 T cells (Fig ##FIG##1##2##). Genes were selected to include those upregulated in memory-phenotype CD4 T cells compared to naive cells (Fig ##FIG##1##2B##, purple text) and those downregulated in memory-phenotype cells compared to naive cells (Fig ##FIG##1##2B##, green text). The selection of genes that were differentially expressed in both directions served to reduce the chance of detecting compounds that caused indiscriminate increases or decreases in expression of all genes in the signature.</p>", "<p>CD4 T cells were chosen for these experiments because of the availability of reagents for magnetic isolation of large numbers of naive or memory-phenotype cells. It is important to note that for these proof of principle experiments, our test populations of lymphocytes were <italic>memory-phenotype </italic>cells, rather than those with known functional properties of memory lymphocytes. Unequivocally identifying human T cells with the functional properties of memory – such as antigen-independent survival and the ability to self-renew – is not feasible; instead we chose a to study cells with a well recognized memory-phenotype as this population is presumably enriched for those cells that have memory function. The method of purifying populations of naive and memory-phenotype cells did not influence the expression levels in the signature, and comparison of results from cells flow-sorted or purified with MACS were highly correlated (data not shown). However, MACS purification allowed more rapid isolation of the number of cells needed for screening applications.</p>", "<title>Quantitation of Differentiation Signature Genes in T Cells</title>", "<p>Gene-specific primers were designed for each of the 55 genes in the differentiation signatures and for four control genes with which to normalize expression level data between replicate wells. Control genes were chosen (<italic>ACTB</italic>, <italic>TUBB</italic>, <italic>TUBG1</italic>, and <italic>HNRPAB</italic>) that showed low signal-to-noise ratios of expression level in Affymetrix data between naive and memory-phenotype CD4 T cells, and which spanned the range of expression levels seen in the transcripts of the differentiation signature. In subsequent analyses each gene in the differentiation signature was normalized to the mean of these four genes.</p>", "<p>Naive and memory-phenotype CD4 cells were flow-sorted using the gating strategy shown in Figure ##FIG##1##2## and plated at various cell densities per well in 96-well U-bottom plates. Following cell lysis, LMA and bead-detection were performed (Fig ##FIG##2##3##). All 59 genes were successfully amplified, indicating that the method can reliably amplify a large number of genes simultaneously. Cell titration experiments demonstrated that optimal gene expression data were obtained at cell numbers of 25,000 per well or above (data not shown); all subsequent experiments were performed using 25,000 cells/well. The absolute transcript expression values spanned a 3-log range (Fig. ##FIG##2##3A##). The most abundant transcripts in the memory-phenotype differentiation signature included LGALS3 and FAS, and in the naive differentiation signature, CCR7 and PECAM1. However, even transcripts that would be expected to be expressed at a low copy number such as the transcription factors KLF10 or BACH2 were amplified reliably suggesting efficient amplification of even low abundance transcripts.</p>", "<p>Comparison of expression levels of naive or memory genes in naive and memory-phenotype cells (Fig. ##FIG##2##3B##) showed that the difference in expression level between naive and memory-phenotype cells was large for some genes, such as LGALS3, ANAX1 (upregulated in memory-phenotype compared to naive), and SATB1 and SCML1 (upregulated in naive compared with memory-phenotype). However, for other genes relatively small differences in expression between each cell type were observed (e.g. KLF10 or SERPINE2). The magnitude of difference in gene expression was not related to the overall transcript abundance: genes such as AHNAK were expressed at high levels but demonstrated small fold-difference in expression between effector/memory phenotype and naive cells. However, while the difference in expression level for individual transcripts was small for many of the genes in the signature, the overall direction of change in expression levels was as predicted for the majority of memory-phenotype marker genes and naive marker genes (Fig ##FIG##2##3B##), i.e. the vast majority of genes expected to be higher in memory-phenotype cells were indeed expressed at higher levels than in naive cells and vice-versa. Overall ~10% of transcripts in the panel were \"contrarian\", or expressed at relative levels opposite to that predicted (e.g. ANXAP2, SMA3). Such genes were not included in subsequent analyses.</p>", "<p>We reasoned that measuring multiple genes representative of a cell state would provide a more robust discrimination of cell state than relying on a single gene because small but consistent differences in expression level of signature genes could be aggregated. To test this, the expression level of the differentiation signature genes were measured in multiple replicates of memory-phenotype and naive CD4 T cells (Fig ##FIG##2##3C##). For each well, the expression of memory-phenotype marker genes or naive marker genes were summed (after conversion to a Z-scores to compensate for the wide range in absolute expression values) and expressed as two vectors or \"scores\" on a bivariate plot. The cumulative scores of expression values for memory-phenotype or naive marker genes separate memory-phenotype (black symbols) or naive (white symbols) CD4 T cells more clearly than does the expression level of any of the individual transcripts.</p>", "<p>To quantify this difference, Z' factors were calculated for individual genes and for the summary score of expression values of all genes. The Z' factor is used to evaluate the ability of assays to detect true positive results in high-throughput screens [##REF##10838414##17##]. The theoretical maximum value is 1, and values &gt; 0.5 suggest excellent ability to detect true positive 'hits'. In contrast, values below 0 do not allow discrimination of positive and negative results. As shown in Table ##TAB##0##1##, the Z' factor for this signature was 0.68, and the highest score was achieved by aggregating all genes in the differentiation signature. No single gene provided as high resolution, and only three individual genes (<italic>S100A4</italic>, <italic>LGALS3</italic>, and <italic>ANAX1</italic>) gave Z' factors above 0. Thus the use of a signature-based assay was superior to any single marker gene in distinguishing naive and memory-phenotype CD4 T cells.</p>", "<title>Comparison of LMA with Affymetrix gene expression profiling</title>", "<p>We next considered how well the method recapitulated the changes in gene expression measured by Affymetrix microarrays. We compared the difference in gene expression levels between memory-phenotype and naive CD4 T cells measured by LMA/bead-based detection with the original Affymetrix microarray data shown in Figure ##FIG##0##1##. The values were highly correlated between platforms (Fig ##FIG##3##4##). This is especially striking because samples from different healthy donors were used to generate the expression data on each platform. Thus the changes detected by LMA/bead-based detection are similar to those measured by Affymetrix microarray.</p>", "<title>Differentiation signature is comparable between donors</title>", "<p>We next evaluated the variability of the gene expression level detection across multiple replicates of naive and memory-phenotype CD4 T cells from two donors. Being able to perform screens involving large numbers of compounds would require pooling data from multiple donors. To identify the extent of donor-to-donor variability, we compared the expression levels of all genes in naive and memory-phenotype CD4 T cells in two donors (Fig ##FIG##4##5##). The expression levels were highly correlated (Rs = 0.99), suggesting that there is little variability in the relative expression levels of this set of transcripts between populations of phenotypically similar CD4 T cells in the two donors studied.</p>", "<title>Assay is precise and robust</title>", "<p>To function as a screening tool that can be scaled up to high-throughput, the assay should ideally be robust and precise, even when used at high-throughput with robotic fluid handling. We therefore performed the assay in large numbers of replications (~100 per cell type) in CD4 T cells obtained from a leukapheresis product from a healthy donor.</p>", "<p>After MACS purification of naive and memory-phenotype CD4 T cells, cells were plated into a 384 well plate. The cells were incubated for 18 hours (to simulate exposure to test compounds), followed by harvest, LMA, and bead detection using robotic fluid handling (Fig ##FIG##5##6##). Replicate measurements of genes in the differentiation signature were highly correlated, and all data points fell within two-fold of their corresponding means (Fig ##FIG##5##6A##). The Z' Factor for the memory-phenotype versus naive comparisons in scaled up and automated experiments were ≥ 0.5, suggesting that the assay can be performed on a scale necessary to permit high-throughput screens.</p>" ]
[ "<title>Discussion</title>", "<p>The ability to identify compounds that direct the cellular differentiation of naive T cells into memory cells could have significant therapeutic impact. However, the lack of reporter assays suitable for high-throughput that can accurately distinguish between discrete differentiation states in T cell development has limited this approach. Here we describe the application of a gene expression-based assay that detects complex, multigene signatures and distinguishes samples of naive and memory-phenotype CD4 T cells from human peripheral blood at high-throughput.</p>", "<p>Traditionally, reporter gene assays measure the transcriptional activity of a single gene that is characteristic of the differentiated state. However, in the case of memory lymphocyte differentiation, no single gene can accurately distinguish between naive and memory-phenotype cells. For instance, the naive markers CCR7 and CD62L are both re-expressed by central memory cells preventing these genes in isolation from being used to separate the two cellular states [##REF##15032595##18##]. Similarly, genes that are not expressed by naive T cells such as granzyme B or perforin are expressed by both effector and memory-phenotype cells making it difficult to screen for compounds that direct differentiation only towards a memory state [##REF##12526810##11##]. We took the approach of identifying a signature of genes that are differentially expressed during memory differentiation both in humans and mice, regardless of their functional role. This cross-species signature readily discriminated between the test populations of cells in these experiments – phenotypically naive cells that would not be expected to manifest the memory differentiation signature, and memory-phenotype cells that, as a population, would be expected to show increased expression of a memory differentiation signature. The assay that we used simultaneously measured the expression level of a panel of 55 genes that included transcripts increased in memory-phenotype CD4 T cells compared to their naive counterparts and an equivalent number that show the opposite profile. We found that, as expected, the expression level of each gene in isolation was often not markedly different between naive and memory-phenotype CD4 T cells. However, most memory-phenotype marker genes were consistently expressed at higher levels in memory-phenotype cells than in naive cells; similarly, naive-phenotype genes were consistently expressed in greater abundance in naive CD4 T cells than in memory-phenotype cells. When the resulting, albeit small, difference in expression level of each of a gene in the panel was aggregated, naive and memory-phenotype cells could be easily distinguished with a Z' factor score better than any gene in isolation. This demonstrates that a signature-based screening platform can provide a powerful degree of resolution in distinguishing differentiation states that lack clear-cut differentiation markers.</p>", "<p>We developed this assay to allow screening to be done using primary human lymphocytes. The use of primary human cells as starting material is highly unusual for differentiation-based screens but offers significant advantages over cell lines. The analysis of primary cells <italic>ex vivo</italic> more closely recapitulates the cellular state <italic>in vivo</italic> than does the analysis of cell lines. Moreover, primary cells may be more sensitive to toxicities of compounds identified within a screen allowing earlier detection of potentially harmful drugs. Three factors suggest that using primary human T cells for memory differentiation screens will prove feasible. First, T cells are relatively abundant and can be accessed in normal volunteers with minimal morbidity. Second, selection of subsets within the peripheral T cell compartment is technically straightforward with a number of available cell separation techniques. Third, we found a remarkable degree of consistency in the relative expression level of genes in the differentiation signature between two different donors. This fact is of great practical significance as several different donors would be needed to contribute naive T cells to a screen for a high-complexity compound library. Further evaluation across a larger number of normal donors will be necessary to define rigorously how significant an obstacle donor-to-donor variability in gene-expression will be in the conduct of large screens spanning more than one donor. However, the lack of donor-to-donor variability in these initial studies suggests that analysis of pooled screening data from multiple donors may be possible.</p>", "<p>There are clearly caveats associated with a signature-based approach to HTS in primary cells. The signature of genes was defined empirically, and so the biological relevance of any of the genes in the signature to the differentiation states of interest is unknown. However, the differentiation signature used in this study is highly evolutionarily conserved, shared by memory differentiation in both T and B cell lineages, and disrupted in functionally exhausted T cells [##REF##18641323##12##]. This suggests that this gene signature <italic>in toto </italic>is highly correlated with the differentation state of functional memory cells. Never-the-less, future studies will be required to determine whether it is possible to elicit the complex gene expression profile representative of a given differentiation state without inducing its attendant functional properties. Second, the use of primary human cells is technically challenging. Like all primary cells we found that increasing time in culture altered the gene expression profiles, presumably due to the lack of input signals from the normal milieu found <italic>in vivo</italic> (data not shown). However, data from mouse models suggests that the lineage commitment step for naive T cells differentiating after antigen-encounter occurs within hours or days of initial TCR signaling [##REF##12960304##19##, ####REF##10073942##20##, ##REF##11323695##21##, ##REF##10376601##22####10376601##22##]. This suggests that a brief exposure to compound may be sufficient to direct differentiation of naive T cells to a memory fate before adverse <italic>ex vivo</italic> effects are apparent in the differentiation signature.</p>", "<p>Our data suggests that it is now possible to screen for compounds that induce memory differentiation in naive human T cells. Compounds identified in such a screen would have significant biological and therapeutic uses. First, as tool compounds, hits in a memory differentiation screen could afford new insights into the mechanisms underlying memory development. Second, as therapeutic agents, memory-differentiating drugs could be used to accelerate the <italic>ex-vivo</italic> expansion of antigen-specific T cells or improve the efficiency of current vaccination approaches. Although widescale differentiation of naive T cells into memory cells would be undesirable in a clinical situation, antigen-specificity on promoting memory differentiation could be achieved by co-administration with vaccines, by optimization of dose and/or timing, or by the use of <italic>ex-vivo</italic> treatment of antigen-specific T cells.</p>" ]
[ "<title>Conclusion</title>", "<p>The use of LMA with bead-based detection offers a generic solution for T cell differentiation-based screens where no single-gene or functional assay is available. The same experimental platform can be easily adapted to other screening applications by using a panel of genes specific to the biology of interest. For instance, screens to detect the differentiation of effector memory T cells into those with a central memory gene expression signature could be designed based on their different expression profiles. Screens to convert conventional CD4 T cells into regulatory T cells could be developed by using a transcriptional profile of genes differentially expressed between conventional CD4 T cells and Tregs [##REF##18024188##23##,##REF##12612578##24##]. Similarly, screens to identify compounds that differentiate exhausted CD8 T cells into functional T cells can be designed using a panel of genes representing each state [##REF##17950003##25##]. Assaying primary human T cells or mouse cells can be accommodated by the design of species-specific probes corresponding to orthologous genes. The ability to perform differentiation screens in T cell immunology with previously \"unscreenable\" phenotypes, and without the need to develop entirely new screening assays each time could significantly accelerate the discovery of compounds, genes or soluble factors that influence the development of T cell immunity in humans.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The differentiation of naive T and B cells into memory lymphocytes is essential for immunity to pathogens. Therapeutic manipulation of this cellular differentiation program could improve vaccine efficacy and the in vitro expansion of memory cells. However, chemical screens to identify compounds that induce memory differentiation have been limited by 1) the lack of reporter-gene or functional assays that can distinguish naive and memory-phenotype T cells at high throughput and 2) a suitable cell-line representative of naive T cells.</p>", "<title>Results</title>", "<p>Here, we describe a method for gene-expression based screening that allows primary naive and memory-phenotype lymphocytes to be discriminated based on complex genes signatures corresponding to these differentiation states. We used ligation-mediated amplification and a fluorescent, bead-based detection system to quantify simultaneously 55 transcripts representing naive and memory-phenotype signatures in purified populations of human T cells. The use of a multi-gene panel allowed better resolution than any constituent single gene. The method was precise, correlated well with Affymetrix microarray data, and could be easily scaled up for high-throughput.</p>", "<title>Conclusion</title>", "<p>This method provides a generic solution for high-throughput differentiation screens in primary human T cells where no single-gene or functional assay is available. This screening platform will allow the identification of small molecules, genes or soluble factors that direct memory differentiation in naive human lymphocytes.</p>" ]
[ "<title>Authors' contributions</title>", "<p>WNH conceived and designed the experiments, analyzed the data and wrote the manuscript. JA, KB, KR, CH, and SN performed the experiments and acquired the data. KR analyzed the data. KS helped design the experiments, analyze the data and write the manuscript.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>The authors are grateful to the volunteers who agreed to take part in this study.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Schema representing steps involved in ligation-mediated amplification.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Gene-expression profiling of naive and memory-phenotype human CD4 T cells</bold>. (A) Peripheral blood T cells, gated on CD4 T cells, stained to identify naive (green gate), central memory (CM) or effector memory (EM) cells. (B) Expression profile of genes differentially expressed in naive and memory-phenotype CD4 T cells sorted using the gates shown in (A). Each row represents an individual gene, and each column a different sample of naive, EM or CM CD4 T cells from seven healthy donors. Genes upregulated in memory-phenotype cells are shown in purple, and those upregulated in naive cells in green.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Amplification of differentiation signature genes in primary human T cells</bold>. (A) Expression level (aribrary units) of memory-phenotype marker genes (upper panel) or naive marker genes (lower panel) measured in sorted populations of naive (white bars) or memory-phenotype (black bars) peripheral blood CD4 T cells (25,000 cells/well). Each bar represents mean of ~6 replicate wells. Standard deviation was &lt; 8% (not shown) in all cases. (B) Difference in expression level for naive (white bars) or memory-phenotype (black bars) marker genes between memory-phenotype and naive CD4 T cells. Values &gt; 0 show relative increase in expression in memory-phenotype CD4 T cells; &lt; 0 show relative increase in naive CD4 T cells. (C) Cumulative score of memory-phenotype (plotted on Y-axis) or naive marker genes (X-axis) in effector/memory phenotype (black symbols) or naive (white symbols) CD4 T cells. Values expressed as Z scores to compensate for wide variation in expression level of individual genes.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Comparison of gene expression levels of differentiation signature genes measured by different platforms</bold>. Difference in expression level of naive (white symbols) or memory-phenotype (black symbols) marker genes or control genes (grey symbols) between memory-phenotype and naive CD4 T cells. Values &gt; 0 show relative increase in expression in memory-phenotype CD4 T cells; &lt; 0 show relative increase in naive CD4 T cells. Expression levels for corresponding genes measured by LMA are plotted on Y axis; by Affymetrix U133A microarray on X-axis. Each point represents the mean values from cells sorted from ~4 – 6 subjects. R<italic>s </italic>and <italic>P </italic>value refer to Spearman correlation coefficient.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Minimal donor variation in relative expression of differentiation signature in different donors</bold>. Correlation of expression level for each transcript in naive or memory-phenotype CD4 T cells measured by the method in two different healthy donors. Rs and P value refer to Spearman correlation coefficient.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Evaluation of LMA/Luminex detection assay reproducibility and scalability</bold>. (A) Mean expression level for each transcript in naive or memory-phenotype CD4 peripheral blood T cells were calculated and the deviation of that data point from its corresponding mean were calculated. The fraction of data points in each of 12 bins of fold deviation values is shown, representing 1100 data points (two differentiation states × 55 transcripts × 6 replicates). (B) Cumulative Z-score for multiple replicates of memory-phenotype (purple) or naive (green) CD4 T cells measured by the method carried out using robotic automation. Values &gt; 0 show relative increase in expression of memory-phenotype signature genes; &lt; 0 show relative increase of naive signature genes.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Z' Factor scores for the aggregate genes in the signature (summary score) or for the highest scoring three individual genes.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Parameter</bold></td><td align=\"center\"><bold>Z' Factor</bold></td></tr></thead><tbody><tr><td align=\"center\">Weighted summary score (all genes)</td><td align=\"center\">0.65</td></tr><tr><td align=\"center\"> S100A4</td><td align=\"center\">0.45</td></tr><tr><td align=\"center\"> LGALS3</td><td align=\"center\">0.36</td></tr><tr><td align=\"center\"> ANAX1</td><td align=\"center\">0.3</td></tr><tr><td align=\"center\">All other genes individually</td><td align=\"center\">&lt;0</td></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Supplementary Table 1. Sequence information for gene-specific probes.</p></caption></supplementary-material>" ]
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[ "<media xlink:href=\"1471-2172-9-44-S1.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>" ]
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{ "acronym": [], "definition": [] }
25
CC BY
no
2022-01-12 14:47:25
BMC Immunol. 2008 Aug 1; 9:44
oa_package/8c/3f/PMC2529265.tar.gz
PMC2529266
18657265
[ "<title>Background</title>", "<p>The evolution of the vertebrate globin superfamily has been extensively studied for many decades by comparing the structure and function of members of the gene families. These are principally haemoglobin, myoglobin, cytoglobin and neuroglobin and, more recently, globin X (in fish and amphibians [##REF##15356282##1##]) and globin Y (specific to amphibians [##REF##16484786##2##]).</p>", "<p>Haemoglobin genes (alpha- and beta-globin) are of particular interest because of their critical role in oxygen transportation from the respiratory surfaces to the inner organs, and because of the dire effects of mutations in human globin genes that cause haemoglobinopathies [##UREF##0##3##]. The genes contained in the alpha (α)- and beta (β)-globin clusters are expressed at different stages of development and in different tissues. Together, gene products from both clusters form the functional tetrameric haemoglobin molecules needed to fulfil oxygen requirements.</p>", "<p>The evolutionary history of α- and β-globin genes can be traced back to the common ancestors of fish, amphibians and amniotes (reptiles, birds and mammals), by comparing gene structure and composition of α- and β-globin clusters across vertebrates. In the amphibians <italic>Xenopus laevis </italic>and <italic>X. tropicalis</italic>, α- and β-globin genes are tightly juxtaposed as 5'-α-β-3' [##REF##16484786##2##,##REF##510792##4##, ####REF##7407927##5##, ##REF##7001356##6####7001356##6##]. In the Antarctic notothenioid fish (<italic>Notothenia coriiceps, N. angustata, Trematomus hansoni, T. pennellii</italic>), there is also a single 5'-α-β-3' locus [##REF##14516071##7##], although in pufferfish (<italic>Fugu rubripes</italic>) there are two globin clusters (one with α-globin genes and the other with both α- and β-globin genes), which are located on different chromosomes [##REF##12517812##8##].</p>", "<p>In amniotes, α- and β-globin clusters are located on different chromosomes. It was proposed that the ancestral α- and β-globin genes were located together in the common ancestor of amniotes, as they are in fish and amphibians, but became separated, either by chromosome fission or translocation between α- and β-genes, or by chromosome/genome or <italic>in trans </italic>duplication and gene loss [##REF##7407927##5##].</p>", "<p>Further duplications then occurred in amniote lineages. The ancestral α-globin gene is thought to have duplicated twice before the divergence of the bird-mammalian lineages, to produce progenitors of embryonic globin genes π/ζ, and adult α<sup>D </sup>and α<sup>A</sup>, all of which are present in birds (for example, the chicken <italic>Gallus gallus</italic>) [##REF##6273837##9##, ####REF##711749##10##, ##REF##6248855##11####6248855##11##] and mammals [##REF##16024261##12##,##REF##15855277##13##]. The order and timing of these duplications is still debated, as is their origin: for instance, α<sup>D </sup>may have evolved by duplication either of adult α<sup>A </sup>(see [##REF##16024261##12##]), or of an embryonic α-like gene [##REF##17586601##14##]. After the avian and mammalian lineages diverged, there were further tandem duplications of the π/ζ and α<sup>A </sup>lineages to produce more complex marsupial and eutherian ('placental') mammalian α-globin clusters, 5'-ζ-ψζ'-α<sup>D</sup>-ψα<sup>3</sup>-α<sup>2</sup>-α<sup>1</sup>-θ-3' (see [##REF##16024261##12##,##REF##3952001##15##, ####REF##6446404##16##, ##REF##282616##17##, ##REF##7407925##18####7407925##18##]). The timing of these duplication events is also uncertain, because we do not know whether these seven α-like globin genes all existed at the stem of the mammalian radiation.</p>", "<p>As for many other gene families [##REF##15892878##19##], comparisons of globin genes between distantly related mammals have provided unique insight into the evolution and function of the mammalian globins. Marsupials diverged from eutherian mammals about 148 million years ago (MYA), and mammalian Subclass Theria that contains these groups diverged from monotremes (Subclass Prototheria) about 166 MYA [##REF##17392779##20##], so comparisons between these major mammal groups provide depth for evolutionary comparisons. Monotremes retain many anatomical and developmental features shared with birds and reptiles. Their small genome, too, and disjunct chromosome size classes are reminiscent of reptile genomes, and the 10 sex chromosomes in a karyotype of 52 chromosomes is unique among mammals [##REF##18185982##21##, ####REF##15502814##22##, ##REF##15534209##23####15534209##23##]. Their importance for comparative studies is now increasingly recognised after the sequencing of the genome of a monotreme, <italic>Ornithorhynchus anatinus </italic>(platypus), to a depth of six to eight times by the Washington University Genome Centre, St Louis [##REF##18464734##24##].</p>", "<p>Indeed, studies of marsupial globins have clarified the timing of some of the duplications. The finding of single ε- (embryonic) and β-globin (adult) genes together in the marsupial β-globin cluster indicated that a two-gene cluster (ε-β) was present in the common therian ancestor [##REF##8265626##25##, ####REF##8752008##26##, ##REF##3375246##27##, ##REF##10381323##28####10381323##28##]. Genes in the cluster were further duplicated to produce the ancestral eutherian β-globin cluster of 5'-ε-γ-η-δ-β-3' (see [##REF##6527390##29##, ####REF##8383794##30##, ##REF##6599973##31##, ##UREF##1##32####1##32##]), which then underwent further tandem duplication events. In contrast, the bird (<italic>G. gallus</italic>) β-like globin genes (ε-β<sup>H</sup>-β<sup>A</sup>-ρ) show very little homology to the mammalian β-like globin genes [##REF##3454296##33##,##REF##8307571##34##].</p>", "<p>The discovery of a β-like globin gene (ω <italic>-</italic>globin) adjacent (3') to the α-globin cluster in marsupials led to a re-interpretation of globin evolution in birds and mammals [##REF##11158601##35##,##REF##15461421##36##]. Comparative sequence and phylogenetic analysis suggested that the ω-globin gene was more closely related to bird β-like globin genes than to other mammalian β-like globin genes. The specific function of the ω-globin gene is not yet known, but it is expressed just before birth and in the early stages of pouch young development [##REF##9342240##37##]. In addition, the ω-globin product binds to α-like globin chains to form functional haemoglobin, so it is likely to be involved in oxygen transportation [##REF##11158601##35##, ####REF##15461421##36##, ##REF##9342240##37####9342240##37##].</p>", "<p>This finding of a remnant β-like globin gene (ω <italic>-</italic>globin) beside the α-globin cluster in marsupials [##REF##11158601##35##,##REF##15461421##36##] provided some support for the alternative hypothesis [##REF##7407927##5##] that the α- and β-globin clusters in birds and mammals arose by <italic>in trans </italic>duplication of a chromosomal region, rather than simply by separation of the ancestral α-β globin cluster by chromosome fission or translocation. Wheeler et al. [##REF##11158601##35##,##REF##15461421##36##] proposed that before the divergence of birds and mammals (&gt;315 MYA), the chromosome region bearing the ancestral α-β clusters duplicated to form two clusters (α1-β1 and α2-β2) on different chromosomes, and their contents diverged independently in mammals and birds by silencing of some genes within each cluster (Figure ##FIG##0##1##). To account for the apparent orthology of the marsupial ω-globin gene and bird β-like globin genes, Wheeler et al. [##REF##11158601##35##,##REF##15461421##36##] suggested that the α1 and β2 were silenced in the eutherian lineage, but β2 was retained in marsupials as the ω-globin. In contrast, α2 and β1 were silenced in the bird lineage (Figure ##FIG##0##1##). On this hypothesis, then, both the α clusters and the β clusters of birds and mammals are paralogous (that is, evolved independently from ancient duplicates in an amniote ancestor) rather than orthologous (that is, diverged from the same ancestral cluster in an amniote ancestor).</p>", "<p>This paralogy hypothesis (which rests on the rather weak orthology between the chicken β and marsupial ω), as well as the dates and types of other duplications, could be further tested by studying globin genes of monotreme mammals, and using comparative data to infer the ancestral globin gene arrangement of a mammal ancestor 166 MYA. The availability of platypus genomic sequences now provides an efficient way to discover all of the globin genes and regulatory signals, and to understand their function and evolution. Studies of globin genes in monotremes are also interesting because the specialized features and lifestyle of these unique mammals may have given rise to special adaptations of globin genes to fulfil unusual oxygen requirements. These features include the need for oxygen by diffusion through the egg membrane to the embryo after birth and the physiological response to hypoxic conditions during hibernation, burrowing and diving [##UREF##2##38##, ####REF##6067227##39##, ##REF##5119901##40####5119901##40##].</p>", "<p>Little is known about monotreme α- and β-globin families. More than 30 years ago, studies of adult blood revealed a single adult α and β globin protein in the platypus [##REF##4464824##41##,##REF##1191127##42##] and echidna (<italic>Tachyglossus aculeatus </italic>[##REF##4798231##43##,##REF##4663350##44##]). Lee et al. [##REF##10381323##28##] later isolated an adult β-globin gene in the echidna that encoded a polypeptide identical to the previously isolated echidna β-globin [##REF##4663350##44##]. To date, there is no evidence of any monotreme embryonic ζ- or ε-globin genes.</p>", "<p>We used platypus genomic sequences from bacterial artificial chromosomes (BACs) to characterise the α- and β-globin gene families of the platypus and investigate their molecular evolution. In particular, we searched for embryonic and ω-globin genes and any novel globin genes that might fulfil the requirements for oxygen transport under hypoxic conditions. We investigated the genome context in order to infer the structure and origin of the ancestral α- and β-globin clusters at the stem of the mammalian radiation. Our results strongly support the hypothesis that the mammalian α- and β-globin clusters are orthologous to the avian α- and β-globin clusters, respectively, and that the β cluster evolved by transposition of a copy of the beta-like ω-globin gene in an amniote ancestor.</p>" ]
[ "<title>Methods</title>", "<title>Isolation and purification of probes to screen for platypus β-globins</title>", "<p>At the start of this project there were no trace sequences available for any globin genes in the platypus trace archive. We therefore designed probes to screen the platypus male Oa_Bb BAC library (Clemson University Genomic Institute, USA). The platypus β-globin-specific primers OaBGF (5'-tggacccagaggttctttgac-3') and OaBGR (5'-tgcaattcactcagcttggag-3') were designed from the reference tammar β-globin sequence [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"AY450928\">AY450928</ext-link>] using Primer3 [##REF##10547847##64##]. Amplification by PCR was performed in a final volume of 25 μl, with 40 ng genomic DNA, 1× Buffer (Roche, Australia), 0.2 mM dNTPs, 0.05 U Taq (Roche, Australia) and 1 μM each of forward and reverse primers. PCR cycling conditions were: 94°C for 2 minutes, then 35 cycles of 94°C for 30 seconds, 50 to 60°C for 30 seconds, 72°C for 1 minute, followed by 72°C for 10 minutes. The PCR products were sub-cloned according to the TOPO TA cloning<sup>® </sup>Kit Protocol (Invitrogen, Australia) and the resulting plasmids were purified according to the centrifugation protocol of Wizard<sup>® </sup>Plus SV Minipreps DNA Purification System (Promega, Australia). The purified plasmids were confirmed to contain PCR products of a partial platypus β-globin gene (167 bp) by sequencing at the Australian Genome Research Facility (AGRF, Brisbane, Australia) using M13 forward (5'-gtaaaacgacggccag-3') and M13 reverse (5'-caggaaacagctatgac-3') primers. Once confirmed, they were used as probes to screen the platypus BAC library.</p>", "<title>Screening the platypus BAC library for β-like globin genes</title>", "<p>The platypus BAC library filters were pre-hybridised at 65°C with Church Buffer (1 mM EDTA, 0.5 M phosphate buffer, 7% (w/v) SDS) including 1% BSA for 4 hours. The platypus β-globin probes (25 ng) were labelled with <sup>32</sup>P-dATP using the Megaprime DNA labelling System (GE Healthcare, Australia) following the manufacturer's instructions. The probes were allowed to hybridise to the filters at stringent conditions (65°C with the above buffer) for 24 hours and then washed twice for 15 minutes each in 2 × SSC/0.1%SDS and 1 × SSC/0.1%SDS. Autoradiography was carried out for 14 days at -80°C with an intensifying cassette.</p>", "<title>Identification of platypus BAC clones containing α-like globin genes</title>", "<p>Unlike β, BACs were not screened for α-like globin genes. Instead they were identified directly from the Encyclopaedia Of DNA Elements Project [##REF##15499007##65##], in which the α-globin cluster is one of the targeted regions [##REF##16024261##12##,##UREF##5##66##]. Two platypus BAC clones (Oa_Bb-2L7 and Oa_Bb-131M24), which were sequenced but not yet annotated, were identified by computational analysis (below) to contain parts of the α-globin cluster and a ω-globin gene.</p>", "<title>Isolation and purification of DNA from BAC clones</title>", "<p>DNA from the identified BAC clones (including those that were screened) was extracted using Wizard<sup>® </sup>Plus SV Minipreps DNA Purification System (Promega, Australia). The purified BAC clones were then subjected to Dot or Southern Blot to confirm the presence of α- or β-globin genes respectively.</p>", "<title>Confirmation of BACs containing globin genes</title>", "<p>Dot blot methods were used to verify the presence of the α-like globin genes. In a plate containing Luria broth agar with chloramphenicol, a Hybond N<sup>+ </sup>(GE Healthcare, Australia) filter was placed and multiple 1 μl of liquid culture BAC clones were spotted onto the filter. The plate was incubated at 37°C overnight and then the filter was soaked in Denaturation Solution (0.5 M NaOH and 1.5 M NaCl) for 5 minutes, followed by soaking twice in Neutralisation Solution (0.5 M Tris-Cl pH 7.4 and 1.5 M NaOH) for 5 minutes each. The filter was then rinsed in 2 × SSC, soaked in 0.4 M NaOH for 20 minutes and washed with 6 × SSC to remove all cellular debris. The filters were then screened with the platypus α-globin probes using the standard library screening procedure (above).</p>", "<p>Southern blotting was used to verify the presence of the β-like globin genes. In a 40 μl reaction, 20 to 40 ng BAC DNA was digested with 10 U of restriction enzyme,<italic>HIND III </italic>(Roche, Australia). The reaction was incubated at 37°C for at least 4 hours and separated by electrophoresis on a 0.8% agarose gel overnight at 40 V. The DNA fragments were transferred onto a Hybond N<sup>+ </sup>(GE Healthcare, Australia) nylon filter overnight by capillary action following the manufacturer's instructions, and cross-linked in 0.4 M NaOH for 20 minutes. These filters were then screened with the platypus β-globin probes using the standard library screening procedure (above).</p>", "<title>Fluorescence <italic>in situ</italic> hybridisation (FISH)</title>", "<p>Male platypus metaphase spreads were prepared and <italic>in situ </italic>mapping was performed using two-colour FISH as described previously by McMillan et al. [##REF##18185982##21##]. The verified BACs containing the α-like globin genes (ζ and ζ': Oa_Bb-2L7) and β-like globin genes (ε and β: Oa_Bb-484F22) were labelled with different fluorochromes and then hybridised to the chromosomes. The signals were detected by fluorescent microscopy, where at least twenty metaphase images were captured and analysed.</p>", "<title>Sequence data of the platypus BAC clones containing the α- and β-globin clusters</title>", "<p>Information about the platypus BAC clones containing the α-like globin genes along with the ω-globin gene was obtained directly from the ENCODE Project [##UREF##5##66##]. Their sequence information was obtained from GenBank; accession numbers: <ext-link ext-link-type=\"gen\" xlink:href=\"AC195438\">AC195438</ext-link> (Oa_Bb-2L7) and <ext-link ext-link-type=\"gen\" xlink:href=\"AC203513\">AC203513</ext-link> (Oa_Bb-131M24).</p>", "<p>The BAC clone containing the β-like globin genes that were found from the library screening procedure were sequenced at the Washington University Genome Sequencing Centre (St Louis, USA). The sequence information for this BAC clone was obtained from GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"AC192436\">AC192436</ext-link> (Oa_Bb-484F22).</p>", "<title>Computational characterisation of the α- and β-globin clusters in the platypus</title>", "<p>Using sequence information of <ext-link ext-link-type=\"gen\" xlink:href=\"AC195438\">AC195438</ext-link>, <ext-link ext-link-type=\"gen\" xlink:href=\"AC203513\">AC203513</ext-link> and <ext-link ext-link-type=\"gen\" xlink:href=\"AC192436\">AC192436</ext-link>, genes were predicted by GENSCAN [##REF##9149143##46##] and GenomeScan [##REF##11337476##47##] using default settings. All predicted gene sequences were then subjected to a BLAST search of the translated nucleotide acid (BlastX) and protein (BlastP) databases to confirm their identities.</p>", "<title>Promoter analyses</title>", "<p>Transcription factor binding motifs were predicted in the 200 bp promoter region located 5' to the predicted platypus α- and β-like genes and <italic>GBY </italic>by rVista 2.0 [##REF##15215384##67##] using user-defined consensus sequences for 'CACCC', 'CAAT', 'TATA', GATA1 ('WGATAR' [##REF##8262042##51##]) and EKLF ('NGNGTGGGN' [##REF##8262042##51##]). The same criteria were used to predict the same motifs in marsupials (<italic>Didelphis virginiana </italic>[ζ and ψζ': <ext-link ext-link-type=\"gen\" xlink:href=\"AC139599\">AC139599</ext-link>] and <italic>Sminthopsis macroura </italic>[α<sup>D</sup>, ψα<sup>3</sup>, α<sup>2</sup>, α<sup>1</sup>, ω: <ext-link ext-link-type=\"gen\" xlink:href=\"AC146781\">AC146781</ext-link>; and ε, β: <ext-link ext-link-type=\"gen\" xlink:href=\"AC148754\">AC148754</ext-link>]) and in humancs [ζ, ψζ', α<sup>D</sup>, ψα<sup>3</sup>, α<sup>2</sup>, α<sup>1</sup>: <ext-link ext-link-type=\"gen\" xlink:href=\"NG_000006\">NG_000006</ext-link>; and ε, β: <ext-link ext-link-type=\"gen\" xlink:href=\"NG_000007\">NG_000007</ext-link>] for consistency in comparison.</p>", "<title>Confirmation of α1 and α3 by BLAST search and Southern blot</title>", "<p>To confirm that the presence of two almost identical genes (α<sup>1 </sup>and α<sup>3</sup>) was real rather than an assembly error, the boundaries (~300 bp) of the homologous regions were investigated by a BLAST search against the platypus WGS database. The raw sequences of best hits were extracted from NCBI GenBank, cleaned and aligned in Sequencher v4.8 (Gene Codes Corporation, Michigan) using default settings.</p>", "<p>Southern blotting was also used to verify the presence of α<sup>1 </sup>and α<sup>3 </sup>genes. In a 30 μl reaction, 100 μg BAC DNA (Oa_Bb: 131M24, 130N2, 150K14 and 223I12) was digested with 10 U of restriction enzyme,<italic>EcoRV </italic>(Roche, Australia). The reaction was incubated at 37°C for at least 4 hours and separated by electrophoresis on a 0.8% agarose gel overnight at 40 V. The DNA fragments were transferred onto a Hybond N<sup>+ </sup>(GE Healthcare, Australia) nylon filter overnight by capillary action following the manufacturer's instructions, and cross-linked in 0.4 M NaOH for 20 minutes. These filters were then screened with the platypus α<sup>1</sup>/α<sup>3 </sup>(test) and α<sup>2 </sup>(control) probes using the standard library screening procedure (above).</p>", "<title>RT-PCR analyses</title>", "<p>To remove DNA contamination, RNAs derived from adult male platypus liver, kidney, spleen, testis, brain and lungs were DNase treated using a DNA-<italic>free</italic>™ kit according to the manufacturer's instructions (Applied Biosystems, Australia). Treated RNAs were then reverse transcribed using Superscript III (Invitrogen, Australia) following the manufacturer's instructions. Primers were designed against predicted α- and β- like and <italic>GBY </italic>globin gene sequences using Primer3 [##REF##10547847##64##]. In each case, the region amplified spanned an intron so that the origin of the template (gDNA or cDNA) was immediately obvious. Primer sequences and the expected sizes of amplified cDNA and gDNA bands are shown in Table ##TAB##1##2##. PCR reactions and cycling conditions were the same as for screening for the platypus β-globin genes, above. The positive bands were directly sequenced by AGRF (Brisbane, Australia) to confirm their identities. The blood contamination in the tested samples had minimal effect on the observed expression pattern, as some tissues (for example, lung and liver) showed no amplification despite containing large quantities of blood.</p>", "<title>Phylogenetic analyses</title>", "<p>Phylogenetic analyses were employed to verify the identities of the platypus globin genes and study the evolutionary relationships of the different members of the α- and β-globin gene families. This study was restricted to the coding domains of the α- and β-globin members and the accession numbers of the sequences used are given in the legends of Figures ##FIG##1##2## and ##FIG##2##3##. Phylogenetic analyses were conducted using MP in PAUP* v.4.0b10 [##REF##12504223##68##], and a BI approach using MrBayes v.3.1.2 [##REF##11524383##69##]. Concordance of trees from each of the different methods, bootstrap proportions and posterior probability estimates were used to examine the robustness of nodes.</p>", "<p>MP analyses were conducted for the entire coding sequence matrix and after excluding third codon positions using a heuristic search option and default options (TBR branch swapping), with the exception of using random stepwise addition repeated 100 times. Character state optimisation for MP trees used the DELTRAN option. MP bootstrap analyses [##UREF##6##70##] were carried out using 1000 bootstrap pseudoreplicates, employing a heuristic search option with random stepwise addition.</p>", "<p>The program MODELTEST [##REF##9918953##71##] and the Akaike information criterion (AIC) were used to assess the most appropriate model for BI analyses. The MODELTEST analyses were facilitated using the program MrMTgui v1.0 [##UREF##7##72##]. The MODELTEST analysis was carried out on separate codon positions for α- and β-globin data sets. For α-globin sequences, a general time reversible (GTR) model [##REF##2338834##73##], with a proportion of invariant sites (I) and unequal rates among sites [##UREF##8##74##], modelled with a gamma distribution (G) was found to be the most appropriate model to use for first and second codon positions, and a GTR+G model was appropriate for third codon positions under the AIC. For β-globin sequences a GTR+I+G model was considered appropriate for first positions, and a GTR+G model was found to be appropriate for second and third codon positions. The MrBayes analysis was carried out applying these different models to each codon position using an unlinked analysis, with default uninformative priors. Four chains were run simultaneously for 2 million generations in two independent runs, sampling trees every 100 generations. The program TRACER (version 1.3; [##UREF##9##75##]) was used to assess tree and parameter convergence. For both the α-globin and β-globin analyses all effective sample sizes for all parameters were &gt;1297, indicating a sufficient sample of the parameter space had been taken. A burn-in of 2000 trees (equivalent to 200,000 generations) was chosen for each independent run of MrBayes, with a &gt;50% posterior probability consensus tree constructed from the remaining 36,002 trees (18,001 trees each run).</p>", "<p>A BI analysis using MrBayes (version 3.1.2) was also carried out using protein sequence data from β-globin genes. A mixed protein model was used, allowing the optimum model of protein evolution to be assessed from a selection of nine fixed-rate models. The optimum model was found to be the Dayhoff model with a posterior probability of 1.0. The analyses were conducted using two million generations in two independent runs, sampling trees every 100 generations. A burn-in of 2,000 trees was used for each run with a 50% consensus tree constructed from the remaining 36,002 trees.</p>" ]
[ "<title>Results</title>", "<title>Identification of BAC clones containing the α- and β-globin clusters</title>", "<p>The draft sequence assembly of platypus [##REF##18464734##24##] is readily available on the University of California Santa Cruz (UCSC) Genome Browser [##UREF##3##45##]. However, currently the assembly is incomplete for the α- and β-globin clusters, as individual globin genes appear on different contigs. There are also sequences of the platypus BAC clones available in NCBI GenBank that are not yet annotated and assembled, nor is part of the platypus genome assembly. Two of these are Oa_Bb-2L7 [GenBank:<ext-link ext-link-type=\"gen\" xlink:href=\"AC195438\">AC195438</ext-link>] and Oa_Bb-131M24 [<ext-link ext-link-type=\"gen\" xlink:href=\"AC203513\">AC203513</ext-link>], which were identified from the Encyclopaedia of DNA Elements Project to contain parts of the α-globin cluster (see Methods). The BAC clone Oa_Bb-484F22 [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"AC192436\">AC192436</ext-link>] containing the β-globin cluster was obtained by screening a male platypus BAC library (Clemson University Genomic Institute, USA) and was subsequently fully sequenced and assembled by the Washington University Genome Sequencing Centre (St Louis, USA). These sequences were therefore used in this study to characterise the whole α- and β-globin clusters in the platypus.</p>", "<p>Genes in these sequenced BAC clones were predicted by programs GENSCAN [##REF##9149143##46##] and GenomeScan [##REF##11337476##47##]. Many genes were predicted, which were then used for BLAST searches of nucleotide (BlastN) and amino acid (BlastP) databases to help identify them (data not shown). Phylogenetic analyses were also conducted for the platypus α- and β-like globin genes to further verify the identity of each gene (see below and also Figures ##FIG##1##2##, ##FIG##2##3## and ##FIG##3##4## below). With only one exception (platypus ε-globin, see below), the identities of all of the genes inferred by BLAST analyses were supported by phylogenetic analyses with high posterior probabilities and bootstrap support values.</p>", "<title>Predictions and characterisation of genes in the platypus α-globin cluster</title>", "<p>One BAC (Oa_Bb-2L7) contained two embryonic α-like globin genes, and a second BAC (Oa_Bb-131M24) contained six α-like globin genes and a β-like globin gene (see Additional file ##SUPPL##0##1##). These two BACs were found to overlap by 10,066 base pairs (bp), resulting in a contig of 330,126 bp that contained the entire platypus α-globin cluster and flanking genes.</p>", "<p>The 330,126 bp α-globin contig was found to contain six α-like globin genes, a β-like globin gene, and a gene that bore little similarity to α- and β-like globin genes but some similarity to cytoglobins (Figure ##FIG##4##5A##). These six α-like globin genes have a three-exon/two-intron structure and conserved donor/acceptor splice sites (GT/AG) typical of all vertebrate α-like globin genes. They are separated from each other by 2 to 6 kilobase pairs (kb). Full details of the exon/intron lengths, location of the putative poly-A addition site (AATAAA) and the lengths of the coding domains with the predicted encoded polypeptide for each predicted gene are given in Table ##TAB##0##1##. Figure ##FIG##4##5B## shows the predictions for some of the well-characterised protein-binding sites in the 5' promoter region (about 200 bp 5' to the cap site of each gene). These include CACCC [##REF##8428663##48##], CAAT [##REF##2376599##49##], TATA [##REF##9642099##50##], GATA 1 [##REF##8262042##51##], EKLF (Erythroid Krüppel-like Factor; [##REF##9744863##52##]) and have been experimentally shown to control the stage- and tissue-specific expression of α- and β-like globin genes in other mammals [##REF##9642099##50##,##REF##17042767##53##, ####REF##8161819##54##, ##REF##9461381##55####9461381##55##].</p>", "<p>Two genes at the 5' end of the α-globin cluster were both identified as ζ-like (referred to here as ζ and ζ') and predicted to encode polypeptides of 142 amino acids (aa), which are typical of known functional mammalian α-like globin genes. The amino acid sequence alignment of ζ and ζ' shows 95% identity. In the promoter region of both genes, CACCC and CAAT consensus boxes are conserved at similar positions, and in comparable order to that of human ζ and ζ' (Figure ##FIG##4##5B##).</p>", "<p>Adjoining the two ζ-like globin genes, four other α-like globin genes were identified. One was an orthologue of bird and reptilian α<sup>D</sup>, and the other three were orthologues of adult α genes (here called α<sup>3</sup>, α<sup>2 </sup>and α<sup>1</sup>). The long and uninterrupted open reading frame (ORF) of α<sup>D </sup>strongly suggests that it encodes a functional polypeptide of 141 aa, typical of known functional α<sup>D </sup>globin genes. The platypus α<sup>D </sup>globin gene contains introns of 1450 bp (intron 1) and 1610 bp (intron 2) that are very large compared with those of other α-like globins, which are usually less than 1000 bp.</p>", "<p>Analyses of the platypus adult α-like globin genes reveal three adult (α<sup>3</sup>, α<sup>2 </sup>and α<sup>1</sup>) globin genes in the α-globin cluster. The sequence of α<sup>3 </sup>(the most 5' gene, adjacent to α<sup>D</sup>) was found to be almost identical to α<sup>1 </sup>(the most 3' gene) in their exon and intron regions, as well as in flanking regions of about 130 bp on both sides. The coding region was 100% identical, and just two sites in intron 1 were found to be different between the two genes. In order to confirm that identification of these two identical genes was not due to an error in the assembly of the original sequence data, the boundaries of the region containing the homology between α<sup>1 </sup>and α<sup>3 </sup>was further analysed by a BLAST search of the platypus whole-genome shotgun (WGS) database (data not shown). Two contigs were identified with homology to α<sup>1 </sup>and α<sup>3</sup>; these had identical sequences on one side of the boundary but different sequences on the other, confirming the presence of two separate genes. Further confirmation was obtained by performing a Southern blot on the α-globin-containing BACs, digested with an enzyme (<italic>EcoRV</italic>) that does not cut within the α<sup>1</sup>, α<sup>2</sup>and α<sup>3 </sup>(data not shown). Probing with α<sup>1</sup>/α<sup>3 </sup>revealed two bright bands, corresponding to α<sup>1 </sup>and α<sup>3</sup>, and one fainter band between them, corresponding to α<sup>2</sup>. Probing with α<sup>2 </sup>produced the same three bands, but in this case the middle one was brighter, corresponding to α<sup>2</sup>, and the outer bands were fainter, corresponding to α<sup>1 </sup>and α<sup>3</sup>. These analyses confirmed the existence of separate genes α<sup>1 </sup>and α<sup>3 </sup>in the platypus α-globin cluster. The α<sup>2 </sup>gene, located between α<sup>1 </sup>and α<sup>3</sup>, was distinct from both genes in the coding sequence (with 83% homology), in intron lengths (intron 1: 405 bp in α<sup>1</sup>/α<sup>3 </sup>and 720 bp in α<sup>2</sup>; intron 2: 151 bp in α<sup>1</sup>/α<sup>3 </sup>and 155 bp in α<sup>2</sup>) and in the promoter region (Figure ##FIG##4##5B##).</p>", "<p>The amino acid sequence encoded by α<sup>1 </sup>and α<sup>3 </sup>was identical to the platypus adult α-chain previously identified by Whittaker and Thompson [##REF##4464824##41##], implying that at least one of these genes is expressed in the adult platypus. The coding domain of α<sup>1 </sup>and α<sup>3 </sup>is shorter (426 bp) than that of α<sup>2 </sup>(429 bp), because it lacks the first three nucleotides of exon 1. The ORF of α<sup>2 </sup>gives a strong indication that it is translated into a functional polypeptide of 142 aa, typical of known functional mammalian α-like globin genes.</p>", "<p>On the 3' side of the six α-like globin genes, a β-like globin gene was predicted, which was identified as the orthologue of the marsupial ω-globin gene. This platypus ω-globin gene has a typical three-exon/two-intron structure, conserved donor/acceptor splice sites, and encodes a polypeptide of 146 aa, typical of all vertebrate β-like globin genes (Table ##TAB##0##1##). The promoter region located 5' of the ω-globin initiation codon contains conserved sites for CAAT-EKLF-CACCC in an order identical to that of marsupial ω-globin gene.</p>", "<p>Unexpectedly, GenomeScan predicted a gene based on the protein similarities with the α- and β-polypeptide chains, approximately 1.5 kb 3' of the ω-globin gene. Like other α- and β-globins, this gene also has a three-exon/two-intron structure and conserved donor/acceptor splice sites (Table ##TAB##0##1##). The lengths of its exons 1, 2, and 3 are 98, 223 and 144 bp, respectively, compared with 92, 223 and 129 bp in other β-like globin genes. However, it has much larger introns of 3364 bp (intron 1) and 3053 bp (intron 2). The long and uninterrupted ORF of this gene can be translated into a polypeptide of 154 aa, which is atypical of any known α- or β-like globin genes. A BLAST search of the amino acid sequence of this gene obtained the best hit with Globin Y (<italic>gby</italic>) of the amphibian <italic>X. laevis </italic>(identity score of 39%), and weaker identity scores with Cytoglobins (<italic>cygb</italic>) of other species, such as the fish <italic>Danio rerio </italic>(27%), <italic>X. tropicalis </italic>(26%), chicken (28%) and human (25%) at the protein level. We designated this gene '<italic>GBY</italic>' based on similarities with <italic>X. laevis gby</italic>, and its similar position adjoining the globin cluster [##REF##16484786##2##]. The predicted polypeptide of platypus <italic>GBY </italic>(154 aa) was shorter than <italic>X. laevis gby </italic>(156 aa), and quite different from <italic>X. laevis cygb </italic>(179 aa), <italic>D. rerio cygb1 </italic>(174 aa) and <italic>cygb2 </italic>(179 aa), and human <italic>CYGB </italic>(190 aa). Using the Expressed Sequence Tag (EST) database, a BLAST search of the platypus <italic>GBY </italic>also obtained an identity score of 38% with <italic>X. tropicalis gby </italic>that was expressed in both tadpoles and adults, but produced no significant matches with any other mammalian genes. The present work was the first opportunity to analyse the promoter region of any <italic>GBY </italic>gene (Figure ##FIG##4##5B##).</p>", "<title>Predictions and characterisation of genes in the platypus β-globin cluster</title>", "<p>In the platypus, only two β-like globin genes were predicted within the 129,521 bp BAC clone (Oa_Bb-484F22) by GENSCAN and GenomeScan (see Additional file ##SUPPL##0##1##). When the predicted amino acid sequences were subjected to BLAST search, the 5' gene had best hits with mammalian embryonic ε-globin genes. Although the phylogenetic analyses using Bayesian inference (BI; see below) indicated that this gene was more closely related to the platypus and echidna adult β-globin genes than to therian ε-globin genes, the position of this gene on the 5' end of the β-globin cluster and expression data (see below) supports its orthology with mammalian embryonic ε-globin genes, and is henceforth referred to as ε. The 3' gene encoded a protein identical to the previously identified platypus adult β-chain [##REF##1191127##42##], and is henceforth referred to as β.</p>", "<p>Both genes encode polypeptides of 146 aa, typical of known functional mammalian β-like globin genes. The promoter region of the platypus β has conserved sites of CACCC and CAAT in all three extant of mammals. However, the promoter region of the platypus ε appears to be quite different from other mammalian ε-globin genes and even from the platypus β (Figure ##FIG##4##5B##). The promoter of platypus ε contains only one predicted motif (CAAT), whereas the promoters of other mammalian ε, β and the platypus β contain many predicted motifs.</p>", "<title>Expression studies of the platypus α- and β-like globin genes</title>", "<p>Transcription studies were performed to gain insight into the expression and function of all of the predicted platypus globin genes. Adult liver, kidney, spleen, testis, lung and brain were obtained for this project: no embryonic samples were available (or are ever likely to be available) for this vulnerable and iconic species. Observation of the expression of any of the predicted genes would constitute a good indication that the gene is transcriptionally active and functional.</p>", "<p>Reverse-transcriptase polymerase chain reaction (RT-PCR) of all predicted platypus genes showed that they are all expressed in at least some of these adult platypus tissues (Figure ##FIG##5##6##). Platypus genes α<sup>1</sup>/α<sup>3</sup>, α<sup>2 </sup>and β, whose orthologues are usually expressed in the bone marrow of an adult human, were expressed in almost all platypus tissues tested, suggesting a broader expression of these genes in the monotreme lineage. Surprisingly, the genes ζ, ζ' and ε, whose therian orthologues are expressed only at embryonic stages of development, were expressed in adult spleen and testis, but not in the other tissues of adult platypus. This suggests that persistent expression of these genes in some adult tissues was selected for in the platypus, perhaps in response to its aquatic lifestyle and the hypoxic conditions of a confined burrow. Also, the expression pattern of platypus ε is similar to embryonic α-like ζ and ζ' but different from that of adult globin genes (α<sup>1</sup>/α<sup>3</sup>, α<sup>2 </sup>and β). The ω and α<sup>D </sup>globin genes, whose functions are unknown, were also expressed mainly in the spleen. <italic>GBY </italic>was expressed in all adult platypus tissues, most strongly in testis.</p>", "<title>Phylogenetic analyses</title>", "<p>Phylogenetic analyses of the α-like globin genes using BI and maximum parsimony (MP) produced several noteworthy results. The platypus adult α globin genes (α<sup>1</sup>/α<sup>3 </sup>and α<sup>2</sup>) grouped closely together to the exclusion of eutherian and marsupial α- and θ-globin genes for all analyses, although posterior probability (69%) and bootstrap support (66%) for this arrangement were relatively weak (Figure ##FIG##1##2##). This finding suggests that the duplication leading to the marsupial and eutherian θ-globin lineage occurred after the divergence of the monotreme and therian lineages. This is consistent with the absence of a θ-globin gene from the region between platypus α<sup>1</sup>- and ω-globin, its expected location based on its position in marsupial α-globin clusters [##REF##16024261##12##,##REF##15983873##56##].</p>", "<p>Both platypus ζ-globin genes grouped closely together and formed a sister group relationship with chicken π, supported by a high posterior probability of 97% (Figure ##FIG##1##2##). A sister group relationship was also found in MP trees for analyses of the entire platypus coding region (bootstrap support &lt;50%), and when third positions in the codon were excluded, was supported by 73% bootstrap pseudoreplicates (data not shown). This differs from the expectation that platypus ζ-globin genes would group with other mammalian ζ-globin genes to the exclusion of chicken π, suggesting that other factors (for example, purifying selection) operated to maintain a similar sequence in birds and monotremes.</p>", "<p>There is still considerable uncertainty in the phylogenetic position of the α<sup>D</sup>-globin clade. It has recently been proposed that the α<sup>D </sup>globin lineage resulted from duplication of the embryonic α-globin lineage, with phylogenetic analyses supporting a sister lineage relationship of these lineages to the exclusion of the adult α-globin lineage [##REF##17586601##14##]. However, this arrangement was not supported in BI analyses of the data set used here, and the position of the α<sup>D </sup>lineage was different in the different analyses. Analyses using BI (Figure ##FIG##1##2##) supported the sister lineage relationship of the α<sup>D </sup>and adult α-globin lineages (as proposed by Cooper et al. [##REF##16024261##12##]), with 87% posterior probability support. In contrast, all MP analyses supported the sister lineage status of α<sup>D </sup>and embryonic α-globin genes, indicating an uncertainty in the phylogenetic position of the α<sup>D</sup>-globin clade.</p>", "<p>Phylogenetic analyses of the β-globin genes provided results similar to recently reported phylogenetic analyses [##REF##11158601##35##,##REF##15461421##36##], with one notable exception. The BI analyses of coding sequence data (Figure ##FIG##2##3##) provided strong support (99% posterior probability) for the sister relationship of bird and mammalian β-like globin genes, contradicting previously published phylogenies of mammalian β-globin genes showing a sister relationship of marsupial ω-globin and bird β-like globin genes [##REF##11158601##35##,##REF##15461421##36##]. MP analyses (Figure ##FIG##3##4##), excluding third position in the codon, gave a similar tree arrangement, albeit with very low bootstrap support (&lt;50%). In marked contrast to the BI analyses of DNA sequence data, BI protein analyses (data not shown) supported the sister relationship of bird β-like globin and mammal ω-globin lineages with a high posterior probability (99%).</p>", "<p>Lastly, phylogenetic analyses using BI indicated that the platypus ε gene was more closely related to the platypus and echidna adult β-globin genes than to therian ε-globin genes, suggesting it may not be orthologous to marsupial and eutherian ε-globin (Figure ##FIG##2##3##). BI analyses of β-globin protein data and MP analyses of the coding sequence data, with third codon positions excluded, grouped the gene as an ancestral lineage to eutherian and monotreme adult β-globin genes (see Figure ##FIG##3##4##). This ancestral position suggests that the lineage evolved following duplication of an ancestral β-globin gene prior to the divergence of monotremes and therians.</p>", "<title>Location of the α- and β-globin clusters in the platypus</title>", "<p>The location of the verified BAC clones containing the α- (Oa_Bb-2L7) and β-globin (Oa_Bb-484F22) clusters in the platypus was determined by fluorescence <italic>in situ </italic>hybridisation (FISH) (Figure ##FIG##6##7##). The β-globin cluster localised to one of the largest autosomes, giving unambiguous signals on the long arm of chromosome 2 (2q5.1). The α-globin cluster localised to the smallest autosome, 21, whose two arms are not distinguishable by size or DAPI banding pattern [##REF##18185982##21##]. This is the first gene that has been localised on the platypus chromosome 21.</p>", "<title>Loci flanking the α- and β-globin clusters in the platypus and other vertebrates</title>", "<p>To explore the genome context of the α- and β-globin clusters in the platypus and other vertebrates, the platypus BAC sequences and the genomes of other sequenced species were searched for loci residing beside the α- and β-globin clusters.</p>", "<p>As well as globin genes, GENSCAN predicted within the platypus α-globin 330,126 bp contig many genes that flank the platypus α-globin cluster (Figure ##FIG##4##5A##), which were identified by BLAST analyses. These include <italic>IL9RP3-POLR3K</italic>-<italic>C16orf33</italic>-<italic>C16orf8-MPG-C16orf35 </italic>upstream (5') of the α-globin cluster, and, <italic>LUC7L-ITFG3-RGS11</italic>-<italic>ARHGDIG</italic>-<italic>PDIA2</italic>-<italic>AXIN1 </italic>downstream (3') of the α-globin cluster (Figure ##FIG##4##5A##).</p>", "<p>To compare the α-globin flanking loci of the platypus and other vertebrates, the genes closest to the α-globin cluster, <italic>MPG</italic>, <italic>C16orf35 </italic>and <italic>LUC7L </italic>were searched for in the human, opossum (<italic>Monodelphis domestica</italic>), chicken, frog (<italic>X. tropicalis</italic>) and zebrafish (<italic>D. rerio</italic>) genomes that were accessible from Ensembl [##UREF##4##57##]. Figure ##FIG##7##8A## shows that the locations of <italic>MPG</italic>, <italic>C16orf35 </italic>and <italic>LUC7L </italic>are conserved adjacent to the α-globin cluster of birds and mammals, and in the same position adjacent to the α-β cluster of amphibians, and all but <italic>LUC7L </italic>were also present in fish. These results are consistent with the previous analyses of Flint et al. [##REF##11157800##58##] and Hughes et al. [##REF##15998734##59##]. Thus the flanking loci analyses reveal that the genome context of the platypus α-globin cluster is the same as the α-globin clusters in therian mammals and birds, and this is the same as for the α-β cluster of fish and frogs.</p>", "<p>GENSCAN also predicted numerous genes other than globin genes in the platypus β-globin BAC (484F22). These were identified by a BLAST search as members of the olfactory receptor gene (<italic>ORG</italic>) family that are responsible for odour detection. Three conserved <italic>ORG </italic>members were identified at the 5' end of the platypus β-globin cluster and one conserved <italic>ORG </italic>member at the 3' end (Figure ##FIG##4##5A##).</p>", "<p>To compare β-globin flanking loci, <italic>ORG </italic>genes, as well as other genes that are closest to the β-globin cluster in other species, <italic>RRM1</italic>, <italic>CCKBR </italic>and <italic>ILK </italic>were searched for in the human, opossum, chicken and zebrafish genomes that were accessible from Ensembl [##UREF##4##57##]. Data from frog (<italic>X. tropicalis</italic>) was not useful since all of these loci lie on different contigs or scaffolds due to assembly problems. The locations of multiple <italic>ORG </italic>genes, <italic>RRM1</italic>, <italic>CCKBR </italic>and <italic>ILK </italic>were found to be conserved adjacent to β-globin cluster of birds and mammals [##REF##16554811##60##,##REF##10220430##61##], but not for the α-β cluster of fish and frogs, nor beside the second α-β cluster of zebrafish and pufferfish (Figure ##FIG##7##8B##). Thus the genome context of the platypus β-globin cluster is the same as in therian mammals and birds, but this is different from the α-β cluster of fish and frogs.</p>" ]
[ "<title>Discussion</title>", "<p>The phylogenetic position of monotremes makes comparisons with platypus of special value for exploring the organization, function and evolution of mammalian genes and genomes. The availability of platypus genome sequence data now makes many such studies possible, and have been used here to characterise the platypus α- and β-globin gene clusters and explore their evolutionary history.</p>", "<title>The platypus α-globin gene cluster</title>", "<p>The platypus α-globin cluster contains at least eight genes within more than 40 kb, including six α-like globin genes (including the identical α<sup>1 </sup>and α<sup>3</sup>), one β-like globin gene (ω-globin) and a gene belonging to another member of the globin super-family (<italic>GBY</italic>) arranged in the order 5'-ζ-ζ'-α<sup>D</sup>-α<sup>3</sup>-α<sup>2</sup>-α<sup>1</sup>-ω-<italic>GBY</italic>-3' (Figure ##FIG##4##5A##). The cluster maps to chromosome 21, the smallest autosome in platypus. All eight genes are likely to be functional since their expression was detected in tissues of an adult platypus.</p>", "<p>The platypus α-globin cluster is almost identical to the arrangement of α-like globin genes in the ancestral therian cluster reported by Cooper et al. [##REF##16024261##12##]. The one exception is the absence of a θ-globin gene from the platypus cluster. Phylogenetic analyses support the basal position of the monotreme adult α-globin lineage relative to marsupial and eutherian α- and θ-globin lineages, implying that the duplication of an adult α-globin to produce θ-globin occurred in the therian lineage after its divergence from the monotreme lineage (Figure ##FIG##8##9B##). However, although the numbers and arrangements of genes is so similar in platypus and therians, the presence of three adult α-globin genes and two embryonic ζ-globin genes in their common ancestor was not supported by phylogenetic analyses, which showed independent groupings of the three adult and embryonic genes within each separate mammalian lineage (Figure ##FIG##1##2## and see Cooper et al. [##REF##16024261##12##]). This result can be interpreted literally as resulting from independent duplications in each mammalian lineage to produce three adult and two embryonic genes in each. However, this seems unlikely to explain the convergence in gene number of the α-globin cluster in these distantly related mammalian lineages. We suggest that a more parsimonious explanation is that the common ancestor of monotremes and therians contained three adult α-globin genes and two ζ-globin genes, which were homogenised by ongoing gene conversion events, leading to the gene tree that does not match the duplication history of the individual genes. The close similarity of the platypus α<sup>3 </sup>and α<sup>1 </sup>loci suggests a very recent gene conversion event that homogenised their sequences. Therefore, we propose that the platypus α-globin cluster of eight genes (ζ-ζ'-α<sup>D</sup>-α<sup>3</sup>-α<sup>2</sup>-α<sup>1</sup>-ω-<italic>GBY</italic>) represents the ancestral mammalian α-globin cluster arrangement (Figure ##FIG##8##9B##), in which all genes were transcriptionally active.</p>", "<p>Importantly, the platypus α-globin cluster contains a copy of the β-like ω-globin gene, also found in the marsupial α-globin cluster, but absent in humans, supporting the hypothesis that ω-globin was present in the common ancestor of all mammals. Phylogenetic analyses also confirm the ancient ancestry of the ω-globin gene, as concluded by Wheeler et al. [##REF##11158601##35##,##REF##15461421##36##]. Among adult platypus tissues this gene was expressed only in the spleen. In marsupials, expression of the ω-globin gene was detected just prior to birth and during early pouch young development [##REF##9342240##37##], although the site of expression was not studied, and there was no evidence of adult expression in blood cells.</p>", "<title>Discovery of a mammalian GBY globin gene adjoining the α-globin cluster</title>", "<p>We discovered a globin gene <italic>GBY </italic>in the platypus that is adjacent (3') to ω in the α-globin cluster. It has a typical three-exon/two-intron structure like other α/β-globin genes, contains an ORF encoding a polypeptide chain of 154 aa, and is expressed in almost all adult tissues, most strongly in testis. The amino acid sequence is unrelated to any of the other globin genes in the cluster, so it is unlikely to be derived by duplication of α- or ω-globin within the monotreme lineage. Rather, it shows sequence similarity to <italic>gby </italic>of <italic>X. tropicalis </italic>and <italic>X. laevis</italic>, a gene thought to be related to cytoglobins [##REF##16484786##2##].</p>", "<p>Little is known of the function of amphibian <italic>gby</italic>, or its relationship with other globins. Fuchs et al. [##REF##16484786##2##] reported that amphibian <italic>gby </italic>encodes a <italic>bona fide </italic>globin of 156 aa, having all of the sequence features of a functional respiratory protein. <italic>gby </italic>was expressed in all adult tissues tested in <italic>X. laevis</italic>, most strongly in ovary, kidney and eye, and was present in 20 expressed sequence tag clones from different stages of <italic>X. laevis </italic>and <italic>X. tropicalis </italic>embryonic and adult development [##REF##16484786##2##], suggesting that it is expressed in embryonic as well as adult stages. Phylogenetic analysis of all vertebrate globins [##REF##16484786##2##] showed that the <italic>gby </italic>lineage diverged at the base of two separate clades, one comprising all vertebrate cytoglobins, myoglobins, agnathan globins and bird globin E, and the other comprising the haemoglobin α- and β-chains.</p>", "<p>The position of platypus <italic>GBY </italic>adjacent to the α-globin cluster and flanked by <italic>LUC7L </italic>mirrors its position in <italic>X. tropicalis </italic>between the main α-β cluster and <italic>LUC7L </italic>[##REF##16484786##2##]. Another common feature of both was strong expression in gonads (ovary in <italic>X. laevis </italic>[##REF##16484786##2##] and testis in platypus), so <italic>GBY </italic>has sex-related expression in both lineages. Thus <italic>GBY </italic>is not specific to amphibians, as was thought, but was a component of the cluster in an ancient tetrapod, and has been lost, or has diverged beyond recognition, in birds and therian mammals.</p>", "<title>The platypus β-globin gene cluster</title>", "<p>Characterisation of the platypus β-globin cluster revealed two β-like globin genes over about 13.2 kb that are arranged in the same order as marsupials, 5'-ε-β-3' (Figure ##FIG##4##5A##). This cluster is located on platypus chromosome 2q5.1. Both genes appear to be transcriptionally active and are likely to be functional.</p>", "<p>At the time of revising this paper, an independent paper on monotreme β-like globin genes was published by Opazo et al. [##REF##18216242##62##] in which they reported the presence of ω, ε<sup>P </sup>and β<sup>P </sup>in the platypus. Largely on the basis of phylogenetic analyses of flanking and coding sequence data, they proposed that platypus ε<sup>P </sup>and β<sup>P </sup>were not 1:1 orthologues of therian ε and β, respectively, and arose by independent duplication of an ancestral β-globin gene in the monotreme lineage, with a separate duplication event, just prior to the divergence of therians, producing the progenitors of ε and β of therians. This hypothesis was strongly supported by our BI phylogenetic (Figure ##FIG##2##3##) analyses, but not by MP analyses of coding sequence data, with third codon sites excluded (Figure ##FIG##3##4##), or BI analyses of protein sequence data (not shown). These contradictory analyses highlight the difficulty in resolving deep relationships among globin genes, particularly when the time periods between duplication and speciation events are relatively small, the phylogenetic signal at third codon positions is potentially saturated, and non-synonymous sites may be subjected to purifying or positive selection. Despite a very high posterior probability (100%) for the grouping of platypus ε with monotreme β, this value is a Bayesian probability and depends on the model adequately representing the evolution of the gene. Furthermore, although it was reported [##REF##18216242##62##] that the 5' flanking sequences of platypus ε and β were similar, we found no evidence for similarity of the promoter signals of these two genes (Figure ##FIG##4##5B##).</p>", "<p>We consider that a more parsimonious explanation is that the platypus ε is orthologous to the marsupial and eutherian embryonic β-like globin lineages (ε and γ), and arose by duplication of an ancestral β-globin gene prior to the mammalian radiation (166 MYA; Figure ##FIG##8##9B##). The sequence of platypus ε may have been homogenised by some gene conversion events, leading it to group with other monotreme adult β-like globin genes. In addition to the MP analyses reported above, this explanation is further supported by the conserved position of ε to the 5' side of the adult β-globin gene in the platypus cluster, which is similar to that found in other therian β-globin gene clusters [##REF##8752008##26##]; see also [##REF##6527390##29##]). Amino acid sequence analyses (BlastP) also provided additional support for the orthology of platypus ε to other mammalian ε-globin genes. Although we were unable to examine the expression of the genes in embryonic tissues, it was found that the expression profile of the platypus ε was similar to the embryonic α-like globins ζ and ζ' of the platypus, but not to the adult β-globin gene, supporting its potential role as an embryonic β-like globin gene.</p>", "<title>The ω-globin gene and the evolution of the β-globin cluster</title>", "<p>The discovery of the marsupial ω-globin gene in the α-globin cluster [##REF##11158601##35##,##REF##15461421##36##] was critical in re-interpreting the relationships of the α- and β-globin clusters in amniotes (reptiles, birds and mammals) to favour the hypothesis that these clusters in birds and mammals are paralogous, having diverged independently from different ancestral copies of the vertebrate α-β-globin locus [##REF##11171947##63##].</p>", "<p>Our observation of an ω-globin gene in the α-globin cluster in the platypus, as well as in the marsupials, confirms that the ancestral mammal α-globin cluster contained a β-like globin gene that was lost in eutherians, as proposed by Wheeler et al. [##REF##11158601##35##,##REF##15461421##36##]. However, the position of monotreme and marsupial ω in the phylogeny (Figure ##FIG##2##3##) is more consistent with the original hypothesis [##REF##7407927##5##] that mammal and bird β-globin are orthologous, having descended from the same β-globin progenitor in an amniote ancestor, and this is strongly supported by flanking sequence data (see below). Our data support the proposition that the ω <italic>-</italic>globin gene represents an ancient β-like globin gene lineage that is ancestral to a group containing both mammalian and bird β-globins with a high posterior probability (99%). This arrangement, however, was not supported by analyses of amino acid sequence data, indicating that there is uncertainty in the phylogenetic position of ω-globin relative to bird β-globins, or that convergent evolution of bird β-globin genes and ω-globin resulted in their similarity at the protein level. To further resolve the key question of whether bird and mammal β-globin gene clusters are orthologous we carried out comparative analyses of flanking loci of the α- and β-globin clusters.</p>", "<title>Genome context of vertebrate α- and β-globin clusters</title>", "<p>We found that the platypus α-globin cluster is flanked by <italic>MPG</italic>, <italic>C16orf35</italic>, <italic>GBY </italic>and <italic>LUC7L</italic>, and that the same genes (except <italic>GBY</italic>) flank the α-globin cluster in mammals and birds [##REF##11157800##58##,##REF##15998734##59##]. The same genes flank the α-β cluster of frog, and even zebrafish and the α-cluster of pufferfish [##REF##12517812##8##] (except <italic>GBY </italic>and <italic>LUC7L</italic>), implying that a very ancient region containing these genes (5'-<italic>MPG</italic>-<italic>C16orf35</italic>-α-β-<italic>GBY</italic>-<italic>LUC7L</italic>-3'), or perhaps an even larger region, was present in their common ancestor and has been conserved since the evolution of jawed vertebrates more than 450 MYA.</p>", "<p>In contrast, the amniote β-globin clusters reside in a very different genome, sharing none of the flanking loci with the mammal and bird α-globin clusters, or the α-β cluster of frogs and fish. In platypus, as well as in therian mammals [##REF##16554811##60##,##REF##10220430##61##], the β-globin clusters are flanked by numerous <italic>ORG </italic>genes on both sides. In birds, also, the β-globin cluster is embedded in <italic>ORG </italic>genes [##REF##16554811##60##]. Even the outside loci <italic>RRM1</italic>, <italic>CCKBR </italic>and <italic>ILK </italic>lie in the same orientation with respect to the bird and mammalian β-globin clusters [##REF##16554811##60##], suggesting that the 5'-<italic>RRM1-ORG-β </italic>(cluster)<italic>-ORG-CCKBR-ILK</italic>-3' arrangement has been conserved since before the divergence of birds and mammals, more than 315 MYA. Therefore, the bird β-globin cluster is orthologous to the β-globin clusters of mammals.</p>" ]
[ "<title>Conclusion</title>", "<title>New model for the evolution of α- and β-globin clusters in amniotes</title>", "<p>This analysis of flanking loci, in addition to the phylogenetic analyses reported above, refutes the prevailing hypothesis that mammal and bird α- and β-globin clusters evolved from different (paralogous) copies of an ancestral α-β-globin region containing <italic>MPG-C16orf35-α </italic>(cluster)-β (cluster)<italic>-GBY-LUC7L</italic>. Rather, the context of β-globin clusters within olfactory receptor genes in birds as well as mammals suggests that a copy of a β-globin locus was moved into a region replete with <italic>ORG </italic>genes before the divergence of birds and mammals 315 MYA. The precise mechanism for this translocation is unknown, but is likely to be either by transposition of a tandem duplicate of an ancestral β-globin gene, or retrotransposition of an intron-containing primary transcript. Phylogenetic analyses suggest that this ancestral β-globin gene within the α-globin cluster is represented by the platypus and marsupial ω-globin gene. The transposed β-globin gene then independently duplicated several times within the avian and mammalian lineages to form the different clusters of differentially expressed β-globin genes. Full details of this new model are given in Figure ##FIG##8##9A## and ##FIG##8##9B##.</p>", "<p>This hypothesis could be further tested by investigating the gene organization of the α- and β-globin clusters in reptiles such as lizards and snakes, which form a sister group to birds. Our hypothesis predicts that reptiles should possess a <italic>MPG-C16orf35</italic>-α (cluster)-β (cluster)<italic>-GBY-LUC7L </italic>cluster, and an unlinked <italic>RRM1-ORG-β </italic>(cluster)<italic>-ORG-CCBKR-ILK </italic>cluster like birds and mammals. The full genome sequence of the first reptilian species,<italic>Anolis carolinensis</italic>, will provide an opportunity to test this hypothesis.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (<italic>Ornithorhynchus anatinus</italic>), to explore haemoglobin evolution at the stem of the mammalian radiation.</p>", "<title>Results</title>", "<p>The platypus α-globin cluster (chromosome 21) contains embryonic and adult α- globin genes, a β-like ω-globin gene, and the <italic>GBY </italic>globin gene with homology to cytoglobin, arranged as 5'-ζ-ζ'-α<sup>D</sup>-α<sup>3</sup>-α<sup>2</sup>-α<sup>1</sup>-ω-<italic>GBY</italic>-3'. The platypus β-globin cluster (chromosome 2) contains single embryonic and adult globin genes arranged as 5'-ε-β-3'. Surprisingly, all of these globin genes were expressed in some adult tissues. Comparison of flanking sequences revealed that all jawed vertebrate α-globin clusters are flanked by <italic>MPG</italic>-<italic>C16orf35 </italic>and <italic>LUC7L</italic>, whereas all bird and mammal β-globin clusters are embedded in olfactory genes. Thus, the mammalian α- and β-globin clusters are orthologous to the bird α- and β-globin clusters respectively.</p>", "<title>Conclusion</title>", "<p>We propose that α- and β-globin clusters evolved from an ancient <italic>MPG-C16orf35-α</italic>-β-<italic>GBY-LUC7L </italic>arrangement 410 million years ago. A copy of the original β (represented by ω in marsupials and monotremes) was inserted into an array of olfactory genes before the amniote radiation (&gt;315 million years ago), then duplicated and diverged to form orthologous clusters of β-globin genes with different expression profiles in different lineages.</p>" ]
[ "<title>Authors' contributions</title>", "<p>VSP designed and performed most of the experiments and analysed the data. VSP also drafted the main manuscript. SJBC conducted phylogenetic analyses and contributed to the writing of the manuscript. JED helped in designing the experiments and trouble-shooting experiments. BF, TG, WCW and RKW were involved in sequencing the platypus BAC clone (Oa_Bb-484F22). JAMG conceived and supervised the research and contributed to the writing of the manuscript. All authors read and approved the final manuscript.</p>", "<title>Competing Interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>We thank Dr Frank Grützner (The University of Adelaide) and Tim Hore (The Australian National University) for providing platypus tissues and RNA preparations. We also thank Dr Paul Waters for providing computational help with the identification and confirmation of two identical but different genes α<sup>1 </sup>and α<sup>3</sup>.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Current proposed model for the evolution of α- and β-globin clusters from paralogous clusters in different lineages</bold>. The unlinked α- and β-globin clusters in birds and mammals evolved from an ancient <italic>in trans </italic>duplication of the ancestral linked α-β cluster, followed by differential gene silencing (marked with X). This resulted in bird β-like globin genes (β2) orthologous to the marsupial ω-globin gene (β2 beside the α-globin cluster) but paralogous to mammalian β-like globin genes (β1). Adapted from Wheeler et al. [##REF##15461421##36##].</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Evolutionary relationships among vertebrate α-like globin genes using a 50% majority rule consensus phylogram from an analysis using Bayesian Inference</bold>. The tree was constructed using mixed models of evolution for each codon position (see methods) and estimated base frequencies in an unlinked analysis using MrBayes (v. 3.1.2). Numbers adjacent to branches refer to % posterior probabilities. GenBank accession numbers for sequences are: Virginian Opossum (<italic>Didelphis virginiana</italic>) ζ<sup>1</sup>, ζ<sup>2</sup>, α<sup>1</sup>, α<sup>2</sup>, θ [<ext-link ext-link-type=\"gen\" xlink:href=\"AC139599.2\">AC139599.2</ext-link>, <ext-link ext-link-type=\"gen\" xlink:href=\"AC148752.1\">AC148752.1</ext-link>]; Stripe-faced Dunnart (<italic>Sminthopsis macroura</italic>) α<sup>D</sup>, α<sup>2</sup>, θ [<ext-link ext-link-type=\"gen\" xlink:href=\"AC146781\">AC146781</ext-link>]; Brazilian Opossum (<italic>Monodelphis domestica</italic>) α [TI# 453585430]; Tammar wallaby (<italic>Macropus eugenii</italic>) θ [<ext-link ext-link-type=\"gen\" xlink:href=\"AY459590\">AY459590</ext-link>], α [<ext-link ext-link-type=\"gen\" xlink:href=\"AY459589\">AY459589</ext-link>]; ζ [<ext-link ext-link-type=\"gen\" xlink:href=\"AY789121\">AY789121</ext-link>], ζ' [<ext-link ext-link-type=\"gen\" xlink:href=\"AY789122\">AY789122</ext-link>]; Horse (<italic>Equus caballus</italic>) θ (ψ α) [<ext-link ext-link-type=\"gen\" xlink:href=\"Y00284\">Y00284</ext-link>], α<sup>1 </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"M17902\">M17902</ext-link>], ζ [<ext-link ext-link-type=\"gen\" xlink:href=\"X07051\">X07051</ext-link>]; pig (<italic>Sus scrofa</italic>) α<sup>D </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"AC145444\">AC145444</ext-link>]; cat (<italic>Felis catus</italic>) α<sup>D </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"AC130194\">AC130194</ext-link>]; cow (<italic>Bos taurus</italic>) α<sup>D </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"AC150547\">AC150547</ext-link>]; Goat (<italic>Capra hircus</italic>) α [<ext-link ext-link-type=\"gen\" xlink:href=\"J00043\">J00043</ext-link>]; Human (<italic>Homo sapiens</italic>) α1 [<ext-link ext-link-type=\"gen\" xlink:href=\"V00491\">V00491</ext-link>], θ [<ext-link ext-link-type=\"gen\" xlink:href=\"X06482\">X06482</ext-link>], ζ [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_005332\">NM_005332</ext-link>]; <italic>mu</italic>/α<sup>D </sup>chain [<ext-link ext-link-type=\"gen\" xlink:href=\"AY698022\">AY698022</ext-link>]; Mouse (<italic>Mus muscularis</italic>) α<sup>1 </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"NM_008218\">NM_008218</ext-link>], ζ [<ext-link ext-link-type=\"gen\" xlink:href=\"X62302\">X62302</ext-link>]; Rabbit (<italic>Oryctolagus cuniculus</italic>)α [<ext-link ext-link-type=\"gen\" xlink:href=\"X04751\">X04751</ext-link>]; Eastern Quoll (<italic>Dasyurus viverrinus</italic>) α [<ext-link ext-link-type=\"gen\" xlink:href=\"M14567\">M14567</ext-link>]; Chicken (<italic>Gallus gallus) </italic>α<sup>A</sup>, π, α<sup>D </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"AF098919\">AF098919</ext-link>]; Duck (<italic>Cairina moschata</italic>) α<sup>D </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"X01831\">X01831</ext-link>]; Pigeon (<italic>Columba livia</italic>) α<sup>D </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"AB001981\">AB001981</ext-link>]; Turtle (<italic>Geochelone nigra</italic>) α<sup>D </sup>[SEG# <ext-link ext-link-type=\"gen\" xlink:href=\"AB1165195\">AB1165195</ext-link>]; Zebrafish (<italic>Danio rerio</italic>) α<sup>1 </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"NM_131257\">NM_131257</ext-link>]; Salamander (<italic>Hynobius retardatus</italic>) larval α [<ext-link ext-link-type=\"gen\" xlink:href=\"AB034756\">AB034756</ext-link>]; Salamander (<italic>Pleurodeles waltlii</italic>) α [<italic>M</italic>13365]; Frog (<italic>Xenopus laevis</italic>) α <italic>I </italic>[<ext-link ext-link-type=\"gen\" xlink:href=\"X14259\">X14259</ext-link>], larval (tadpole) α T5 [<ext-link ext-link-type=\"gen\" xlink:href=\"X02798\">X02798</ext-link>]; Yellowtail (<italic>Seriola quinqueradiata</italic>) α<sup>A </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"AB034639\">AB034639</ext-link>]; Salmon (<italic>Salmo salar</italic>) α [<ext-link ext-link-type=\"gen\" xlink:href=\"X97289\">X97289</ext-link>]; Southern Puffer (<italic>Sphoeroides nephelus</italic>) α<sup>2 </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"AY016023\">AY016023</ext-link>]; Platypus (<italic>Ornithorhynchus anatinus</italic>) ζ, ζ', α<sup>D</sup>, α<sup>3</sup>, α<sup>2</sup>, α<sup>1 </sup>[<ext-link ext-link-type=\"gen\" xlink:href=\"AC203513\">AC203513</ext-link>].</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Evolutionary relationships among vertebrate β-like globin genes using a 50% majority rule consensus phylogram from an analysis using Bayesian inference</bold>. The tree was constructed using mixed models of evolution for each codon position (see methods) and estimated base frequencies in an unlinked analysis using MrBayes (v. 3.1.2). Numbers adjacent to branches refer to % posterior probabilities. GenBank accession numbers for sequences are: Fat-tailed Dunnart (<italic>Sminthopsis crassicaudata</italic>) β [<ext-link ext-link-type=\"gen\" xlink:href=\"Z69592\">Z69592</ext-link>], ε [<ext-link ext-link-type=\"gen\" xlink:href=\"Z48632\">Z48632</ext-link>], ω [<ext-link ext-link-type=\"gen\" xlink:href=\"AY014770\">AY014770</ext-link>]; Stripe-faced Dunnart (<italic>S. macroura</italic>) β, ε [<ext-link ext-link-type=\"gen\" xlink:href=\"AC148754\">AC148754</ext-link>]; Virginian Opossum (<italic>Didelphis virginiana</italic>) β [<ext-link ext-link-type=\"gen\" xlink:href=\"J03643\">J03643</ext-link>], ε [<ext-link ext-link-type=\"gen\" xlink:href=\"J03642\">J03642</ext-link>]; Brazilian Opossum (<italic>Monodelphis domestica</italic>) β [<ext-link ext-link-type=\"gen\" xlink:href=\"XM_001365299\">XM_001365299</ext-link>], ε [<ext-link ext-link-type=\"gen\" xlink:href=\"XM_001364448\">XM_001364448</ext-link>], ω [<ext-link ext-link-type=\"gen\" xlink:href=\"XM_001364828\">XM_001364828</ext-link>]; Tammar Wallaby (<italic>Macropus eugenii</italic>) β [<ext-link ext-link-type=\"gen\" xlink:href=\"AY450928\">AY450928</ext-link>], ε [<ext-link ext-link-type=\"gen\" xlink:href=\"AY450927\">AY450927</ext-link>], ω [<ext-link ext-link-type=\"gen\" xlink:href=\"AY014769\">AY014769</ext-link>]; African clawed frog (<italic>Xenopus laevis</italic>) larval β <italic>I </italic>[<ext-link ext-link-type=\"gen\" xlink:href=\"NM_001086273\">NM_001086273</ext-link>], larval βII [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_001088028\">NM_001088028</ext-link>]; Western clawed frog (<italic>X. tropicalis</italic>) β [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_203528\">NM_203528</ext-link>], larval ε1 [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_001016495\">NM_001016495</ext-link>]; Chicken (<italic>Gallus gallus</italic>) β (β<sup>A</sup>) [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_205489\">NM_205489</ext-link>], ε [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_001004390\">NM_001004390</ext-link>], γ (β<sup>A</sup>) [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_001031489\">NM_001031489</ext-link>]; Duck (<italic>Cairina moschata</italic>) β [<ext-link ext-link-type=\"gen\" xlink:href=\"J00926\">J00926</ext-link>], ε [<ext-link ext-link-type=\"gen\" xlink:href=\"X15740\">X15740</ext-link>]; Human (<italic>Homo sapiens</italic>) β [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_000518\">NM_000518</ext-link>], γ [<ext-link ext-link-type=\"gen\" xlink:href=\"BC130459\">BC130459</ext-link>], ε [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_005330\">NM_005330</ext-link>]; Mouse (<italic>Mus musculus</italic>) β (β1) [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_008220\">NM_008220</ext-link>], γ (β h0) [<ext-link ext-link-type=\"gen\" xlink:href=\"NW_001030869\">NW_001030869</ext-link>], ε (ε<sup>y</sup>) [<ext-link ext-link-type=\"gen\" xlink:href=\"M26897\">M26897</ext-link>]; Goat (<italic>Capra hirus</italic>)<italic>β </italic>(β<sup>A</sup>) [<ext-link ext-link-type=\"gen\" xlink:href=\"DQ350619\">DQ350619</ext-link>], ε (ε<sup>I</sup>) [<ext-link ext-link-type=\"gen\" xlink:href=\"X01912\">X01912</ext-link>], γ [<ext-link ext-link-type=\"gen\" xlink:href=\"M15388\">M15388</ext-link>]; Rabbit (<italic>Oryctolagus cuniculus</italic>) β, γ, ε [<ext-link ext-link-type=\"gen\" xlink:href=\"M18818\">M18818</ext-link>]; Echidna (<italic>Tachyglossus aculeatus) </italic>β [<ext-link ext-link-type=\"gen\" xlink:href=\"L23800\">L23800</ext-link>]; Pufferfish (<italic>Fugu rubripes</italic>) β [<ext-link ext-link-type=\"gen\" xlink:href=\"AY170464\">AY170464</ext-link>]; Zebrafish (<italic>Danio rerio</italic>) ε1 [<ext-link ext-link-type=\"gen\" xlink:href=\"NM_001103130\">NM_001103130</ext-link>]; Platypus β, ε [<ext-link ext-link-type=\"gen\" xlink:href=\"AC192436\">AC192436</ext-link>], ω [<ext-link ext-link-type=\"gen\" xlink:href=\"AC203513\">AC203513</ext-link>].</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Evolutionary relationships among vertebrate β-like globin genes analysed by maximum parsimony (MP) trees of length 926 (one of eight trees)</bold>. Third position in codons were excluded in the MP analyses, which were conducted using a heuristic search in PAUP* v.4.0b10 [##REF##15499007##65##]. The tree is rooted using pufferfish β-globin. Numbers adjacent to branches represent % bootstrap values (&gt;50%) from MP heuristic analyses of 1000 pseudoreplicates. Accession numbers for sequences are given in the caption of Figure 3.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Gene structure of the platypus α- and β-globin clusters and flanking loci, and comparisons of their promoter regions with other mammals</bold>. (A) The platypus α-globin cluster contains six α-like globin genes (red), a β-like (ω) globin gene (blue) and a distantly related globin gene, <italic>GBY </italic>(green), which are flanked by <italic>IL9RP3-POLR3K</italic>-<italic>C16orf33</italic>-<italic>C16orf8-MPG-C16orf35 </italic>on the 5' end and <italic>LUC7L-ITFG3-RGS11</italic>-<italic>ARHGDIG</italic>-<italic>PDIA2</italic>-<italic>AXIN1 </italic>on the 3' end (black). The platypus β-globin cluster contains only two genes, ε and β (blue), which are flanked on both sides by <italic>ORG </italic>genes (black). (B) Relative positions of the putative transcription factor binding sites in the 200 bp promoter region located upstream of the predicted platypus, marsupial (<italic>Didelphis virginiana </italic>ζ and ψζ', and <italic>Sminthopsis macroura </italic>α<sup>D</sup>, ψα<sup>3</sup>, α<sup>2</sup>, α<sup>1</sup>, ω, ε and β) and human α- and β-like globin genes. For the platypus <italic>GBY </italic>no data was available from other species, including <italic>Xenopus tropicalis</italic>, for comparisons.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Expression of all predicted α- and β-like globin genes including <italic>GBY </italic>in an adult platypus</bold>. For each of the platypus predicted genes, expression was investigated by reverse transcriptase polymerase chain reaction in adult liver, kidney, spleen, testis, brain and lung. Primers for each gene were designed between two exons so that it would result in a product distinguishable from genomic contamination of cDNA. The negative control (last lane) contained no cDNA. All genes were expressed in one or more tissues, indicating that they are transcriptionally active and might be functional.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p><bold>Chromosomal location of the platypus α- and β-globin clusters</bold>. Two-colour fluorescence in situ hybridisation showing the location of the α-globin cluster on chromosome 21 (green) and the β-globin cluster on chromosome 2q5.1 (red). The chromosomes are counterstained with DAPI (blue).</p></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Figure 8</label><caption><p><bold>Loci flanking vertebrate α-globin (A) and β-globin (B) clusters</bold>. The relative locations of flanking loci (A) <italic>MPG</italic>, <italic>C16orf35</italic>, <italic>LUC7L </italic>and <italic>GBY </italic>and (B) <italic>RRM1</italic>, <italic>CCKBR</italic>, <italic>ILK </italic>and <italic>ORG </italic>genes were searched for beside the α-β globin cluster in zebrafish (<italic>Danio rerio</italic>) and frog (<italic>Xenopus tropicalis</italic>), and beside the separate α-globin and β-globin clusters in chicken (<italic>Gallus gallus</italic>), opossum (<italic>Monodelphis domestica</italic>) and human (<italic>Homo sapiens) </italic>from Ensembl [##UREF##4##57##]. The pufferfish (<italic>Fugu rubripes</italic>) flanking loci shown here were adapted from Gillemans et al. [##REF##12517812##8##]. For the platypus, the α-globin flanking loci were characterised in this study, and <italic>ORG </italic>genes surrounding the platypus β-globin cluster were discovered: however, the BAC clone (484F22) was too small to cover the region containing the loci <italic>RRM1</italic>, <italic>CCKBR </italic>and <italic>ILK</italic>. In <italic>X. tropicalis LUC7L </italic>was found on another scaffold (466 from Ensembl) but sequence analyses by Fuchs et al. [##REF##16484786##2##] suggested that <italic>LUC7L </italic>resides 3' to the frog α-β-<italic>GBY </italic>cluster. The flanking loci as well as the α- and β-globin clusters are differentiated by colour.</p></caption></fig>", "<fig position=\"float\" id=\"F9\"><label>Figure 9</label><caption><p><bold>Proposed model for the evolution of the α- and β-globin clusters in vertebrate lineages</bold>. (A) A region containing <italic>MPG-C16orf35</italic>-α-β-<italic>GBY</italic>-<italic>LUC7L </italic>represented the ancient α-β globin cluster of jawed vertebrates (&gt;450 MYA), which is seen in the amphibian lineage. This region further duplicated and underwent some gene silencing in teleost fish. In an amniote ancestor of reptiles, birds and mammals (&gt;315 MYA), a copy of an ancestral β-globin gene from this region was inserted into a different chromosome within a region replete with multiple copies of <italic>ORG </italic>genes. The original amniote β-globin gene survives as the ω-globin gene (β1) in the α-globin cluster of marsupials and monotremes, whereas the transposed β-globin gene (β2) duplicated several times to form different clusters in the different lineages. (B) Tandem duplications of the ancestral amniote α-globin gene produced a three-gene (π-α<sup>D</sup>-α<sup>A</sup>) cluster in the avian lineage. In the mammalian lineage, further duplications gave rise to a six-gene (ζ-ζ'-α<sup>D</sup>-α<sup>3</sup>-α<sup>2</sup>-α<sup>1</sup>) cluster with ongoing gene conversion events homogenising the embryonic and adult genes. In monotremes, the ancestral ω (β1) and <italic>GBY </italic>are retained. After the divergence of monotreme and therian mammals, there was an additional duplication of α<sup>2 </sup>to form θ, giving rise to the seven-gene cluster (ζ-ζ'-α<sup>D</sup>-α<sup>3</sup>-α<sup>2</sup>-α<sup>1</sup>-θ) in marsupials and eutherians. Marsupials also retain the ancestral ω but may have lost <italic>GBY </italic>gene; eutherians retain no identifiable remnant of either gene. Furthermore, the ancestral transposed β2-globin gene duplicated independently in birds and mammals. Before the mammalian radiation, we propose that the ancestral β2 gene duplicated to form a two-gene β-globin cluster (ε-β) as seen in monotremes and marsupials, except that ongoing gene conversion events homogenised platypus ε to group with monotreme β genes. After the divergence of marsupial and eutherian mammals, there were further tandem duplications of these two genes to produce complex β-globin cluster (ε-γ-η-δ-β) in eutherians.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Gene-structure of the predicted platypus α- and β-like globin genes and <italic>GBY</italic></p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Length/</bold><break/><bold>Genes</bold><break/></td><td align=\"left\"><bold>Exon 1 </bold><break/><bold>(bp)</bold><break/></td><td align=\"left\"><bold>Intron 1 </bold><break/><bold>(bp)</bold><break/></td><td align=\"left\"><bold>Exon 2 </bold><break/><bold>(bp)</bold><break/></td><td align=\"left\"><bold>Intron 2 </bold><break/><bold>(bp)</bold><break/></td><td align=\"left\"><bold>Exon 3 </bold><break/><bold>(bp)</bold><break/></td><td align=\"left\"><bold>Position of </bold><break/><bold>Poly-A</bold><break/></td><td align=\"left\"><bold>CDS </bold><break/><bold>(bp)</bold><break/></td><td align=\"left\"><bold>Poly- </bold><break/><bold>peptide </bold><break/><bold>(aa)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>ζ</bold></td><td align=\"left\">95</td><td align=\"left\">337</td><td align=\"left\">205</td><td align=\"left\">114</td><td align=\"left\">129</td><td align=\"left\">+119</td><td align=\"left\">429</td><td align=\"left\">142</td></tr><tr><td align=\"left\"><bold>ζ<sup>1</sup></bold></td><td align=\"left\">95</td><td align=\"left\">336</td><td align=\"left\">205</td><td align=\"left\">102</td><td align=\"left\">129</td><td align=\"left\">+133</td><td align=\"left\">429</td><td align=\"left\">142</td></tr><tr><td align=\"left\"><bold>α<sup>D</sup></bold></td><td align=\"left\">92</td><td align=\"left\">1450</td><td align=\"left\">205</td><td align=\"left\">1610</td><td align=\"left\">129</td><td align=\"left\">+77</td><td align=\"left\">426</td><td align=\"left\">141</td></tr><tr><td align=\"left\"><bold>α<sup>1</sup></bold>/<bold>α</bold><sup><bold>3</bold></sup></td><td align=\"left\">92</td><td align=\"left\">405</td><td align=\"left\">205</td><td align=\"left\">151</td><td align=\"left\">129</td><td align=\"left\">+94</td><td align=\"left\">426</td><td align=\"left\">141</td></tr><tr><td align=\"left\"><bold>α<sup>2</sup></bold></td><td align=\"left\">95</td><td align=\"left\">720</td><td align=\"left\">205</td><td align=\"left\">155</td><td align=\"left\">129</td><td align=\"left\">+115</td><td align=\"left\">429</td><td align=\"left\">142</td></tr><tr><td align=\"left\"><bold>ω</bold></td><td align=\"left\">92</td><td align=\"left\">256</td><td align=\"left\">223</td><td align=\"left\">111</td><td align=\"left\">129</td><td align=\"left\">+69</td><td align=\"left\">444</td><td align=\"left\">147</td></tr><tr><td align=\"left\"><bold><italic>GBY</italic></bold></td><td align=\"left\">98</td><td align=\"left\">3364</td><td align=\"left\">223</td><td align=\"left\">3053</td><td align=\"left\">144</td><td align=\"left\">+141</td><td align=\"left\">465</td><td align=\"left\">154</td></tr><tr><td align=\"left\"><bold>ε</bold></td><td align=\"left\">92</td><td align=\"left\">143</td><td align=\"left\">223</td><td align=\"left\">474</td><td align=\"left\">129</td><td align=\"left\">+96</td><td align=\"left\">444</td><td align=\"left\">147</td></tr><tr><td align=\"left\"><bold>β</bold></td><td align=\"left\">92</td><td align=\"left\">153</td><td align=\"left\">223</td><td align=\"left\">438</td><td align=\"left\">129</td><td align=\"left\">+71</td><td align=\"left\">444</td><td align=\"left\">147</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>PCR primers used for amplification of the α- and β-like globin genes including <italic>GBY </italic>from the platypus gDNA and cDNA</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Gene</bold><break/></td><td align=\"left\"><bold>Forward Primer</bold><break/></td><td align=\"left\"><bold>Reverse Primer</bold><break/></td><td align=\"left\"><bold>gDNA</bold><break/><bold>(bp)</bold></td><td align=\"left\"><bold>cDNA </bold><break/><bold>(bp)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>ζ</bold></td><td align=\"left\">GGCCGACAAGACCGCAGTCATCTCCC</td><td align=\"left\">CCCGATGGCGCTGATGACT</td><td align=\"left\">527</td><td align=\"left\">190</td></tr><tr><td align=\"left\"><bold>ζ<sup>1</sup></bold></td><td align=\"left\">TGACCAAAGGCGACAAGACCT</td><td align=\"left\">CCCCGATGGCACCGATGACC</td><td align=\"left\">534</td><td align=\"left\">198</td></tr><tr><td align=\"left\"><bold>α<sup>D</sup></bold></td><td align=\"left\">GAGGCTGTGAAGAACCTGGA</td><td align=\"left\">GGTGTACTCCCCTTGCAGAT</td><td align=\"left\">1793</td><td align=\"left\">153</td></tr><tr><td align=\"left\"><bold>α<sup>2</sup></bold></td><td align=\"left\">TGGCCCACCTCGATGACCTGG</td><td align=\"left\">GGGAAGGTGTCTGGCCACC</td><td align=\"left\">289</td><td align=\"left\">134</td></tr><tr><td align=\"left\"><bold>α<sup>1</sup></bold>/<bold>α</bold><sup><bold>3</bold></sup></td><td align=\"left\">GCAAGGCCGCCGGTCACGGC</td><td align=\"left\">CGCTGTCCATGTCATCGAAGTGCC</td><td align=\"left\">597</td><td align=\"left\">192</td></tr><tr><td align=\"left\"><bold>ω</bold></td><td align=\"left\">ATTGTGTCCATCTGGGGAAA</td><td align=\"left\">GCTTGGCAAAGTTGCTCTTC</td><td align=\"left\">488</td><td align=\"left\">232</td></tr><tr><td align=\"left\"><bold><italic>GBY</italic></bold></td><td align=\"left\">CTGGAAACAGGTGTGCAAGA</td><td align=\"left\">CTATCTCCGGGGTGTAGCAG</td><td align=\"left\">3202</td><td align=\"left\">149</td></tr><tr><td align=\"left\"><bold>ε</bold></td><td align=\"left\">ATCTGAGCGCTGAGGAGAAG</td><td align=\"left\">GACAGGTTGCCGAAGGAGTCA</td><td align=\"left\">285</td><td align=\"left\">142</td></tr><tr><td align=\"left\"><bold>β</bold></td><td align=\"left\">CTGTGGGGGAAAGTGAACAT</td><td align=\"left\">GGTCAGCACCTTAGCACCAT</td><td align=\"left\">321</td><td align=\"left\">168</td></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p><bold>Annotation of the platypus α- and β-like and <italic>GBY </italic>globin genes</bold>. This table shows the predicted positions of six α-like, ω and <italic>GBY </italic>globin genes in the platypus BAC clone Oa_Bb-131M24 [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"AC203513\">AC203513</ext-link>], two α-like globin genes in BAC clone Oa_Bb-2L7 [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"AC195438\">AC195438</ext-link>], and two β-like globin genes in BAC clone Oa_Bb-484F22 [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"AC192436\">AC192436</ext-link> reverse direction].</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><p>For each predicted gene, the length of the exons and introns, position of poly-A addition site (AATAAA) from the stop codon, and the length of their putative coding domain (CDS) and encoded polypeptide are shown. All genes contained consensus splice sites (GT/AG) in both introns.</p></table-wrap-foot>" ]
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[{"surname": ["Dickerson", "Geis"], "given-names": ["RE", "I"], "source": ["Hemoglobin: Structure, Function, Evolution and Pathology"], "year": ["1983"], "publisher-name": ["Menlo Park, CA: Benjamin Cummings"], "fpage": ["65"], "lpage": ["116"]}, {"surname": ["Weatherall"], "given-names": ["DJ"], "source": ["The Structure, Organization, and Regulation of Human Genes"], "year": ["1991"], "edition": ["3"], "publisher-name": ["Oxford: Oxford University Press"]}, {"surname": ["Andersen", "Mesch", "Lovell", "Nicol"], "given-names": ["NA", "U", "DJ", "SC"], "article-title": ["The effects of sex, season and hybernation on haematology and blood viscosity of free-ranging echidnas ("], "italic": ["Tachyglossus aculeatus"], "source": ["Can J Zool"], "year": ["2000"], "volume": ["78"], "fpage": ["174"], "lpage": ["181"], "pub-id": ["10.1139/cjz-78-2-174"]}, {"article-title": ["University of California Santa Cruz (UCSC) Genome Browser"]}, {"article-title": ["Ensembl Genome Browser"]}, {"article-title": ["NIH Intramural Sequencing Center (NISC)"]}, {"surname": ["Felsenstein"], "given-names": ["J"], "article-title": ["Confidence limits on phylogenies: an approach using the bootstrap"], "source": ["Evolution"], "year": ["1985"], "volume": ["39"], "fpage": ["783"], "lpage": ["791"], "pub-id": ["10.2307/2408678"]}, {"article-title": ["MrMTgui v1.0"]}, {"surname": ["Yang"], "given-names": ["Z"], "article-title": ["Among-site rate variation and its impact on phylogenetic analyses"], "source": ["Trends Ecol Evol"], "year": ["1996"], "volume": ["11"], "fpage": ["367"], "lpage": ["372"], "pub-id": ["10.1016/0169-5347(96)10041-0"]}, {"surname": ["Rambaut", "Drummond"], "given-names": ["A", "AJ"], "article-title": ["Traver v1.3"], "year": ["2004"]}]
{ "acronym": [], "definition": [] }
75
CC BY
no
2022-01-12 14:47:25
BMC Biol. 2008 Jul 25; 6:34
oa_package/bc/af/PMC2529266.tar.gz
PMC2529267
18598348
[ "<title>Background</title>", "<p>Duchenne Muscular Dystrophy (DMD) is a lethal X-linked disorder that affects 1 in 3,500 newborn human males. It is caused by mutations in a large gene located at Xp21 that encodes the muscle protein dystrophin [##REF##16989735##1##]. One third of DMD cases result from new mutations, whereas the remaining are maternally inherited. Affected boys are confined to a wheelchair around age 10 to 12 and later develop respiratory and cardiac problems that lead to death usually in the third or fourth decade.</p>", "<p>Satellite cells are spare stem-cells responsible for muscle growth and regeneration. Replacement of defective muscle cells by the patient's satellite cells has been pursued for a long time [##REF##13768451##2##,##REF##11776472##3##]. Other alternative sources of stem cells (SC) such as bone marrow, cord-blood or adipose tissue have been tested in the mdx mouse, which is the murine model for human DMD. These experiments resulted in the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin expression in the affected muscle [##REF##10517639##4##]. However, the mdx mouse does not present evident muscle weakness, with the exception of significant histopathological alterations in the diaphragm [##REF##1865908##5##]. Myoblasts transplantation has also been tried for skeletal muscle tissue engineering [##REF##15601570##6##] but it failed due to the immunogenic properties of these cells. A new strategy to overcome this obstacle has been recently developed through myoblast transplantation tolerance with anti-CD45RB, anti-CD154 and mixed chimerism [##REF##15268726##7##].</p>", "<p>The closest model for human DMD is the golden retriever muscular dystrophy (GRMD) dog, which has a splice acceptor site mutation in intron 6, causing a frameshift due to deletion of exon 7 from the mature mRNA [##REF##1577476##8##]. This mutation results in the absence of the muscle protein dystrophin [##REF##1536178##9##].</p>", "<p>In the first reported trial using GRMD dogs, bone marrow hematopoietic SC were transplanted from normal litter mates to immunosuppressed GRMD dogs. Nevertheless, dystrophin expression was not restored [##REF##15328150##10##]. In another study, it was suggested that mesoangioblast multipotent cells from dorsal aorta improved muscle function when transplanted into dystrophic dogs [##REF##17108972##11##]. The latter authors used both heterologous wild-type (WT) and autologous genetically modified canine mesoangioblasts in their experiments. In their study, the authors suggested that mesoangioblasts are promising cells to be used in SC therapy for DMD patients. However, because these studies were performed under different regimes of immunosuppression, one cannot rule out that immunosuppression alone may be responsible for clinical improvement as in DMD boys, as it has been suggested in other studies [##REF##17306535##12##].</p>", "<p>We recently reported the successful isolation of a population of hIDPSC from dental pulp of non-exfoliated deciduous teeth. Under standard culture conditions, these cells express both the embryonic stem (ES) cells transcriptional factors Oct4 and Nanog as well as surface markers of mesenchymal stem cells (MSC) such as CD105, CD73, and CD13. Nevertheless, they lack the expression of CD45, CD34, CD14, CD43, and of HLA-DR. These cells are able to undergo spontaneous and induced <italic>in vitro </italic>differentiation into osteoblasts, adipocytes and chondroblasts, muscle cells, and into neurons <italic>in vitro</italic>. After transplantation into normal mice, these cells show significant engraftment in liver, spleen, brain and kidney, among others [##REF##17409736##13##].</p>", "<p>Recently, hIDPSC, which are constituted by a homogeneous population positive for MSC markers, were shown to contribute for the reconstruction of large cranial defects produced in non immunosuppressed rats after their transplantation onto collagen membrane, without presenting any graft rejection [##REF##18216690##14##]. Furthermore, populations of dental pulp MSC (DP-MSC) similar to those of hIDPSC, with immunosuppressive activity were described [##REF##16210973##15##]. Analysis of their proliferation activity demonstrated that it was significantly higher in DP-MSC, when compared to those from bone marrow. Similarly to bone marrow MSC, these cells inhibited the proliferation of phytohemagglutinin stimulated T-cells, presenting an even stronger effect than in BM-MSC [##REF##16210973##15##,##REF##11986244##16##]. Other authors reported that DPSC can survive and engraft in ischemic environments in non immunosuppressed rats with acute myocardial infarction [##REF##18079433##17##]. Taken together these data led us to investigate the myogenic potential of these cells in GRMD dogs.</p>", "<p>Herein, we show results obtained after early transplantation of hIDPSC in four affected litter-mate GRMD dogs at an early age (two males and two females) with no use of immune suppression based on recent investigation. With this study we aimed at analyzing the cells ability for migration, engraftment, myogenic potential and expression of human dystrophin in affected muscles. Additionally, the efficiency of single and serial early transplantation were compared with the subsequent evaluation of the dog's clinical condition without the interference of immunosuppression protocols.</p>" ]
[ "<title>Materials and Methods</title>", "<title>Dogs genotyping</title>", "<p>Clinical studies were approved by the ethical committee of the School of Veterinary Medicine and Animal Science of São Paulo University. The animals were identified by numbers and experimental procedures were described in Table ##TAB##0##1##. DMD genotyping was done from blood genomic DNA extracted with GFX Genomic Blood DNA Purification Kit (GE Healthcare, Piscataway, NJ – USA/Canada) and established as previously reported [##REF##10376903##18##]. DMD diagnosis was confirmed by restriction digestion of PCR products of the dystrophin gene with Sau96I and by elevated serum creatine kinase (CK).</p>", "<title>Mouse model</title>", "<p>Normal mice were used to investigate the ability of hIDPSC migration by intraperitoneal injections and engraftment capacity of these cells without immunosuppression.</p>", "<title>Cell culture</title>", "<p>Cells were obtained and characterized as described previously [##REF##17409736##13##]. Shortly, dental pulp was extracted from normal exfoliated human deciduous teeth of 5- to 7-year-old children under local anesthetic. Tissue explant of dental pulp was used to isolate the cells. Human IDPSC cultures were maintained in DMEM/F12 (Dulbecco's-modified Eagle's medium/Ham's F12, 1:1, Invitrogen, Carlsbad, CA) supplemented with 15% fetal bovine serum (FBS, Hyclone, Washington), 100 units/ml penicillin, 100 units/ml streptomycin, 2 mM L-glutamine, and 2 mM nonessential amino acids. Cells were maintained semi-confluent to prevent their differentiation and replaced every 4 or 5 days. Medium was replaced daily. Human IDPSC were incubated at 37°C in a high humidity environment with 5% CO2.</p>", "<title>FACS analysis</title>", "<p>Monoclonal anti-human SH2 (CD105, Serotec, Oxford, UK), SH3 and SH4 (CD73) (Case Western Reserve University, Cleveland, Ohio, USA) antibodies against cell surface molecules and their respective isotype controls were used in flow cytometry analysis. About 10<sup>6 </sup>cells were incubated with primary antibody for 30 minutes at 4°C and washed in PBS with 2% FBS and 1 M sodium azide (buffer) followed by addition of secondary anti-mouse-PE – conjugated antibody according to manufacturer's instructions (Becton Dickinson, NJ, USA; Guava Technologies, CA). Negative controls were also performed incubating the cells in PBS followed by incubation with respective secondary antibody only. Results were analyzed using Guava Express<sup>®</sup>Plus software (Guava Technologies, Hayward, CA, USA). Flow cytometry was analyzed using a fluorescence-activated cell sorter (FACS, Becton, Dickinson, San Jose, CA) with CELL Quest program (Becton, Dickinson, San Jose, CA).</p>", "<title>In vitro myogenic differentiation of hIDPSC</title>", "<p>Cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum (FBS, Hyclone, Washington), 5% horse serum, 0.1 M dexamethasone, 50 nM hydrocortisone (Sigma Aldrich, São Paulo, SP), 1% antibiotic (100 units/ml penicillin and 100 mg/ml streptomycin; Invitrogen, São Paulo, SP) for 60 days.</p>", "<title>Fluorescent dye staining of hIDPSC</title>", "<p>The culture cell was washed twice in calcium and magnesium-free Dulbecco's phosphate-buffered solution (DPBS, Invitrogen) and dissociated with 0.25% trypsin/EDTA solution (Invitrogen). The suspension was centrifuged and the cell pellet was ressuspended in DMEM (Invitrogen) with 10% FBS (Invitrogen) containing the fluorescent dye (Vybrant CM-Dil Cell-Labelling Solution; Molecular Probes, Invitrogen). Cells were incubated for 15 minutes at 37°C, washed twice in DPBS immediately prior to intraperitoneal injection in mice.</p>", "<title>Muscle biopsy and histology</title>", "<p>Biceps femoralis biopsy samples were taken before the hIDPSC transplantation experiment on day 0 (d 0) and after 47, 107 and 117 days in different animals as summarized in Table ##TAB##0##1##. A second biopsy was taken from the male under systemic SC, delivery (MT1-S), after one year of treatment. Each biopsy from <italic>biceps femuralis </italic>was divided into 3 fragments for histological, immunofluorescence/WB and FISH analyses. For IF analysis, tissue samples were embedded in Jung Tissue Freezing Medium (Leica Mycrosystems Nussloch GmbH, Nussilosh, Germany) and frozen in liquid nitrogen. For WB analysis, 2 mm<sup>2 </sup>fragments were frozen directly in liquid nitrogen in small flasks. For histological analysis routine hematoxylin and eosin (HE) staining was applied.</p>", "<title>Immunohistochemistry and Confocal microscopy</title>", "<p>Differentiated cells were fixed in 4% paraformaldehyde for 2 h at 4°C, washed three times in wash buffer (150 mM NaCl, 1 mg/ml BSA, 0.5% Nonidet P- 40, 50 mM Tris pH 6.8) and membranes were permeabilized with two 10 min incubations in RIPA (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycolate, 0.1% SDS, 1 mM EDTA, 50 mM Tris pH 8.0). Cells were subsequently re-fixed in 4% PFA for 30 min at 4°C, blocked in wash buffer for 1 h at 4°C and incubated overnight at 4°C with primary antibodies. Cy3-conjugated goat anti-(mouse IgG) secondary antibody and FITC-conjugated goat anti-(rabbit IgG) secondary antibody, normal mouse IgG and normal rabbit IgG were used. The primary antibody was omitted from some slides to serve as a negative control.</p>", "<p>Myogenic differentiation was characterized using mouse anti-human muscle α-actinin antibody and rabbit anti-human muscle myosin antibody (Santa Cruz Biotechnology, CA, USA) and by RT-PCR using MyoD1(MD1) forward primer 5'AAGCGCCATCTCTTGAGGTA3' and reverse primer 3'GCCTTTATTTTGATCACC5' following the protocol described previously[##REF##17409736##13##].</p>", "<p>Tissues samples were excised, immediately frozen in liquid nitrogen and then stored at -80°C. Cryostat tissue sections with 5 μm thickness were mounted on a glass slide. According to the antibody used, slides were fixed and dehydrated with cold methanol (Merck, Darmstadt, Germany). To detect the presence of hIDPSC mouse, anti-hIDPSC antibody previously obtained [##REF##17409736##13##] was used at a 1:500 dilution, whereas mouse anti-human nuclei antibody (Monoclonal, Chemicon International, California, USA) was used at a 1:100 dilution followed by FITC-conjugated secondary antibodies of respective isotype at a 1:1000. Dystrophin analysis was done using IF by applying the methodologies from our group [##REF##12491947##19##]. Human specific anti-dystrophin monoclonal Mandys106 2C6 antibody was kindly provided by Dr. Glenn E. Morris at Center for Inherited Neuromuscular Diseases, Oswestry, UK [##REF##7887428##20##]. Antibody DYS2 (Vector Laboratories – Burlingame, CA) against the C-terminal domain of dystrophin and anti-mouse IgG Cy3- conjugated secondary antibody were also used to confirm the presence of some positive dystrophin fibers. Microscope slides were mounted in Vectashield mounting medium (Vector Laboratories) with or without 4',6-Diamidino-2-phenylindol (DAPI). IF analysis was done in a Zeiss Imager Z1 Apotome microscope with epi-fluorescence, or using an argon ion laser scan microscope LSM 410 (Zeiss – Jena, Germany).</p>", "<p>Confocal microscopy: An argon ion laser set at 488 nm for FITC and at 536 for rodamine excitation were used. The emitted light was filtered with a 505 nm (FITC) and 617 nm (rodamine) long pass filter in a laser scan microscope (LSM 410, Zeiss – Jena, Germany). Sections (5 mm) were taken approximately at the mid – height level of tissues. Photo-multiplier gain and laser power were kept constant throughout each experiment.</p>", "<title>Fluorescent in situ hybridization (FISH)</title>", "<p>FISH analysis was done with specific centromere probes for human chromosomes X (green) and Y (red) (Aquiarius-Cytocell, Cambridge, UK). Tissue samples were fixed in 4% paraformaldehyde, embedded in Jung Tissue Freezing Medium and 5 μm sections were made.</p>", "<p>The slides with sections were then fixed with cold methanol (Merck, Darmstadt, Germany) and dehydrated in series of ethanol. FISH reactions were done according to the manufacturer's protocol. Microscope slides were mounted in antifade (Vectashield mounting medium) with or without Propidium Iodide (PI, Vectashield mounting medium/PI). FISH analysis was made using confocal microscope as described above.</p>", "<title>Biochemical analysis</title>", "<p>Dogs underwent periodic veterinary examinations during treatment. Blood samples were collected monthly by jugular venipuncture. Hematological and serum biochemical testing were performed with an automated cell counter (Baker System 9000, Serno-Baker Diagnostics Inc, Allentown, Pennsylvania). Urea, alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK) and creatinine were measured in serum with an automated analyzer (Labtest<sup>® </sup>– LABTEST Diagnóstica S.A. – Lagoa Santa, MG). Data are shown in Table ##TAB##1##2##.</p>", "<title>Physical score exams</title>", "<p>We established a physical exam score (Table ##TAB##2##3##), adapted from literature [##REF##17475492##21##] that evaluated dogs motility and posture, and which gave values between 0 (normal) to 24 (severely affected).</p>" ]
[ "<title>Results</title>", "<title>Evaluation of hIDPSC skeletal myogenic potential and migrating ability</title>", "<p>Isolated hIDPSC (2n = 46, XY) were used at passages 6 or 7 [##REF##17409736##13##]. Because cells have been cryopreserved, we checked their morphology (Figure ##FIG##0##1A##), proliferation efficiency (Figure ##FIG##0##1B##) and expression of principal MSC markers such as SH2, SH3 and SH4 (Figure ##FIG##0##1C–F##), prior to their use. Additionally, cells were re-analyzed prior to transplantation. Their <italic>in vitro </italic>ability to differentiate into myotubes was confirmed by immunostaining of α-actinin (sarcomeric) (Figure ##FIG##0##1G##) and of myosin (Figure ##FIG##0##1H##) and by RT-PCR of the MyoD1 gene (Figure ##FIG##0##1I##). After thawing, migration ability of CM-DiI-fluorescently labeled hIDPSC (10<sup>5</sup>) was confirmed by its detection in the heart muscle of intraperitoneal injected normal mice after 2 weeks (Figure ##FIG##0##1J–M##). No prior immunosuppression was done.</p>", "<title>hIDPSC transplantation in GRMD dogs</title>", "<p>To minimize the effects of inter and interfamilial variability previously observed in GRMD dogs we used animals from the same litter. Six affected animals (3 males and 3 females) born after the breading of an affected male with a carrier female through artificial insemination were used in the experiment. The dystrophin mutation in these puppies was confirmed by PCR (Figure ##FIG##1##2##). Cells were injected in four dogs, two MT1-S (male transplanted 1- systemic) and MT2-IM (male transplanted 2- intramuscular) and two females FT1-S (female transplanted 1-systemic) and FT2-IM (female transplanted 2- intramuscular). Each gender received 6 × 10<sup>7 </sup>hIDPSC via arterial or muscular injection. One non-injected male (MC – male control) and one non-injected female (FC – female control) were analyzed as age-matched controls (Table ##TAB##0##1##). The first systemic and local injections were done respectively in two 28- days-old female dogs. To verify the potential effect of short- and long-term treatment with hIDPSC, females received one unique injection, whereas males were treated with monthly injections, starting with 44 days old.</p>", "<p>One male (MT2-IM) received six intramuscular (<italic>biceps femularis</italic>) injections, whereas another subject (MT1-S) received nine arterial (femoral artery) systemic injections. No immunosuppression was used before or after cell transplantation. Muscle biopsies were obtained at the beginning of the experiment (d 0) from FC only; after 107 days in the three females and after 47 and 117 days only from the injected male dogs. After one year, a muscle biopsy was collected from MT1-S.</p>", "<title>Engraftment of hIDPSC in muscles of GRMD dogs after transplantation</title>", "<p>Serial frozen sections of canine muscles were analyzed for engraftment. Scattered human cells were identified in females who received one injection of hIDPSC each. Only some of the sections obtained from these animals showed a few human anti-hIDPSC antibody positive fibers along with the presence of human cells in muscles and connective tissues, and probably in the region of injection from dog FT2-IM, representative by Figure ##FIG##2##3A##. MT1-S presented an apparently higher engraftment of hIDPSC in muscle fibers, as demonstrated by anti-hIDPSC antibody, which were detected in several sections of the biopsies (Figure ##FIG##2##3B–J##). Transversal sections (serial-section analysis) from canine/human chimeric muscle fibers showed variable engraftment within different fibers as well as within one fiber, which indicates the occurrence of fusion between both cell types (Figure ##FIG##2##3B–D,I,J##). FISH analysis with human Y and X DNA probes confirmed these results and showed their presence in chimeric muscle fibers. Interestingly, FISH experiments showed human nuclei in the central part of the fibers; in a pattern resembling those of immature myotubes (Figure ##FIG##2##3K## and inset). Further analysis using FITC-conjugated anti-human nuclei specific antibody also showed the presence of human nuclei in several MT1-S muscle fibers (Figure ##FIG##2##3L–P##).</p>", "<title>Human dystrophin expression in muscles of GRDM dogs</title>", "<p>After 107 and 117 days, immunofluorescence (IF) analysis of muscle biopsies from the four injected dogs detected few fibers labeled with human specific anti-dystrophin antibody (Mandys1062C6), (data not shown). However, scattered large fibers positive for this antibody were observed after one year of consecutive injections in MT1-S (Figure ##FIG##3##4A–D##). The expression of dystrophin in this animal was also confirmed with a C-terminal antibody for dystrophin (Figure ##FIG##3##4E–J##). However, the expected band of 427 kDa was not observed in western blot (WB), which may indicate that only a small amount of the expressed protein was present in the sample (data not shown).</p>", "<title>Histological characterization of GRMD dogs after treatment with hIDPSC</title>", "<p>Muscle degeneration showing necrosis, splitting, centrally located nuclei, and significant endomysial and perimysial connective tissue replacement was observed in all injected and not injected muscles. Interestingly, muscle histopathological analysis of MT1-S done one year after the first injection showed a high number of very large fibers, with several centrally located nuclei associated to a significant proportion of splitting fibers which is suggestive of segmental necrosis (Figure ##FIG##3##4K,L##). FISH and anti-human nuclei specific antibodies results supported that hIDPSC can contribute for the formation of these large centrally nucleated fibers (Figure ##FIG##2##3K,M–O##).</p>", "<title>Clinical and laboratorial assessment</title>", "<p>Several biochemical parameters were assessed in all dogs monthly and analyzed at times 30, 75, 180, 360 days (Table ##TAB##1##2##). Mean serum CK showed a peak between 75 to 180 days with an apparent stabilization afterwards. Other biochemical parameters summarized in Table ##TAB##1##2## suggest that all treated dogs maintained liver and kidney functions and apparently did not show any evident alteration in response to cells transplantation such as white blood cell counting (WBC).</p>", "<p>The progression of the dystrophic process was analyzed based on a mobility score which is summarized in Table ##TAB##0##1##. One affected female control (FC) and two dogs under treatment died during the experiment (FT1-S and MT2-IM). The motility score showed a moderate disease progression in dogs MT1-S and FT2-IM. Although when comparing male and female phenotypes, it should be taken into consideration that females locomotion apparatus differs from males since they have a lighter bone structure, a smaller body and a more delicate locomotion pattern. MT1-S, at one year old, showed a good performance with moderate scores mainly in postural tone, standing up, crossing barriers and hoping. Currently, at age of 18 (Additional file ##SUPPL##0##1##) and 25 months (Additional files ##SUPPL##1##2## and ##SUPPL##2##3##) his motility is stable and he is doing well. In our experience almost 90% of the GRMD dogs die before 24 months of age. However, Chokito, the dog in which we found a greater amount of muscle dystrophin is showing a better course. The movies show this dog at 18 and 26 months old and so far he showed no decline.</p>", "<p>FT2-IM had a comparable performance but she died suddenly of cardiac arrest at age of 16 months (Table ##TAB##0##1##, ##TAB##1##2##, ##TAB##2##3##).</p>" ]
[ "<title>Discussion</title>", "<p>Herein we showed that hIDPSC were capable to migrate and engraft into GRDM dog muscle as confirmed by FISH and immunohystochemical analyses. Due to our ethical committee rules which is against dogs euthanasia or taking multiple biopsies we did not quantify the engraftment of these cells into different muscles, however all analyzed biopsy fragments demonstrated the presence of hIDPSC. We observed that these cells were able to form chimeric canine/human fibers with recipient muscles, although the expression of human dystrophin was observed only in limited number of the fibers following early, multiple cell transplantation. Additionally, these cells persisted in the host muscle for at least 1 year. Moreover, it has been suggested that thawing might modify the properties of SC [##REF##17518650##22##].</p>", "<p>Two independent groups have previously reported the transplantation of different types of canine stem cells in the GRMD model. In the first study, 7 dogs received canine allogenic hematopoietic cell transplantation at ages 4.5 to 5.5 months [##REF##15328150##10##]. However, donor cells did not show any significant contribution to the injured skeletal muscle nor significant increase of dystrophin positive fibers, although near-complete or complete donor hematopoietic chimerism was detected in physically active dogs. The authors concluded that allogenic bone marrow cell transplantation is not a viable therapy in its current form due to the absence of clear clinical benefits [##REF##15328150##10##]. The second group used canine heterologous wild type (WT) mesoangioblasts and allogenic genetically modified mesoangioblasts [##REF##17108972##11##]. The authors reported GRMD muscle function improvement and high level of dystrophin expression in particular after heterologous wild-type (WT) mesoangioblasts transfer, but less with autologous genetically modified SC. Both early and late SC transplantation showed very promising results [##REF##17108972##11##]. However, since immunosuppression was applied in both studies; clinical interpretation of the results is difficult because the use of anti-inflammatory molecules may improve muscle function in affected muscular dystrophy patients [##REF##17306535##12##].</p>", "<p>The number of animals, which received SC transplantation in present work, was just the same used in previous publications [##REF##15328150##10##,##REF##17108972##11##]. However, our study differs in several aspects. First, we used in our experiments an exceptional litter with 3 affected females and 3 affected males. Second we transplanted human, not canine SC without the interference of immunosuppression protocols. Third, clinical monitoring of dogs showed no signs of fever, skin eruption, arthralgia or even glottis edema suggesting, that human cells were well accepted by the dog organism.</p>", "<p>As canine mesoangioblasts and hIDPSC are derived from different tissues and species, it is difficult to compare their potential benefits after transplantation in the GRMD model. However, both cell types proliferate efficiently and present a good migrating capacity in host tissues [##REF##15328150##10##,##REF##17108972##11##]. In our experiment, in contrast to the study reported by Cossu group [##REF##17108972##11##] we did not observe significant increase of dystrophin positive fibers in the 4 GRDM dogs after SC transplantation. Although all dogs showed engraftment of hIDPSC in muscles, only one of them (MT1-S) showed human dystrophin expression. However, this dog, which received the largest amount of human stem cells, is still alive and well at 25 months of age after the experiment was over. It showed a slower rate of progression of the dystrophic process than his five litter-mate dogs or to most of the dogs from the Brazilian GRMD colony, since the majority of them died before 18 months of age (unpublished observation).</p>", "<p>At the end of the experiment, MT1-S had no difficulties in swallowing or chewing hard food, maintained jaw strength as well as the ability to run while carrying objects. The female FT2-IM had also good clinical scores despite the fact we could not detect dystrophin in her muscle biopsy. It died suddenly of cardiac failure at 16 months of age. Due to females having a more delicate structure of the locomotion apparatus and other anatomical variation in the thorax and muscle structures [##UREF##0##23##], gender could indeed have interfered with muscle functions and motility performance. Furthermore, since female GRMD dogs are very rare, there are no parameters to evaluate the natural history of their disease, not to mention the absence of studies that use such dogs. FC, FT1-S, FT2-IM and MT2-IM died from dystrophy complications at the ages 10, 8, 16 and 11 months, respectively.</p>", "<p>In DMD patients, serum CK is elevated since birth and shows a peak of activity around 2 to 4 years when there is massive muscle degeneration [##REF##2072118##24##]. In accordance with Sampaolesi studies [##REF##17306535##12##] we observed that mean serum CK activities in GRMD dogs were higher between 75 to 180 days followed by an apparent stabilization (Table ##TAB##1##2##).</p>", "<p>Significant immune modulatory function of MSC has been demonstrated in hematopoietic stem cell transplantation. They strongly inhibit alloantigen-induced dendritic cell differentiation, down-regulate alloantigen-induced lymphocyte expansion, decrease alloantigen-specific cytotoxic capacity mediated by either cytotoxic T lymphocytes or NK cells. Interestingly, a more effective suppressive activity on mixed lymphocyte culture-induced T-cell activation was observed when MSC were heterologous rather than autologous. It was suggested that due to these properties, MSC can be used to prevent immune complications related to both hematopoietic stem cells and solid organ transplantation and it was proposed that MSC are \"universal\" suppressors of immune reactivity. Moreover, it was shown that regulatory CD4+ or CD8+ lymphocytes were generated in co-cultures of peripheral blood mononuclear cells with MSC. This strongly indicates that these regulatory cells may amplify the reported MSC-mediated immunosuppressive effect [##REF##12589164##25##, ####REF##15820948##26##, ##REF##17606437##27####17606437##27##]. In our experiments the dogs did not show any immune reaction to hIDPSC transplantation. This could be explained by the fact that according to previous publication immunosuppressive activity of human DPSC was significantly higher, when compared to those from bone marrow [##REF##16210973##15##]. More recently, similar results were observed with different populations of stem cells isolated from dental pulp [##REF##18216690##14##,##REF##16210973##15##,##REF##18079433##17##].</p>", "<p>The absence of dogs immune response to hIDPSC transplantation here reported for the first time is an important observation, because anti-inflammatory drugs could occult the benefits of stem cells therapy for dystrophy [##REF##17137828##28##]. Indeed, white blood cells counting did not present any important changes in response to cell transplantation and no lymphocytes infiltration was observed in muscle cells. We also did not observe immune reactions using the same cells in experiment on reconstruction of cranial defects in rats [##REF##18216690##14##]. This result is also supported by other authors, which demonstrated that human stem cell isolated from dental pulp have immunosuppressive activity [##REF##16210973##15##]. In conclusion, we believe that rejection of hIDPSC did not occur since they present all basic characteristic of mesenchymal stem cells [##REF##16923606##29##].</p>", "<p>It is very tempting to suggest that the presence of some dystrophin in muscle as a result of repeated systemic hIDPSC transplantation ameliorated the clinical condition of MT1-S since this disease in dogs causes their death usually between 11 and 18 months [##REF##1536178##9##,##REF##11944998##30##]. Interestingly, after transplantation of human DPSC in non immunosuppressed rats with acute myocardial infarction, surviving and engraftment of the cells in ischemic environments have been observed. Although no differentiation into cardiac or smooth muscle as well as into endothelial cells was observed, these cells apparently contributed for the improvement of left ventricular function, induced angiogenesis and reduced infarct size. According to the authors, the benefits observed after DPSC transplantation might be due to secretion of paracrine factors by these cells [##REF##18079433##17##]. Since the expression of dystrophin was modest, clinical improvement in this dog might be the result of the immune modulatory effect of hIDPSC transplantation rather than the presence of dystrophin, or rather than the great clinical variability observed among GRMD dogs [##UREF##1##31##]. However, our preliminary results, which should be validated in a larger sample, suggested that multiple systemic transplantations of hIDPSC in GRMD dogs could slow down the progression of the dystrophic process, through similar mechanisms described by Gandia and co-workers [##REF##18079433##17##].</p>", "<p>In short, our investigation confirmed that early systemic delivery of SC in GRMD model is more effective than local injections. Although we showed that donor human SC can engraft, differentiate, and persist in the host, it seems that the apparent clinical benefit observed in treated animals is not due to dystrophin expression in the host muscles, but due to their immune modulatory effect of SC from dental pulp [##REF##18216690##14##,##REF##16210973##15##,##REF##18079433##17##,##REF##17685479##32##]. However, both observations that cell engraftment increases with consecutive transplantation and the absence of immunological response, have very important implications in designing future therapeutic trials. The potential clinical effect of SC in GRMD model should be supported by further investigation involving a larger number of animals and the efficacy of later as compared to early transplantation.</p>" ]
[]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression.</p>", "<title>Methods</title>", "<p>Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes.</p>", "<title>Results and Discussion</title>", "<p>We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent <italic>in situ </italic>hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age.</p>", "<title>Conclusion</title>", "<p>Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>All authors have read and approved the final manuscript. The specific contributions of each author are: DSM, TPG, ACM, MPB assisted clinical support to the dogs, conduce the clinical trial with laboratory analyses and physical scores establishment. EZ, NV conducted genotyping process, material collections and also immunohistochemistry and blotting dystrophy analyses. SASF, CMCM, OASA conduce the stem cells preparation and FISH analyses; AK conducted immunohistochemistry and confocal microscopy; RMC co-conducted the clinical trial and cell applications and assisted with dogs analyses; IK, CEA, MAM and MZ conceived, designed and directed the entire study, interpreted all data and wrote the manuscript.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>This research was supported by FAPESP, CEPID Process n°: 1998/14254-2 and CNPq Process n°: 55.2210/2005–6. FAPESP support other two projects: 2004/15370-9 and 2005/50082-7. We would like to thank Dr. Glenn Morris from Center for Inherited Neuromuscular Disease (CIND), RJAH Orthopaedic Hospital, Oswestry, Shropshire, UK for providing anti-human dystrophin antibodies and Dr. Mariz Vainzof for helpful suggestions. Also, to Prof. Joe N. Kornegay and Dr. Janet Bogan from Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, and the Dalton Cardiovascular Research Center, University of Missouri, Columbia, USA who were responsible for the donation of the first GRMD carrier female and the establishment of the Brazilian colony of GRMD dogs. We are very grateful also to Marta Canovas, for technical support for dystrophin analysis, Alex S.Souza for an excellent technical assistance with Confocal Microscopy, to T. Kawano chief of the Laboratory of Parasitology of Butantan Institute, São Paulo, Brasil and to Constancia Gotto for secretarial assistance and invaluable support. Finally to Dr. Marcia Triunfol for final English review.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Characterization, myogenic differentiation <italic>in vitro </italic>and migrating capacity in vivo of hIDPSC.</bold> (A) Fibroblast-like morphology of hIDPSC. (B) Proliferating potential of these cells during 16 successive passages. (C-E) Flow cytometry showing expression of CD105 (SH2), CD73 (SH3 and SH4), respectively. (F) Negative control for respective isotype. (G,H) Myogenic differentiation <italic>in vitro</italic>: fused hIDPSC forming myotubes. Positive immunostaining with α-actinin (G) and myosin (H): insets in (G and H) show details of anti-bodies localization within myotubes, higher magnification. (I) RT-PCR analysis of the expression of human dystrophin (MyoD1) observed hIDPSC and human muscles, used as a positive control. (J-M). Migrating capacity of hIDPSC visualized 30 days after their intraperitoneal injection into normal mice (J-M): (J) Cells stained with DiI-Vybrant (red) in mouse, (K) positive reaction with primary human anti-IDPSC antibody in mice (secondary antibody FITC-conjugated was used (green), (L) Morphology of mouse cardiac muscles. (M) merged image of J-L. A= light microscopy, phase contrast, G-H = epi-fluorescence (EF), J-M= confocal microscopy: J, K = fluorescent microscopy (Fcm), L = DIC (Differential interference contrast) M = Fcm+DIC. Scale bars: G,H = 10 μm, A,J-M = 100 μm, N-P = 50 μm</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>DMD genotyping.</bold> GRMD puppies from a colony of dogs with X-linked muscular dystrophy were genotyped within 48 hs after birth. The genomic PCR product digested with Sau96I produces the wild type band (310 pb) and the mutant band (150 pb) labelled with arrows. ■ = Affected male. ● = Affected female. ◉ = Carrier female.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Representative figures of hIDPSC engraftment observed within canine skeletal muscles (GRMD).</bold> (A) FT2-IM, after 107 days of single transplantation: Positive immunostaining with anti-hIDPSC antibody (green) was observed in several muscle fibers (white arrowhead) and in the nuclei (white arrows, blue, DAPI stained superposition with anti-IDPSC antibody, green) of hIDPSC localized in the periphery of canine muscle fibers. (B-I) One year after multiple hIDPSC transplantation. Positive immunostaining with anti-IDPSC antibody was observed in MT1-S muscles: (B-D) transversal and (E-H) longitudinal sections. Inset in (F) demonstrate (higher magnification) skeletal muscle Z-bands (red arrowhead) observed in the local of positive immunostaining with anti-hIDPSC antibody (DIC). (H) Higher magnification of (G) demonstrating positive reaction of hIDPSC antibody (green) with skeletal muscle Z-bands (red arrowhead). (I) Chimeric human/canine muscle fiber only a half of which presents positive green fluorescent immunostaining (green). (J) Control: affected male without hIDPSC transplantation immunostained with anti-hIDPSC antibody did not present any labeling. (K) FISH analysis of dystrophic male's muscles using specific human probe for chromosome: Y (red) and in inset X (yellow, as a result of merged images of PI (red) stained nucleus and probe of chromosome X (green) are presented). (L-P) Immunostaining using FITC conjugated anti-human nucleus (anti-HN) antibody (green). (L) Positive control. Merged image of positive staining with anti-HN antibody and nuclei stained with DAPI in normal human muscles. (M-O) Positive immunostaining with anti-HN antibody observed in the nuclei of hIDPSC (green) engrafted into canine muscle fibers of MT1-S. They (white arrowhead) can be seen within canine muscle fibers and in perimysium (white arrows). Canine nuclei (group of 4 nuclei indicated by red arrow) did not present any reaction with anti-HN antibody. (P) Negative control. Muscles of normal dog did not react with anti-HN antibody. Only nuclei stained with DAPI can be observed. Confocal microscopy, A,D,G-L,P = Fcm+DIC; C,F,M,N = Fcm, B,E, Inset in (F) = DIC, Scale bars: A-H, L,P = 50 μm, K = 100 μm, and M-O = 20 μm, Inset in (K) = 10 μm</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Immunofluorescence analysis using the specific human anti-dystrophin monoclonal antibodies: Mandys106 2C6 (A-D) and C-terminal (E-J) antibodies one year after the hIDPSC transplantation.</bold> (A) Positive control: expression of Mandys106 2C6 antibody in normal human muscles. (B) Negative control: lack of expression of same antibody in the muscles of normal dog. (C, D) MT1-S shows positive staining with Mandys106 2C6 in large dystrophic fibers (white arrows). (E) Positive control: expression of C-terminal in normal human muscles. (F) C-terminal antibody in the muscles of normal dog presents weak labeling. (G) Negative control: lack of its expression in the muscles of affected dog. (H-J) MT1-S shows positive staining with C-terminal antibody in some large dystrophic fibers (arrow). K) Toluidine blue and L) HE staining shows very large fibers with multiple centrally located nuclei and splitting. A, C, E-H = Fcm + DIC, B = DIC, D,I,= EF, J,K = Light microscopy, Scale bars = 50 μm</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>GRMD dogs, born April, 2006 used for IDPSC transplantation and respective controls. </p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Dog n°</td><td align=\"center\">Dog name</td><td align=\"center\">Cell Treatment</td><td align=\"center\">Onset treatment</td><td align=\"center\">Biopsy after 1<sup>a </sup>injection</td><td align=\"center\">Progression/motility</td><td align=\"center\">Physical score</td><td/><td align=\"center\">Outcome of experiment at time ...</td></tr></thead><tbody><tr><td/><td/><td/><td/><td/><td/><td align=\"center\">180d</td><td align=\"center\">365d</td><td/></tr><tr><td align=\"center\">♀</td><td align=\"center\">Laka 2L5</td><td align=\"center\">Control</td><td align=\"center\">-</td><td align=\"center\">0 d</td><td align=\"center\">Severe/major decline</td><td align=\"center\">4</td><td align=\"center\">†</td><td align=\"center\">Died (300d)</td></tr><tr><td align=\"center\">FC</td><td/><td/><td/><td align=\"center\">107 d</td><td/><td/><td/><td align=\"center\">Ascite</td></tr><tr><td align=\"center\">♀</td><td align=\"center\">Lancy 2L6</td><td align=\"center\">hlDPSC – S</td><td align=\"center\">Just with</td><td align=\"center\">107 d</td><td align=\"center\">Severe/major decline</td><td align=\"center\">3</td><td align=\"center\">†</td><td align=\"center\">Died (240d)</td></tr><tr><td align=\"center\">FT1-S</td><td/><td/><td align=\"center\">28 d</td><td/><td/><td/><td/><td align=\"center\">Ascite</td></tr><tr><td align=\"center\">♀</td><td align=\"center\">Amandita 2L4</td><td align=\"center\">hlDPSC – IM</td><td align=\"center\">Just with</td><td align=\"center\">107 d</td><td align=\"center\">Mild/modest decline</td><td align=\"center\">4</td><td align=\"center\">†</td><td align=\"center\">Died (480d)</td></tr><tr><td align=\"center\">FT2-IM</td><td/><td/><td align=\"center\">28 d</td><td/><td/><td/><td/><td align=\"center\">Cardiac failure</td></tr><tr><td align=\"center\">♂</td><td align=\"center\">Bis 2L2</td><td align=\"center\">Control</td><td align=\"center\">-</td><td align=\"center\">117 d</td><td align=\"center\">Severe/major decline</td><td align=\"center\">2</td><td align=\"center\">17</td><td align=\"center\">Alive</td></tr><tr><td align=\"center\">MC</td><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"center\">♂</td><td align=\"center\">Chokito 2L3</td><td align=\"center\">hlDPSC – S</td><td align=\"center\">Start with 44 d</td><td align=\"center\">47 d</td><td align=\"center\">Mild/modest decline</td><td align=\"center\">5</td><td align=\"center\">11</td><td align=\"center\">Alive and well</td></tr><tr><td align=\"center\">MT1-S</td><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td/><td/><td/><td align=\"center\">Followed by 8 injections/month</td><td align=\"center\">117 d</td><td/><td/><td/><td/></tr><tr><td/><td/><td/><td/><td align=\"center\">365 d</td><td/><td/><td/><td/></tr><tr><td align=\"center\">♂</td><td align=\"center\">Toddy 2L7</td><td align=\"center\">hlDPSC – IM</td><td align=\"center\">Start with 44 d</td><td align=\"center\">47 d</td><td align=\"center\">Severe/major decline</td><td align=\"center\">6</td><td align=\"center\">† (24)</td><td align=\"center\">Died (370d)</td></tr><tr><td align=\"center\">MT2-IM</td><td/><td/><td align=\"center\">Followed by 5 injections/month</td><td align=\"center\">117 d</td><td/><td/><td/><td align=\"center\">Gastric Malabsorption deficiency</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Serum biochemical analyses in four GRMD dogs under hIDPSC therapy (MT1, MT2, FT1 and FT2) and matched age control (MC and FC).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>ANIMALS 30 days</bold></td><td align=\"center\"><bold>CK (U/L)</bold></td><td align=\"center\"><bold>ALT (U/L)</bold></td><td align=\"center\"><bold>FA (U/L)</bold></td><td align=\"center\"><bold>Urea (mg/dL)</bold></td><td align=\"center\"><bold>Creat (mg/dL)</bold></td><td align=\"center\"><bold>RBC (mlh/mm<sup>3</sup>)</bold></td><td align=\"center\"><bold>Ht (%)</bold></td><td align=\"center\"><bold>Hb (g/dL)</bold></td><td align=\"center\"><bold>WBC (mil/mm<sup>3</sup>)</bold></td></tr></thead><tbody><tr><td align=\"center\"><italic>Reference values*</italic></td><td align=\"center\"><italic>0–200</italic></td><td align=\"center\"><italic>until 50</italic></td><td align=\"center\"><italic>until 130</italic></td><td align=\"center\"><italic>until 40</italic></td><td align=\"center\"><italic>until 1.5 – 2</italic></td><td align=\"center\"><italic>3.5 – 6</italic></td><td align=\"center\"><italic>26 – 39</italic></td><td align=\"center\"><italic>8.5 – 13</italic></td><td align=\"center\"><italic>8,5 – 17,3</italic></td></tr><tr><td align=\"center\">FC</td><td align=\"center\">9,275</td><td align=\"center\">162</td><td align=\"center\">500</td><td align=\"center\">31</td><td align=\"center\">1</td><td align=\"center\">3.3</td><td align=\"center\">27</td><td align=\"center\">7.1</td><td align=\"center\">19,300</td></tr><tr><td align=\"center\">FT1-S</td><td align=\"center\">9,274</td><td align=\"center\">159</td><td align=\"center\">478</td><td align=\"center\">28.6</td><td align=\"center\">1</td><td align=\"center\">3.6</td><td align=\"center\">25</td><td align=\"center\">7.6</td><td align=\"center\">26,300</td></tr><tr><td align=\"center\">FT2-IM</td><td align=\"center\">4,647</td><td align=\"center\">134</td><td align=\"center\">352</td><td align=\"center\">22</td><td align=\"center\">0.98</td><td align=\"center\">4</td><td align=\"center\">26</td><td align=\"center\">8.8</td><td align=\"center\">32,000</td></tr><tr><td align=\"center\">MC</td><td align=\"center\">3,618</td><td align=\"center\">110</td><td align=\"center\">260</td><td align=\"center\">32.8</td><td align=\"center\">1</td><td align=\"center\">4.2</td><td align=\"center\">30</td><td align=\"center\">8.8</td><td align=\"center\">32,300</td></tr><tr><td align=\"center\">MT1-S</td><td align=\"center\">7,716</td><td align=\"center\">230</td><td align=\"center\">174</td><td align=\"center\">30</td><td align=\"center\">1</td><td align=\"center\">4.6</td><td align=\"center\">34</td><td align=\"center\">11</td><td align=\"center\">24,800</td></tr><tr><td align=\"center\">MT2- IM</td><td align=\"center\">5,668</td><td align=\"center\">163</td><td align=\"center\">217</td><td align=\"center\">32.9</td><td align=\"center\">1.1</td><td align=\"center\">4.7</td><td align=\"center\">33</td><td align=\"center\">11.5</td><td align=\"center\">23,600</td></tr><tr><td align=\"center\"><bold>ANIMALS 75 days</bold></td><td align=\"center\"><bold>CK (U/L)</bold></td><td align=\"center\"><bold>ALT (U/L)</bold></td><td align=\"center\"><bold>FA (U/L)</bold></td><td align=\"center\"><bold>Urea (mg/dL)</bold></td><td align=\"center\"><bold>Creat (mg/dL)</bold></td><td align=\"center\"><bold>RBC (mlh/mm<sup>3</sup>)</bold></td><td align=\"center\"><bold>Ht (%)</bold></td><td align=\"center\"><bold>Hb (g/dL)</bold></td><td align=\"center\"><bold>WBC (mil/mm<sup>3</sup>)</bold></td></tr><tr><td align=\"center\">FC</td><td align=\"center\">32,599</td><td align=\"center\">294</td><td align=\"center\">300</td><td align=\"center\">46</td><td align=\"center\">0.85</td><td align=\"center\">4.8</td><td align=\"center\">29</td><td align=\"center\">9.2</td><td align=\"center\">28,200</td></tr><tr><td align=\"center\">FT1-S</td><td align=\"center\">15,100</td><td align=\"center\">245</td><td align=\"center\">161</td><td align=\"center\">43.5</td><td align=\"center\">0.92</td><td align=\"center\">4.3</td><td align=\"center\">26</td><td align=\"center\">8.7</td><td align=\"center\">34,800</td></tr><tr><td align=\"center\">FT2-IM</td><td align=\"center\">24,784</td><td align=\"center\">192</td><td align=\"center\">117</td><td align=\"center\">37.8</td><td align=\"center\">0.9</td><td align=\"center\">4.6</td><td align=\"center\">28</td><td align=\"center\">9.2</td><td align=\"center\">30,200</td></tr><tr><td align=\"center\">MC</td><td align=\"center\">15,257</td><td align=\"center\">189</td><td align=\"center\">138</td><td align=\"center\">37.8</td><td align=\"center\">0.9</td><td align=\"center\">4.1</td><td align=\"center\">27</td><td align=\"center\">8.9</td><td align=\"center\">36,600</td></tr><tr><td align=\"center\">MT1-S</td><td align=\"center\">30,729</td><td align=\"center\">293</td><td align=\"center\">181</td><td align=\"center\">42.8</td><td align=\"center\">0.94</td><td align=\"center\">4.6</td><td align=\"center\">29</td><td align=\"center\">9.9</td><td align=\"center\">25,900</td></tr><tr><td align=\"center\">MT2- IM</td><td align=\"center\">15,291</td><td align=\"center\">237</td><td align=\"center\">191</td><td align=\"center\">46.9</td><td align=\"center\">0.92</td><td align=\"center\">4.6</td><td align=\"center\">28</td><td align=\"center\">9.7</td><td align=\"center\">39,100</td></tr><tr><td align=\"center\"><bold>ANIMALS 180 days</bold></td><td align=\"center\"><bold>CK (U/L)</bold></td><td align=\"center\"><bold>ALT (U/L)</bold></td><td align=\"center\"><bold>FA (U/L)</bold></td><td align=\"center\"><bold>Urea (mg/dL)</bold></td><td align=\"center\"><bold>Creat (mg/dL)</bold></td><td align=\"center\"><bold>RBC (mlh/mm<sup>3</sup>)</bold></td><td align=\"center\"><bold>Ht (%)</bold></td><td align=\"center\"><bold>Hb (g/dL)</bold></td><td align=\"center\"><bold>WBC (mil/mm<sup>3</sup>)</bold></td></tr><tr><td align=\"center\"><italic>Reference values*</italic></td><td align=\"center\"><italic>0–200</italic></td><td align=\"center\"><italic>until 50</italic></td><td align=\"center\"><italic>until 130</italic></td><td align=\"center\"><italic>until 40</italic></td><td align=\"center\"><italic>until 1.5 – 2</italic></td><td align=\"center\"><italic>5.5 – 7</italic></td><td align=\"center\"><italic>34 – 40</italic></td><td align=\"center\"><italic>11 – 15.5</italic></td><td align=\"center\"><italic>8 – 16,000</italic></td></tr><tr><td align=\"center\">FC</td><td align=\"center\">56,736</td><td align=\"center\">416</td><td align=\"center\">58</td><td align=\"center\">40</td><td align=\"center\">0.4</td><td align=\"center\">5.5</td><td align=\"center\">39</td><td align=\"center\">12.4</td><td align=\"center\">16,200</td></tr><tr><td align=\"center\">FT1-S</td><td align=\"center\">48,960</td><td align=\"center\">396</td><td align=\"center\">28</td><td align=\"center\">23</td><td align=\"center\">0.5</td><td align=\"center\">5.4</td><td align=\"center\">38</td><td align=\"center\">12.1</td><td align=\"center\">15,500</td></tr><tr><td align=\"center\">FT2-IM</td><td align=\"center\">51,296</td><td align=\"center\">228</td><td align=\"center\">8</td><td align=\"center\">38</td><td align=\"center\">0.4</td><td align=\"center\">6.0</td><td align=\"center\">39</td><td align=\"center\">13.1</td><td align=\"center\">14,100</td></tr><tr><td align=\"center\">MC</td><td align=\"center\">56,736</td><td align=\"center\">524</td><td align=\"center\">91</td><td align=\"center\">51</td><td align=\"center\">0.5</td><td align=\"center\">5.6</td><td align=\"center\">37</td><td align=\"center\">12</td><td align=\"center\">20,400</td></tr><tr><td align=\"center\">MT1-S</td><td align=\"center\">25,632</td><td align=\"center\">440</td><td align=\"center\">66</td><td align=\"center\">55</td><td align=\"center\">0.4</td><td align=\"center\">5.5</td><td align=\"center\">36</td><td align=\"center\">12.5</td><td align=\"center\">18,600</td></tr><tr><td align=\"center\">MT2- IM</td><td align=\"center\">36,512</td><td align=\"center\">500</td><td align=\"center\">62</td><td align=\"center\">40</td><td align=\"center\">0.4</td><td align=\"center\">5.5</td><td align=\"center\">38</td><td align=\"center\">12</td><td align=\"center\">15,600</td></tr><tr><td align=\"center\"><bold>ANIMALS 360 days</bold></td><td align=\"center\"><bold>CK (U/L)</bold></td><td align=\"center\"><bold>ALT (U/L)</bold></td><td align=\"center\"><bold>FA (U/L)</bold></td><td align=\"center\"><bold>Urea (mg/dL)</bold></td><td align=\"center\"><bold>Creat (mg/dL)</bold></td><td align=\"center\"><bold>RBC (mlh/mm<sup>3</sup>)</bold></td><td align=\"center\"><bold>Ht (%)</bold></td><td align=\"center\"><bold>Hb (g/dL)</bold></td><td align=\"center\"><bold>WBC (mil/mm<sup>3</sup>)</bold></td></tr><tr><td align=\"center\">reference values*</td><td align=\"center\">0–200</td><td align=\"center\">until 50</td><td align=\"center\">until 130</td><td align=\"center\">until 40</td><td align=\"center\">until 1.5 – 2</td><td align=\"center\">5 – 8</td><td align=\"center\">37 – 54</td><td align=\"center\">12 – 18</td><td align=\"center\">6 – 15,000</td></tr><tr><td align=\"center\">FC**</td><td/><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"center\">FT1-S **</td><td/><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"center\">FT2-IM</td><td align=\"center\">35,712</td><td align=\"center\">764</td><td align=\"center\">8</td><td align=\"center\">35</td><td align=\"center\">0.4</td><td align=\"center\">6.1</td><td align=\"center\">41</td><td align=\"center\">13.1</td><td align=\"center\">14,000</td></tr><tr><td align=\"center\">MC</td><td align=\"center\">41,920</td><td align=\"center\">376</td><td align=\"center\">49</td><td align=\"center\">48</td><td align=\"center\">0.6</td><td align=\"center\">5.6</td><td align=\"center\">36</td><td align=\"center\">13</td><td align=\"center\">20,000</td></tr><tr><td align=\"center\">MT1-S</td><td align=\"center\">40,384</td><td align=\"center\">604</td><td align=\"center\">41</td><td align=\"center\">34</td><td align=\"center\">0.5</td><td align=\"center\">5.2</td><td align=\"center\">33</td><td align=\"center\">12</td><td align=\"center\">18,000</td></tr><tr><td align=\"center\">MT2- IM***</td><td align=\"center\">9,280</td><td align=\"center\">208</td><td align=\"center\">33</td><td align=\"center\">50</td><td align=\"center\">0.7</td><td align=\"center\">5.1</td><td align=\"center\">34</td><td align=\"center\">11.7</td><td align=\"center\">15,000</td></tr><tr><td align=\"center\"><bold>GRMD Control Data (n:6)</bold></td><td align=\"center\">35,710</td><td align=\"center\">551</td><td align=\"center\">43.25</td><td align=\"center\">35.75</td><td align=\"center\">0.52</td><td align=\"center\">5.75</td><td align=\"center\">37.75</td><td align=\"center\">12.87</td><td align=\"center\">14,6</td></tr><tr><td align=\"center\"><bold>Normal Dogs Data (n: 6)</bold></td><td align=\"center\">145</td><td align=\"center\">15</td><td align=\"center\">82</td><td align=\"center\">28</td><td align=\"center\">1.8</td><td align=\"center\">4.2</td><td align=\"center\">30.7</td><td align=\"center\">14.2</td><td align=\"center\">11,7</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Physical exams score for motility and postural features from dystrophic dogs</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\" colspan=\"4\"><bold>Scores</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>CRITERIA</bold></td><td align=\"left\"><bold>0</bold></td><td align=\"left\"><bold>1</bold></td><td align=\"left\"><bold>2</bold></td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>1. Posture symmetry</bold></td><td align=\"left\">Symmetric animal</td><td align=\"left\">Slight asymmetry: discreet lordosis</td><td align=\"left\">High asymmetry: lordosis posture and tail elevation</td></tr><tr><td align=\"left\"><bold>2. Increase of tarsal normal angle (plantigrady)</bold></td><td/><td/><td/></tr><tr><td/><td align=\"left\">Absent</td><td align=\"left\">modest</td><td align=\"left\">severe (walk on tarsus)</td></tr><tr><td align=\"left\"><bold>3. Increase of carpal normal angle (palmigrady)</bold></td><td/><td/><td/></tr><tr><td/><td align=\"left\">Absent</td><td align=\"left\">modest</td><td align=\"left\">severe (walk on carpus)</td></tr><tr><td align=\"left\"><bold>4. Stifle stiffness while moving</bold></td><td align=\"left\">normal stifle motility</td><td align=\"left\">slight stifle stiffness</td><td align=\"left\">severe stifle stiffness associated with short strides</td></tr><tr><td align=\"left\"><bold>5. Pelvic balance</bold></td><td/><td/><td/></tr><tr><td/><td align=\"left\">Normal</td><td align=\"left\">reduced</td><td align=\"left\">absent</td></tr><tr><td align=\"left\"><bold>6. Postural tone (muscle palpation and inspection)</bold></td><td/><td/><td/></tr><tr><td/><td align=\"left\">normal tone</td><td align=\"left\">hypotonic</td><td align=\"left\">flaccid</td></tr><tr><td align=\"left\"><bold>7. Contractures</bold></td><td align=\"left\">Absent</td><td align=\"left\">decrease in passive range of motion</td><td align=\"left\">contractures limiting passive range of motion</td></tr><tr><td align=\"left\"><bold>8. Hopping</bold></td><td/><td/><td/></tr><tr><td/><td align=\"left\">Normal</td><td align=\"left\">reduced</td><td align=\"left\">inability to achieve postural reaction</td></tr><tr><td align=\"left\"><bold>9. Standing up</bold></td><td align=\"left\">Normal</td><td align=\"left\">ability reduced (perform task slowly/slower)</td><td/></tr><tr><td/><td align=\"left\">inability to stand up alone</td><td/><td/></tr><tr><td align=\"left\"><bold>10. Barrier crossing</bold></td><td align=\"left\">normal</td><td align=\"left\">reduced</td><td align=\"left\">inability to jump</td></tr><tr><td align=\"left\"><bold>11. Food prehension</bold></td><td align=\"left\">Eat normal dry food</td><td align=\"left\">changed for wet food</td><td align=\"left\">laborious swallowing</td></tr><tr><td align=\"left\"><bold>12. Locomotion apparatus structure</bold></td><td align=\"left\">strong</td><td align=\"left\">losing structures components as muscle mass and bone fragilities</td><td align=\"left\">weak and thin</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\">Total score = 24</td><td/><td/><td/></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>MT1-S, at 18 months, showed a good performance with moderate scores. His capacity of running and strength with objects are well after cell therapy.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional file 2</title><p>MT1-S, at 25 months, continues with a good performance. After 16 months of experimental end, Chokito continues with his capacity of running and muscle strength.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional file 3</title><p>MT1-S with 25 months. Chokito presenting his capacity of jumping obstacles.</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><p>All animals were descendant of the GRMD couple Peter (affected) × Lady (carrier)</p></table-wrap-foot>", "<table-wrap-foot><p> Also, control dogs were used from all colony animals (Normal X Affected)</p><p>*Reference values from Veterinary Hospital (HOVET) – FMVZ-USP and FMVZ-UNESP, São Paulo, Brazil.</p><p>** Animal died with 10 months (FC) and 8 months (FT1-S) *** Animal died with 13 months</p></table-wrap-foot>" ]
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[ "<media xlink:href=\"1479-5876-6-35-S1.mpg\" mimetype=\"video\" mime-subtype=\"mpeg\"><caption><p>Click here for file</p></caption></media>", "<media xlink:href=\"1479-5876-6-35-S2.mpg\" mimetype=\"video\" mime-subtype=\"mpeg\"><caption><p>Click here for file</p></caption></media>", "<media xlink:href=\"1479-5876-6-35-S3.mpg\" mimetype=\"video\" mime-subtype=\"mpeg\"><caption><p>Click here for file</p></caption></media>" ]
[{"surname": ["DiDio"], "given-names": ["LJA"], "article-title": ["Tratado de anatomia aplicada"], "source": ["P\u00f3llus Editorial, S\u00e3o Paulo"], "year": ["1998"], "fpage": ["103"], "lpage": ["109"]}, {"surname": ["Ambrosio", "Zucconi", "Martins", "Vanucchi", "Perez", "Vieira", "Valadares", "Jazedje", "Miglino", "Zatz"], "given-names": ["C", "E", "D", "C", "M", "N", "M", "T", "M", "M"], "article-title": ["Extreme clinical variability in GRMD: From neonatal death to asymptomatic carriers. [abstract]"], "source": ["Neuromuscular Disorders"], "year": ["2007"], "volume": ["17"], "fpage": ["776"], "comment": ["doi:10.1016/j.nmd.2007.06.056"], "pub-id": ["10.1016/j.nmd.2007.06.056"]}]
{ "acronym": [], "definition": [] }
32
CC BY
no
2022-01-12 14:47:25
J Transl Med. 2008 Jul 3; 6:35
oa_package/44/60/PMC2529267.tar.gz
PMC2529268
18717994
[ "<title>Background</title>", "<p>Pancreatic cancer is one of the most lethal malignant tumors, and it was estimated that approximately 200,000 people died of this cancer worldwide in the year 2000 [##REF##11668491##1##]. It is the fifth leading cause of cancer death in Japan and the fourth in the United States [##REF##15231857##2##]. Unfortunately, the pancreas is located at an inaccessible site within the abdomen, making the diagnosis of pancreatic cancer more difficult than that of other digestive tract cancers. Therefore, most patients with pancreatic cancer are diagnosed late after progression of their disease. Furthermore, pancreatic cancer frequently infiltrates neighboring tissues or vessels at an early stage, leading to a poor prognosis.</p>", "<p>In the United States, the 1-year and 5-year survival rates of patients with pancreatic cancer are less than 25% and 5%, respectively [##REF##11750846##3##]. At present, surgical resection provides the only chance of cure for these patients, but it has been reported that about 90% of patients do not undergo pancreatic resection and 58% are only given palliative treatment [##REF##10401733##4##]. Moreover, recurrence after surgical resection is very common. We do not have any effective nonsurgical treatments for pancreatic cancer, because it shows strong resistance to the currently available chemotherapy and radiotherapy protocols [##REF##12643012##5##]. In order to improve the clinical outcome, new modalities for the treatment of this disease are required.</p>", "<p>Gene therapy using oncolytic viruses is one of the approaches that should be considered. The strategy of this therapy is to exploits the lytic property of virus replication to kill tumor cells. Recent knowledge of molecular biology makes it possible to modify viruses to target specific molecules or signal transduction pathways in cancer cells. Oncolytic viruses which are developed to be able to infect and replicate selectively in malignant tumor cells can spread and destroy malignant tumors without adverse effects in normal tissues.</p>", "<p>To achieve tumor-selective viral replication, one approach has been the replacement of endogenous viral sequences with a tissue- or tumor-specific promoter. A number of tumor promoter genes such as α-fetoprotein [##REF##8864751##6##], carcinoembryonic antigen [##REF##7578407##7##], erbB-2 [##REF##8986436##8##]and prostate-specific antigen [##REF##8573614##9##,##REF##9205053##10##] have been used to restrict the expression of suicide genes both <italic>in vitro </italic>and <italic>in vivo</italic>.</p>", "<p>Midkine is a heparin-binding growth factor that is induced by retinoic acid in embryonal carcinoma cells, and it may be another candidate for this purpose [##REF##9889592##11##]. The biological roles of midkine are diverse and it is closely linked to neural development [##REF##2270483##12##,##REF##2583087##13##] as well as to the pathogenesis of neurodegenerative diseases. At the same time, midkine is involved in the development of cancer because of its mitogenic effect [##REF##11244508##14##], promotion of angiogenesis [##REF##9135027##15##], anti-apoptotic activity [##REF##10731694##16##], fibrinolytic activity [##REF##7488150##17##], and transforming activity [##REF##9020479##18##].</p>", "<p>Midkine expression is increased in a number of malignant tumors, including esophageal, stomach, colon, hepatocellular, breast and pancreatic carcinoma, when compared with the level of expression in adjacent non-cancerous tissues [##REF##8383007##19##, ####REF##7559083##20##, ##REF##7520350##21##, ##REF##10626184##22####10626184##22##]. In contrast, the expression of midkine in normal human tissues is quite limited, with moderate expression in the kidneys and weak expression in the lungs, colon, and thyroid gland [##REF##8383007##19##,##REF##7559083##20##,##REF##8507561##23##].</p>", "<p>On this basis, the midkine promoter could be a potential candidate for use in suicide gene therapy. Here, we demonstrate that an adenovirus vector encoding the essential adenoviral E1A gene under the control of 0.6 kb midkine promoter showed specific replication in midkine-expressing pancreatic cancer cell lines and not in non-midkine-expressing cells, and that Ad5MK selectively prevented tumor growth both <italic>in vitro </italic>and <italic>in vivo</italic>.</p>" ]
[ "<title>Methods</title>", "<title>Cell culture and tumor samples</title>", "<p>Seven human pancreatic cancer cell lines were used. AsPC-1, BxPC-3, CFPAC-1, HPAC, MIAPaCa-2, and PANC-1 cells were obtained from the American Type Culture Collection (Rockville, MD), and were maintained in the medium recommended by the ATCC at 37°C in a humidified atmosphere of 5% CO<sub>2</sub>. Suit-2 cells were kindly provided by Dr. Tomoda (National Kyushu Cancer Center, Fukuoka, Japan), and were cultured in DMEM (Gibco-BRL, Grand Island, NY) with 10% fetal bovine serum (FBS) (ICN Biomedicals, Aurora, Ohio). Human embryonic kidney (HEK) 293 cells were purchased from RIKEN Bioresourse Center (Tukuba, Ibaragi, Japan). Each culture medium contained 100 units/ml of penicillin and 0.1 mg/ml of streptomycin (Gibco-BRL).</p>", "<p>Pancreatic cancer tissues were obtained from 22 patients who underwent pancreatectomy for ductal carcinoma at our Department. Other pancreatic malignancies were excluded, such as intraductal papillary mucinous adenocarcinoma, acinar cell carcinoma, and endocrine tumors. Informed consent was obtained from each patient according to our Institutional guidelines. A resected specimen was immediately examined by inspection and palpitation at the operation room. A part of malignant or normal tissues considered was cut by surgical knife and it was divided two pieces. One for the tissue samples to extract RNA was immediately frozen in liquid nitrogen and stored at -80°C, the other was fixed in 10% formalin solution to make a paraffin block and performed with HE staining to evaluate pathologically.</p>", "<title>Antibodies</title>", "<p>Rabbit polyclonal anti-midkine antibody was kindly provided by Dr. Kadomatsu (Nagoya University School of Medicine, Nagoya, Japan). The following antibodies were purchased; mouse monoclonal anti-E1A (Ad2/Ad5) antibody (clone M73 #05-599) from Upstate Biotechnology (Lake Placid, NY), goat anti-mouse IgG (#62-6500) and HRP-goat anti-mouse IgG (#81-6520) from Zymed Laboratories (South San Francisco, CA), and mouse monoclonal anti-β-actin (clone AC-15 #A-5441) from Sigma (St. Louis, MO). Anti-adenoviral hexon protein antibody was included in the Adeno-X rapid titer kit (BD Biosciences Clontech, Palo Alto, CA).</p>", "<title>RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)</title>", "<p>Total cellular RNA was prepared using TRIZOL Reagent (Life Technologies, Rockville, MD) and cDNA was obtained from 1 μg of total RNA by the random primer method with a First-Strand cDNA Synthesis kit (Pharmacia Biotech, North Peapack, NJ) according to the manufacturer's instructions. Five microliters of first-strand cDNA solution was subjected to the polymerase chain reaction (PCR) with synthetic oligonucleotide primers (NIPPON EGT, Toyama, Japan). For RT-PCR analysis of human adenovirus type 5 E1A, a pair of primers (5'-ATGAGACATATTATCTGCCACGG-3'/5'-TAGACAAACATGCCACAGGTCC-3') was used and PCR was done for 35 cycles at 54°C, yielding a product of 551 base pairs. The reproducibility of the technique and quality of the total RNA were confirmed by amplifying β-actin as well (primers: 5'-GGCATCGTGATGGACTCCG-3'/5'-GCTGGAAGGTGGACAGCGA-3'; product: 613 base pairs).</p>", "<title>Quantitative RT-PCR</title>", "<p>To assess midkine gene expression, we used quantitative real-time RT-PCR analysis based on the TaqMan fluorescence method, which employs a dual-labeled non-extendable oligonucleotide hydrolysis (TaqMan) probe in addition to the two amplification primers. The probe contains 6-carboxy-fluorescein (FAM) as a fluorescent reporter dye, and 6-carboxytetramethyl-rhodamine (TAMRA) as a quencher for its emission spectrum. During the extension phase of PCR, the probe hybridizes to the target sequence and is then cleaved by the 5' to 3' exonuclease activity of Taq polymerase. The increase in the fluorescence of the reporter is proportional to the amount of specific PCR products, providing highly accurate and reproducible quantification. The level of reporter dye fluorescence is assessed with an automated sequence detector combined with analysis software (ABI Prizm 7700 Sequence Detection System; PE Applied Biosystems, Foster City, CA). Reaction conditions were set according to the manufacturer's protocol. The following primers and TaqMan probe were used for analysis. The midkine-specific primers were 5'-CGACTGCAAGTACAAGTTTGAGAAC-3' (upstream primer) and 5'-TCTCCTGGCACTGAGCATTG-3' (downstream primer), while 5' (FAM)-AAGGCACCCTGAAGAAGGCGCG-(TAMRA) 3' was the TaqMan probe.</p>", "<p>The PCR parameters were 95°C for 10 min (for activation of Taq-Polymerase), followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Amplification of β-actin for quality control and normalization was done with the TaqMan β-actin Control Reagent kit (PE Applied Biosystems), which utilizes standard TaqMan probe chemistry.</p>", "<title>Western blot analysis</title>", "<p>Cells were lysed in RIPA buffer containing 50 mM HEPES (pH 7.0), 250 mM NaCl, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonylfluoride (PMSF), and 20 μg/ml gabexate mesilate, and were incubated on ice for 10 minutes. Then the lysate was sonicated for 10 sec. Total extracts were cleaned by centrifugation at 15,000 rpm for 10 min at 4°C and the supernatants were collected. The protein concentration was measured with the BCA protein assay reagent (Pierce, Rockford, IL). Lysates were resuspended in one volume of gel loading buffer, which contained 50 mM Tris-Hcl (pH 6.7), 4% SDS, 0.02% bromophenol blue, 20% glycerol, and 4% 2-mercaptoethanol, and then were heated at 95°C for 5 min. The extracted protein was subjected to Western blotting. In brief, 50 μg aliquots of protein were size-fractionated in a single dimension by SDS-PAGE (6–10% gels) and transblotted to 0.45 μm polyvinylidine difluoride membranes (IPVH304F0, Millipore, Billerica, MA) with a semi-dry electroblotting apparatus (Bio-Rad, Richmond, CA).</p>", "<p>The blots were then washed three times in TBS with 0.1% Tween-20 (TBST) and incubated for 1 hour at room temperature in blocking buffer (Block Ace, Dainipponseiyaku, Osaka, Japan). Subsequently, the blots were incubated with an appropriate primary antibody for 1 h at room temperature or overnight at 4°C. Excess antibody was removed by washing the membrane with TBST three times for 10 min each. Then the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, followed by an addition of TBST. Reaction products were detected with the enhanced chemiluminescence system (Amersham, Buckinghamshire, United Kingdom). The membranes were treated with chemiluminescence reagents according to the manufacturer's protocol, and were exposed to X-ray films for 5–120 sec.</p>", "<title>Dual luciferase assay</title>", "<p>We prepared the midkne 0.6-luc vector, in which the 609-base pair genomic DNA fragment of the midkne gene was cloned into the pGL2-basic vector (Promega, Madison, WI) and the firefly luciferase gene was included without a promoter sequence[##REF##15251170##24##].</p>", "<p>The transcripitional activity of a number of pancreatic cancer cell lines was measured with this dual luciferase reporter assay system (Promega, Madison, WI). Midkine 0.6-luc and a control vector (the renilla luciferase gene fused with the HSV-TK promoter (pRL-TK, Promega, Madison, WI) at a molar ratio of 10:1) were transfected together into target cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The cells were lysed after 2 days and luciferase activity was measured according to the manufacturer's protocol. The relative firefly luciferase activity of each cell lysate was calculated from the level of luminescence.</p>", "<title>Adenoviruses and adenoviral transduction analysis</title>", "<p>We prepared recombinant adenovirus type 5 containing the 0.6 kb midkne promoter (Ad5MK) for midkne-regulated expression of E1A[##REF##15251170##24##]. Type 5, E1A-deleted, replication-defective adenovirus containing the green fluorescent protein gene (Ad5GFP) was constructed using AdEasy XL Adenoviral Vector System (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Adenoviruses were propagated in HEK 293 cells, purified by two rounds of cesium chloride density centrifugation, dialyzed, and stored at -70°C. Viral titers were determined with an Adeno-X Rapid Titer Kit (BD Biosciences Clontech, Palo Alto, CA).</p>", "<p>To assess the efficiency of adenoviral transduction in human pancreatic cancer cells, we performed fluorescent staining using Ad5GFP. Pancreatic cancer cells were seeded onto coverslips and infected with at recombinant adenovirus at various multiplicities of infection (MOIs). After 48 hours, coverslips were mounted on the glass slides with Vectashield mounting medium (Vector Labolatories, Burlingame, CA), and the cells were examined under a fluorescence microscope (Olympus, Tokyo, Japan).</p>", "<title>Assessment of adenoviral replication</title>", "<p>First, cells were infected with Ad5MK at various MOIs for 1 h and then the virus was removed. The infected cells were lysed in RIPA buffer to extract proteins after culture for 48 h. The proteins were subjected to SDS-PAGE and expression of E1A protein was analyzed by Western blotting. Next, cells were infected with Ad5MK at 1 MOI for 1 h and the medium was then refreshed. After the cells were cultured for 2 days, the cell lysate was prepared with three cycles of freezing and thawing.</p>", "<p>HEK293 cells were infected with serially diluted cell lysates or tissue lysates. After 48 hr, the cells were stained with anti-adenoviral hexon protein antibody by using an Adeno-X rapid titer kit (BD Biosciences Clontech, Palo Alto, CA).</p>", "<title><italic>In vitro </italic>cytotoxicity test</title>", "<p>Pancreatic cancer cells were plated into 12-well plates in triplicate at a density of 1.0 × 10<sup>4 </sup>cells/well. After 24- to 36 h of culture, cells were infected with Ad5GFP or Ad5MK at various MOIs for 1 h, and the infecting medium was replaced with complete medium. After 1, 3 and 5 days, the number of viable cells was counted using a cell counter (Coulter Z1, Beckman-Coulter, Fullerton, CA).</p>", "<title>Animal study</title>", "<p>The animal study was performed in accordance with the guidelines for animal experiments of the Institute of Laboratory Animals at Kyoto University. Six-week-old male BALB/c nude mice were purchased from CLEA Japan (Tokyo, Japan). First, the mice were subcutaneously inoculated with Suit-2 cells (2 × 10<sup>6</sup>/ml) in 100 μl of Hank's balanced salt solution (HBSS) (Gibco-BRL) containing 20% matrigel (BD Biosciences, Bedford, MA). When the tumors reached about 10 mm in diameter, Ad5GFP or Ad5MK (2 × 10<sup>9 </sup>PFU, 0.1 ml/mouse) was injected intratumorally. The mice were sacrificed at 3 or 7 days after adenoviral injection to extract RNA and lysates from the tumors. RT-PCR analysis of human adenovirus type 5 E1A and staining with anti-adenoviral hexon antibody were performed to assess the replication of adenoviruses <italic>in vivo</italic>.</p>", "<p>Next, Suit-2 cells (2 × 10<sup>6</sup>/ml) in 500 μl of sterile PBS were inoculated into the peritoneal cavity of BALB/c nude mice to create a peritoneal dissemination xenograft model. The mice were divided into the following 4 groups: (1) an Ad5MK group, (2) an Ad5GFP group, (3) a PBS group, and (4) an untreated group. Ad5MK (2 × 10<sup>9 </sup>PFU, 0.5 ml/mouse), Ad5GFP (2 × 10<sup>9 </sup>PFU, 0.5 ml/mouse) or PBS (0.5 ml/mouse) was administered intraperitoneally at 4 days after the injection of Suit-2 cells. Survival was measured from the start of treatment.</p>", "<title>Statistical analysis</title>", "<p>Quantitative data are presented as the mean ± SEM. Each <italic>in vitro </italic>experiment was performed independently at least three times. To compare mRNA levels in pancreatic tissue samples, Wilcoxon's rank sum test was used. Survival rates were calculated by the Kaplan-Meier method, and differences between groups were evaluated with the log-rank test and Wilcoxon's test. Statistical analysis was done by using JMP statistical software and statistical significance was considered to be present at p &lt; 0.05.</p>" ]
[ "<title>Results</title>", "<title>Midkine expression by human pancreatic cancer cell lines</title>", "<p>We examined the expression of midkine mRNA in seven human pancreatic cancer cell lines by TaqMan PCR (Table ##TAB##0##1##). Midkine mRNA expression was strong in AsPC-1 and CFPAC-1 cells, but it was weak in MIAPaCa-2 cells.</p>", "<p>Next, midkine protein expression was assessed by Western blot analysis (Figure ##FIG##0##1A##). AsPC-1 and CFPAC-1 cells showed strong expression of midkine protein, whereas BxPC-3, HPAC and Suit-2 cells showed moderate expression. In contrast, expression by PANC-1 cells was weak and MIAPaCa-2 cells showed no midkine band. The extent of midkine protein expression was in parallel to that of midkine mRNA expression. In the following experiments, we therefore designated the MIAPaCa-2 cell line as midkine-negative.</p>", "<title>Midkine expression in human pancreatic cancer</title>", "<p>We assessed the expression of midkine mRNA in 22 pancreatic cancer samples and 18 adjacent non-cancerous pancreatic tissue samples by TaqMan PCR (Figure ##FIG##0##1B##). The midkine mRNA/β-actin mRNA ratio of pancreatic cancer and non-cancerous pancreatic tissue was 0.60 ± 0.55 and 0.22 ± 0.13, respectively. Expression of midkine mRNA was significantly stronger in pancreatic cancer than in non-cancerous tissue (p &lt; 0.001).</p>", "<title>Transcriptional activity of the midkine promoter in human pancreatic cancer cells</title>", "<p>We investigated the transcriptional activity of the midkine promoter by the dual luciferase reporter assay in AsPC-1, CFPAC-1, MIAPaCa-2, PANC-1, and Suit-2 cells (Table ##TAB##1##2##). This assay showed that the relative luciferase activity mediated by the 0.6 kb midkne fragment in AsPC-1, PANC-1, and Suit-2 cells was approximately 6 to 20 times greater than that in midkne-negative MIAPaCa-2 cells. Transcriptional activity in MIAPaCa-2 cells was extremly low. The transcriptional activity in each of the cell lines was usually correlated with the expression of midkne, although CFPAC-1 cells showed low transcriptional activity and high midkne expression.</p>", "<title>Sensitivity of human pancreatic cancer cell lines to adenovirus infection</title>", "<p>We examined the induction of Ad5GFP in human pancreatic cancer cells (Figure ##FIG##1##2##). Cells were infected with Ad5GFP (1, 10, or 25 MOI) at 16 to 18 h after seeding. After 48 h, GFP-expressing cells were detected by fluorescence microscopy. We found that pancreatic cancer cells exhibited a heterogeneous adenoviral transduction profile. Many of the CFPAC-1 and Suit-2 cells infected at an MOI of 25 expressed GFP, whereas AsPC-1 cells showed far lower adenoviral transduction efficiency. We also found that the number of GFP-expressing cells increased in an MOI-dependent manner.</p>", "<title>Ad5MK shows specific replication and infectivity for human pancreatic cancer cell lines</title>", "<p>Since the adenoviral infection cycle is completed within 24 h, E1A expression by infected cells after 48 h reflects viral replication. Therefore, to determine the specificity of Ad5MK replication, we used pancreatic cancer cell lines with different levels of midkine expression and then examined E1A expression by Western blotting (Figure ##FIG##2##3A##). We found that viral replication was dependent on the MOI of infection.</p>", "<p>Next, we examined the infectivity of Ad5MK prepared from pancreatic cell line in HEK293 cells by using an anti-adenoviral hexon antibody (Figure ##FIG##2##3B##). Stained HEK293 cells were found after infection with preparations from midkine-positive cells (PANC-1, Suit-2, AsPC-1, and CFPAC-1), whereas there were no stained cells after infection with a preparation from midkine-negative MIAPaCa-2 cells.</p>", "<title>Specific cell killing effect of Ad5MK <italic>in vitro</italic></title>", "<p>We subsequently examined the ability of Ad5MK to kill pancreatic cancer cells (Figure ##FIG##3##4##). The number of viable cells was counted by using a Coulter counter. Ad5MK showed a much stronger-killing effect against Suit-2 and PANC-1 cells that have moderate and weak midkine expression, respectively, compared with its effect on midkine-negative MIAPaCa-2 cells.</p>", "<p>On the other hand, moderate cell-killing effect against CFPAC-1 cells was found in 10 MOI of Ad5MK. CFPAC-1 cells had high midkine expression and moderate efficiency of adenoviral transduction. Transcriptional activity of midkine promoter in CFPAC-1 cells was low, but it was higher compared to that of MIAPaCa-2 cells. This may be the reason why growth suppression of CFPAC-1 cells was observed by 10 MOI of Ad5MK. In contrast, Ad5MK had no effect on AsPC-1 cells as far as the designated conditions, although these cells showed strong midkine expression. This finding may have reflected low efficiency of adenoviral transduction. Ad5GFP had no influence on the growth of any of the cells in this assay compared with normal control cells (data not shown).</p>", "<title>Adenoviral replication and anti-tumor effect of Ad5MK <italic>in vivo</italic></title>", "<p>We assessed adenoviral replication <italic>in vivo </italic>by using a Suit-2 subcutaneous xenograft model of pancreatic cancer. RNA and tumor lysates were obtained from the subcutaneous xenografts after injection of adenoviruses. RT-PCR analysis for human adenovirus type 5 E1A showed that E1A expression could be detected in the Ad5MK groups (Figure ##FIG##4##5A##). On the other hand, no band was seen in the untreated and Ad5GFP groups. After staining with anti-adenoviral hexon antibody, positive cells were only found in the Ad5MK groups, whereas there were no positive cells in the untreated and Ad5GFP groups (Figure ##FIG##4##5B##).</p>", "<p>Finally, we assessed the anti-tumor effect in a Suit-2 intraperitoneal xenograft model after the intraperitoneal injection of Ad5MK (Figure ##FIG##5##6##). Following the inoculation of 2 × 10<sup>6</sup>/ml Suit-2 cells into the peritoneal cavity of nude mice, 2 × 10<sup>9 </sup>PFU of Ad5MK or Ad5GFP, or 500 μl of PBS, was administered intraperitoneally. In this intraperitoneal xenograft model, our preliminary study revealed that untreated mice died of peritoneal dissemination with bloody ascites after approximately 2 to 3 weeks. Almost all of the mice in the untreated, PBS, and Ad5GFP groups died within 35 days, whereas almost all of the mice in the Ad5MK group survived for more than 50 days. There was a statistically significant difference of the survival time between the Ad5MK group and the other group.</p>" ]
[ "<title>Discussion</title>", "<p>Conditionally replicative adenoviruses, which show tumor-specific replication and oncolysis, are a promising new treatment modality for malignancies resistant to conventional therapies [##REF##10741699##25##,##REF##11960282##26##]. In this study, we demonstrated a possible strategy for pancreatic cancer based on Ad5MK replication-competent adenovirus. Midkine expression is increased in various human tumors, including gastrointestinal cancers [##REF##8383007##19##, ####REF##7559083##20##, ##REF##7520350##21##, ##REF##10626184##22####10626184##22##]. We showed that most human pancreatic cancer cell lines (6 out of 7 lines tested) express midkine to some extent and that the level of midkine mRNA expression in pancreatic cancer tissues is significantly higher than in non-cancerous pancreatic tissues. These findings suggested that the midkine promoter may be a potential candidate for safe suicide gene therapy targeting pancreatic cancer.</p>", "<p>We next examined the promoter activity of midkine in a dual luciferase reporter assay. A 2.3-kb fragment from the 5' region of the midkine gene contains the elements responsible for promoter activity [##REF##10363587##27##]. Within this 2.3-kb fragment, Yoshida et al. have demonstrated the presence of a cis-element with strong promoter activity located at the -559/50 region using the CAT assay, and they also reported that its midkine promoter activity in tumor cells was comparable to that of the SV40 early promoter [##REF##12017272##28##]. Therefore, we examined the promoter activity of this 0.6-kb fragment in human pancreatic cancer cells by using the dual luciferase reporter assay. We showed that the relative luciferase activity in AsPC-1, PANC-1, and Suit-2 cells was greater than that in midkne-negative MIAPaCa-2 cells. Transcriptional activity in the cell lines was generally parallel to the level of midkne expression. In CFPAC-1 cells, however, the level of midkne expression did not correspond well with the transcripitional activity of the midkne promoter. We cannot explain this discrepancy, but it has been reported that the level of endogenous midkne expression and its promoter activity are not always correlated with each other because of various factors such as negative and positive regulatory elements residing outside the region analysed [##REF##12791382##29##]. Another possible explanation may be differences between cell lines with respect to regulation of the proteins involved in transcription [##REF##12791382##29##].</p>", "<p>Because the efficiency of adenoviral transfection varies between cell lines, it was essential to examine the infectivity of Ad5GFP for multiple human pancreatic cancer cell lines. Adenoviral transduction efficiency was far lower for AsPC-1 than the other cell lines. Adenoviral replication in AsPC-1 cells was lower than in any other midkine-positive cell line and no killing of AsPC-1 cells was observed at 10 MOI, although these cells showed high midkine expression and transcriptional activity. Thus, we consider that adenoviral transduction efficiency as well as transcriptional activity is critical for adenovirus-mediated gene therapy. In pancreatic cancer cells, the combined efficiency of transduction and transcription determines the actual cell-killing effect of Ad5MK.</p>", "<p>When clinical application is considered, not only anti-tumor activity but also toxicity for normal tissues should be taken into account. In this regard, Ad5MK showed specific cytotoxicity for midkine-positive cells both <italic>in vitro </italic>and <italic>in vivo</italic>. Despite the target selectivity of Ad5MK, weak E1A expression was found in midkine-negative cells after infection with 5 MOI of Ad5MK. Type 5 adenoviruses are commonly used in viral therapy experiments, but show species-specific replication, and it has been reported that these adenoviruses do not replicate in mice or rats. There are no suitable animal models apart from the cotton rat to assess the toxicity of conditionally replicative adenoviruses, but replicative viruses have already shown at least low-level viral production and/or systemic toxicity in clinical trials[##REF##10741699##25##,##REF##11606381##30##,##REF##16690359##31##]. The precise requirements for selective targeting to prevent damage to normal tissues <italic>in vivo </italic>need to be clarified in the future.</p>", "<p>We demonstrated an anti-tumor effect of Ad5MK on midkine-positive tumor cells <italic>in vivo </italic>as well as <italic>in vitro</italic>. Our animal studies showed that mice treated with Ad5MK survived for significantly longer than the other groups. Even in the Ad5MK group, however, half of the mice died due to peritoneal dissemination after about 50 days. This may have been due to an inadequate dose of adenovirus or because of the regrowth of cancer cells with resistance to adenovirus-mediated gene therapy. In this study, we only gave a single dose of adenovirus intraperitoneally after tumor cell inoculation. In the future, we should investigate the optimum volume and number of adenovirus doses to improve the efficacy of Ad5MK treatment. Another way to improve the results could be employment of fiber-modified adenoviruses, which can enter cancer cells resistant to conventional adenoviral gene transfer in a coxsackievirus and adenovirus receptor (CAR)-independent manner.</p>" ]
[ "<title>Conclusion</title>", "<p>Midkine expression was increased in pancreatic cancer cell lines and pancreatic cancer tissues. Ad5MK showed specific targeting of and cytotoxicity for midkine-positive cells both <italic>in vitro </italic>and <italic>in vivo</italic>. These results suggest that replication-competent adenoviruses based on the midkine promoter might have the potential to be used in gene therapy for pancreatic cancer.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>To develop a novel therapeutic strategy for human pancreatic cancer using a midkine promoter-based conditionally replicating adenovirus.</p>", "<title>Methods</title>", "<p>We examined midkine mRNA expression and midkine protein expression by seven human pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, MIAPaCa-2, PANC-1, and Suit-2), as well as by non-cancerous pancreatic tissue and pancreatic cancers. Midkine promoter activity was measured in cancer cell lines by the dual luciferase reporter assay. Adenoviral transduction efficiency was assessed by fluorescent staining of cancer cell lines using adenovirus type 5 containing the green fluorescent protein gene (Ad5GFP). Replication of adenovirus type 5 containing the 0.6 kb midkne promoter (Ad5MK) was assessed by the detection of E1 protein in cancer cell lines. The cytotoxicity of Ad5MK for cancer cells was evaluated from the extent of growth inhibition after viral infection. Infection and replication were also assessed in nude mice with subcutaneous Suit-2 tumors by intratumoral injection of Ad5MK, Ad5GFP, or vehicle. E1a mRNA expression in the treated tumors and expression of the replication-specific adenoviral hexon protein were evaluated. Finally, the anti-tumor activity of Ad5MK against intraperitoneal xenografts of Suit-2 pancreatic cancer cells was examined after intraperitoneal injection of the virus.</p>", "<title>Results</title>", "<p>Both midkine mRNA expression and midkine protein expression were strong in AsPC-1 and CFPAC-1 cell liens, moderate in BxPC-3, HPAC, and Suit-2 cell lines, and weak in PANC-1 and MIAPaCa-2 cell lines. Expression of midkine mRNA was significantly stronger in pancreatic cancers than in non-cancerous pancreatic tissues. The relative luciferase activity mediated by the 0.6 kb midkne fragment in AsPC-1, PANC-1, and Suit-2 cell lines was approximately 6 to 20 times greater than that in midkne-negative MIAPaCa-2 cell lines. Pancreatic cancer cell lines exhibited a heterogeneous adenoviral transduction profile. E1A expression was higher in cell lines with strong midkine expression than in cell lines with weak midkine expression. Ad5MK showed much greater cytotoxicity for midkine-expressing Suit-2 and PANC-1 cell lines than for midkine-negative MIAPaCa-2 cell lines. In the Suit-2 subcutaneous xenograft model, expression of E1A was detected in Ad5MK-treated tumors, but not in untreated and Ad5GFP-treated tumors. In the Suit-2 intraperitoneal xenograft model, the Ad5MK group survived for significantly longer than the Ad5GFP, PBS, and untreated groups.</p>", "<title>Conclusion</title>", "<p>Ad5MK has an anti-tumor effect against human pancreatic cancer cell lines that express midkine mRNA. Midkine promoter-based conditionally replicative adenovirus might be a promising new gene therapy for pancreatic cancer.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>ET conceived of the study and performed experiments on adenoviruses. RD conceived of the study, and participated in its design and coordination and helped to draft the manuscript. KK conceived of the study and performed experiments on mice. TM conceived of the study and performed experiments on mice. DI conceived of the study and performed experiments on pancreatic cancer cell lines. MK conceived of the study and performed experiments on pancreatic cancer cell lines. AK conceived of the study and performed experiments on pancreatic cancer cell lines. KN conceived of the study and performed experiments on mice. TI conceived of the study and performed experiments on mice. TM conceived of the study and performed experiments on pancreatic cancer cell lines. MW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. MT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. SU conceived of the study, and participated in its design.</p>" ]
[ "<title>Acknowledgements</title>", "<p>This study was supported by a Grant-in-Aid (#20390355) from the Ministry of Education, Culture, Sports, Science and Technology.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Midkine protein expression by human pancreatic cancer cell lines and human pancreatic cancer tissues</bold>. (A) Midkine protein expression by human pancreatic cancer cell lines. AsPC-1 and CFPAC-1 cells showed strong expression of midkine protein, whereas BxPC-3, HPAC, and Suit-2 cells showed moderate expression. In contrast, expression by PANC-1 cells was low and MIAPaCa-2 cells showed no detectable midkine band. (B) Midkine mRNA expression in non-cancerous pancreatic tissues and human pancreatic cancers. Expression of midkine mRNA in the pancreatic cancers was significantly stronger than in the non-cancerous pancreatic tissues (p &lt; 0.001).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Sensitivity of human pancreatic cancer cell lines to adenovirus infection</bold>. Pancreatic cancer cells were infected with Ad5GFP (1, 10 or 25 MOI) at 16 to 18 h after seeding. After 48 h, GFP-expressing cells were detected by fluorescence microscopy. (Original magnification ×100).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Replication specificity and infectivity of Ad5MK for human pancreatic cancer cell lines</bold>. (A) E1A protein expression by pancreatic cancer cells. Pancreatic cancer cell lines were infected with Ad5MK showing different levels of midkine expression, Ad5GFP alone, or the vehicle. (B) Adenoviral hexon staining. HEK293 cells were treated with preparations of Ad5MK-infected pancreatic cancer cells. After 48 hr, HEK293 cells were stained with anti-adenoviral hexon antibody. (Original magnification ×100)</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold><italic>In vitro </italic>cytotoxicity of Ad5MK</bold>. Pancreatic cancer cells were plated in triplicate in 12-well plates at 1.0 × 10<sup>4 </sup>cells/well. After 24 to 36 h, the cells were infected with Ad5GFP or Ad5MK at various MOIs. After 1, 3, and 5 days, the number of viable cells was counted.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Replication of adenovirus in a Suit-2 subcutaneous xenograft model of pancreatic cancer</bold>.(A) Human adenovirus type 5 E1A expression. RNA was extracted from subcutaneous tumors after injection of adenoviruses. Expression of E1A was analyzed by RT-PCR. (B) Adenoviral hexon staining. HEK293 cells were infected with Ad5MK prepared from subcutaneous xenografts after injection of adenoviruses. After 48 hr, HEK293 cells were stained with anti-adenoviral hexon antibody. Positive cells were only found in the Ad5MK groups, whereas there were no positive cells in the untreated and Ad5GFP groups. (Original magnification ×100)</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Anti-tumor effect of Ad5MK in a Suit-2 intraperitoneal xenograft model</bold>. After intraperitoneal inoculation of 2 × 10<sup>6</sup>/ml Suit-2 cells in nude mice, 2 × 10<sup>9 </sup>PFU of Ad5GFP or Ad5MK, or 500 μl of PBS, was administered intraperitoneally. There was a significant difference of survival time between the Ad5MK group and the other groups (p = 0.0002 Log-rank test and p = 0.0002 by Wilcoxson's test).</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Expression of midkine mRNA in human pancreatic cancer cells.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Cell line</td><td align=\"center\">Midkine mRNA/β-actin mRNA</td></tr></thead><tbody><tr><td align=\"center\">AsPC-1</td><td align=\"center\">1.19 ± 0.04</td></tr><tr><td align=\"center\">BxPC-3</td><td align=\"center\">0.37 ± 0.01</td></tr><tr><td align=\"center\">CFPAC-1</td><td align=\"center\">1.42 ± 0.02</td></tr><tr><td align=\"center\">HPAC</td><td align=\"center\">0.31 ± 0.01</td></tr><tr><td align=\"center\">MIAPaCa-2</td><td align=\"center\">0.001 ± 0.0004</td></tr><tr><td align=\"center\">PANC-1</td><td align=\"center\">0.02 ± 0.001</td></tr><tr><td align=\"center\">Suit-2</td><td align=\"center\">0.13 ± 0.003</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Transcriptional activity of MK promoter in human pancreatic cancer cells.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Cell lines</td><td align=\"center\">Relative luciferase activity</td></tr></thead><tbody><tr><td align=\"center\">AsPC-1</td><td align=\"center\">0.57 ± 0.09</td></tr><tr><td align=\"center\">CFPAC-1</td><td align=\"center\">0.06 ± 0.01</td></tr><tr><td align=\"center\">MIAPaCa-2</td><td align=\"center\">0.03 ± 0.01</td></tr><tr><td align=\"center\">PANC-1</td><td align=\"center\">0.16 ± 0.02</td></tr><tr><td align=\"center\">Suit-2</td><td align=\"center\">0.29 ± 0.01</td></tr></tbody></table></table-wrap>" ]
[]
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[ "<table-wrap-foot><p>Data are expressed as the mean ± SEM.</p></table-wrap-foot>", "<table-wrap-foot><p>Relative luciferase activity was defined as firefly luciferase activity per renilla luciferase activity in cancer cells. Data are expressed as the mean ± SEM.</p></table-wrap-foot>" ]
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{ "acronym": [], "definition": [] }
31
CC BY
no
2022-01-12 14:47:25
J Exp Clin Cancer Res. 2008 Aug 21; 27(1):30
oa_package/52/9e/PMC2529268.tar.gz
PMC2529269
18681964
[ "<title>Background</title>", "<p>Aberrant crypt foci (ACF) were first described by Bird and Good. The lesion of ACF is composed of the enlarged crypts that are slightly elevated above the surrounding mucosa and the densely stained crypts with methylene blue [##UREF##0##1##]. ACF are considered as putative preneoplastic lesions of the colon in both humans and experimental animals [##REF##17627293##2##,##REF##18413814##3##].</p>", "<p>Although ACF may share some morphologic, genetic, and biochemical features with colonic tumors [##REF##7923189##4##, ####REF##18172251##5##, ##REF##11017067##6##, ##REF##17234750##7####17234750##7##], conflicting evidence has also been reported. For example, mutations in the K-<italic>ras </italic>gene are relatively common in human ACF, but are only detected at a relatively late stage of colon cancer [##REF##8246286##8##, ####REF##18384435##9##, ##REF##7923190##10##, ##REF##9097977##11####9097977##11##]. In rat models, although hundreds of ACF are induced per animal by azoxymethane (AOM), and K-<italic>ras </italic>mutations are frequently observed in those ACF, only a few colon tumors are observed per animal [##REF##17893236##12##, ####REF##9230276##13##, ##REF##10874009##14####10874009##14##]. In azoxymethane/dimethylhydrazine-treated rats and in patients with sporadic colorectal cancer, the number of tumors is minuscule compared with the large number of ACF [##REF##11309270##15##], demonstrating that only a very small fraction of the ACF in theory has the potential to progress to the stage of a tumor. Therefore, there is a strong need to investigate the role of ACF in colon carcinogenesis. Alexander <italic>et al</italic>. [##REF##11431334##18##] observed a type of small flat dysplastic lesions that was denoted as ACFMin in Min/+ mice, an Apc/familial adenomatous polyposis model. ACFMin exhibited dysplastic crypts similar to those found in adenomas and only those ACFMin showed a continuous development from the monocryptal stage to adenoma with rapid multiplication of crypts and altered expression of β-catenin. In contrast, the classic elevated ACF are hyperplastic, slow-growing, and show normal expression of β-catenin suggesting they are not directly associated with tumorigenesis [##REF##10868458##16##, ####REF##9363998##17##, ##REF##11431334##18####11431334##18##].</p>", "<p>The Wnt pathway controls the cell fate during the embryonic development. It not only drives tumorigenesis but also is required at different stages during the gut development. Activated Wnt signal pathway precludes the posttranslational down-regulation of β-catenin, which consequently accumulates in the cytoplasm and translocates into the nucleus where it complexes with the transcription factor Tcf-4 and activates specific target genes (such as c-MYC, cyclin D1 and MMP-7, etc.) [##UREF##1##19##].</p>", "<p>In this study we evaluated the relationship between ACF and tumorigenesis by observing the sequential development from ACF to tumor in AOM-treated rats. In addition, we histochemically examined the expression of β-catenin and mmp-7.</p>" ]
[ "<title>Methods</title>", "<title>Animals and chemicals</title>", "<p>Sixty male SPF Wistar rats (8 to 9 weeks old; ~180 g weight) were provided by Nanfang Medical University animal center. the animal use is approved by the Animal Care and Use committee (approval number: 2002-009 2004B023 2004A068)and all the protocols are in agreement to the requirement of the committee. AOM (Sigma, St. Louis, MO, USA) was dissolved in 0.9% NaCl immediately before use and adjusted to pH 6.5–7.0. Rats were injected subcutaneously with azoxymethane (15 mg/kg bw/injection) once a week for 2 weeks. Animals were sacrificed sequentially from weeks 8 to 25 after the last injection of azoxymethane. The colons stained with methylene blue were transilluminated in the stereomicroscope, and the colonic lesions were characterized and scored based on the morphology of the tissues. The lesions were dissected and characterized by histopathological (for experimental details, see Table ##TAB##0##1##) and immunohistochemical analysis.</p>", "<title>Scoring of classic ACF, dark ACF, and tumors</title>", "<p>The colons were removed, rinsed in ice-cold PBS, slit open longitudinally, and fixed flat between two wet (PBS) filter papers for 48 hours in 10% neutral buffered formalin followed by 10 seconds staining with 0.2% methylene blue (Chemical Ltd, Guangzhou, China) dissolved in the same formalin solution. Deeply stained crypts were examined by transillumination in stereomicroscopy after staining. Classic ACF were characterized by their obvious enlarged crypts, slight elevation above the surrounding mucosa thickened layer of epithelial cells, increased pericryptal space, and their light blue staining. Dark ACF were characterized by having dark blue staining, moderate enlarged or small crypts that are elevated from the surrounding epithelium and mild enlarged or small compressed crypts that are not elevated from the surrounding epithelium. Thus, darker blue staining and compressed pit pattern were used as the criteria for identification of dark ACF. The size of the lesions was scored by crypt multiplicity (AC/lesion). The crypt multiplicity of lesions was determined by transforming the diameter (mm) scored with an eyepiece to the crypt multiplicity by using the following empirical formula: c = 20 × d<sup>2</sup>. The lesions containing &gt;32 aberrant crypts were defined as tumors.</p>", "<title>Histopathological analysis</title>", "<p>Areas with mucosal lesions identified by examining the surface of the whole colon preparations in the stereomicroscopy were dissected and embedded in paraffin wax. After embedding, the tissue samples were sectioned in parallel with the mucosal surface. If the lesions have more than 32 aberrant crypts, then the tissues samples were sectioned in vertical with the mucosal surface. All sections were stained with H &amp; E. Blind examination of the lesions from different levels of the crypts was performed by two pathologists.</p>", "<p>Histopathological classification was based on the following criteria. Hyperplasia with no dysplasia was characterized as having slightly dilated crypts with normal epithelium. Mild dysplasia was characterized as having elongated, crowded and pseudostratified nuclei with well preserved polarity and normal or slightly reduced number of goblet cells. Moderate dysplasia was characterized as having elongated, more crowded and pseudostratified nuclei with well preserved polarity and more reduced number of goblet cells than in mild dysplasia. Severe dysplasia was characterized as having enlarged, round or ovoid nuclei with prominent nucleoli. The polarity of the nuclear in severe dysplasia is partially lost and the number of the goblet cells is significantly reduced or completely lost.</p>", "<title>Immunohistochemical analysis</title>", "<p>Paraffin-embedded formalin-fixed sections were prepared, deparaffinized, and rehydrated in xylene, graded alcohol, and water. Demasking was performed in microwave oven for 12 minutes in Tris-EDTA solution. To determine the expression of β-catenin, sections were stained with monoclonal anti-β-catenin antibody (Wuhan Boster Ltd, Cat. No. BA0426, Wuhan, China) at the dilution of 1:200 and counterstained with hematoxylin using a SP kit. To determine the expression of MMP-7, sections were stained with monoclonal anti-MMP-7 antibody (Wuhan Boster Ltd, Cat. No. BA2110) at the dilution of 1:200 and counterstained with hematoxylin using a SP kit. β-catenin expression was evaluated separately for the cell membrane, cytoplasmic and nuclear compartments. If more than 70% of the membranes in the lesion cells were positively stained by the antibody, the membranous expression of β-catenin in the lesions was judged to be normal. If less than 70% of membranes in the lesion cells were positively stained, the membranous expression of β-catenin was judged to be reduced. On the other hand, if more than 10% of the cells were positive for the β-catenin staining in cytoplasm/nucleus, it was considered as cytoplasm/nucleus β-catenin expression because normal colonic mucosal cell cytoplasm/nucleus shows negative β-catenin expression. Thus, if more than 10% of the lesion cells showed elevated β-catenin labeling in the cytoplasm and/or distinct nuclear staining, the lesion was recorded as having cytoplasmic overexpression and/or nuclear accumulation of β-catenin. Reduced cell membrane expression or cytoplasmic overexpression/nuclear accumulation are recognized as altered expression. Cytoplasmic overexpression/nuclear accumulation are also called ectopic expression. For MMP-7, the criterion for positive expression was the presence of prominent cytoplasmic staining.</p>", "<title>Statistical analysis</title>", "<p><italic>X</italic><sup>2 </sup>test was used to calculate the statistical difference of proportions between groups. Student's <italic>t </italic>test was used to compare two groups and one-way ANOVA was used to compare ranks between multiple groups.</p>" ]
[ "<title>Results</title>", "<title>Stereomicroscopy and histopathological analysis revealed two types of ACF:classic ACF and dark ACF</title>", "<p>Examination of the surface in the colon with stereomicroscopy revealed two types of ACF: classic ACF and dark ACF (Fig. ##FIG##0##1A##). Dark ACF were characterized by their darker blue staining than classic ACF. Furthermore, dark ACF have mild enlarged or small compact crypts that are not elevated from the surrounding epithelium. Histopathological examination showed that the lesions were classified into four categories: (1) hyperplasia without dysplasia; (2) mild dysplasia; (3) moderate dysplasia; and (4) severe dysplasia. All the classic ACF (165 cases) showed hyperplasia without dysplasia (Fig. ##FIG##0##1B##). The average crypt multiplicity for classic ACF slightly increased from 3.93 crypt/lesion at weeks 8~14 to 4.52 crypts/lesion at week 22~25 (F = 3.17, P = 0.278) (Table ##TAB##1##2##). In contrast, all the 22 dark ACF cases showed dysplasia with different degree (Fig. ##FIG##0##1C##) and the crypt with moderate or severe dysplasia accounted for 95.4% (21/22) of the cases. The average focal crypt multiplicity of dark ACF increased significantly between different categories (F = 15.4, P = 0.000), In addition, crypt multiplicity of dark ACF increased significantly faster than those of classic ACF.</p>", "<title>Dark ACF is phenotypically similar to tumor</title>", "<p>Nascent tumors exhibited characteristic of dark blue staining and contained branched or gyrus-like pit pattern of compressed crypts (Fig. ##FIG##0##1D##). In addition, nascent tumors displayed the same surface morphology as dark ACF. All the tumors showed moderate or severe dysplasia (10 of 10). There was no significant difference between the proportion of morderate/severe dysplasia in dark ACF (95.4%) and that in tumors (100%). The rapid increase of crypt multiplicity in dark ACF is similar to the characteristic of tumors.</p>", "<title>Expression of β-catenin in classic ACF, dark ACF and tumors</title>", "<p>The immnoreactivity of β-catenin in the classic ACF, dark ACF and tumors is summarized in Table ##TAB##2##3##. Expression of β-catenin is normal in most of the classic ACF (157 of 165, Fig. ##FIG##1##2A##). Four of the classic ACF showed reduced expression in cell membrane and the other four classic ACF showed cytoplasmic expression of β-catenin. In contrast, 72.72% (16/22) of dark ACF showed cytoplasmic overexpression or nuclear accumulation (Fig. ##FIG##1##2C##). All the ten tumor cases showed cytoplasmic overexpression or nuclear accumulation of β-catenin. Interestingly, the expression pattern of β-catenin for dark ACF or tumors was mainly characterized by reduced expression in membrane during the early period (week 8~14) and then transited to nuclear accumulation during the later period (week 22~25) (Table ##TAB##3##4##).</p>", "<title>Expression of mmp-7 in classic ACF, dark ACF and tumors</title>", "<p>The immnoreactivity of mmp-7 in the classic ACF, dark ACF and tumors is summarized in Table ##TAB##2##3##. The expression rate of mmp-7 in dark ACF (Fig. ##FIG##1##2D##) was 81.82% (18/22), which was significantly higher than that in classic ACF (7.87%, 13/165) (Fig. ##FIG##1##2B##). All the tumor cases exhibited cytoplasmic overexpression of mmp-7. Furthermore, there was no significant difference between expression of mmp-7 in dark ACF and tumors.</p>" ]
[ "<title>Discussion</title>", "<p>By examining the sequential development of early lesions in the colon of Wistar rats 8 to 25 weeks after AOM treatment, we identified two types of lesions: classic ACF and dark ACF. Dark ACF were characterized by their darker blue staining than the classic ACF. In addition, dark ACF has mildly enlarged or small crypts that are not elevated from the surrounding epithelium. The crypts of dark ACF is compact and similar to a compressed luminal opening. Dark ACF shares similarities with tumors with regard to the surface morphology of the colon, higher crypt multiplicity, pathologic classification and expression pattern of β-catenin and mmp-7. However, classic ACF are different to tumors. These results may explain why only a few colon tumors are observed although hundreds of ACF are induced per animal. Activation of β-catenin, a core protein in Wnt signal transduction pathway in nucleus, correlates with the tumorigenesis and abnormal accumulation of β-catenin plays an important role in the development of tumors [##UREF##2##20##, ####REF##12613197##21##, ##REF##15054482##22####15054482##22##]. One of the studies found that β-catenin is less expressed in the membrane of ACF, but more expressed in cytoplasm and nucleus when ACF variation increases [##REF##10910031##23##]. Our study showed that the ectopic expression ratio of β-catenin was 72.7% in dark ACF, which had no significant difference compared with that in tumors. In addition, expression pattern of β-catenin transited from reduced cell membrane expression in the early period to nuclear accumulation in the later period. Simultaneously, activation of oncogene encoding β-catenin could stimulate the downstream target, MMP-7, to suppress tumor cell apoptosis and degradation of basic membrane. Duan <italic>et al</italic>. [##UREF##3##24##] found that expression of β-catenin in cytoplasm and nucleus directly correlated with the expression of MMP-7 protein, suggesting that abnormal expression of β-catenin could initiate Wnt signal transduction pathway leading to the expression of MMP-7. Our study found that β-catenin ectopic expression ratio was positively correlated with the MMP-7 positive ratio, and meanwhile, there was no statistical difference between the expression of MMP-7 in dark ACF and that in tumors. These results suggested that increased β-catenin accumulation in dark ACF activated the Wnt pathway leading to the loss of cell adhesion and separation. Loss of cell adhesion and separation resulted in more contact between cells and high expression of MMP-7, leading to further degradation of the outer membrane and unlimited cell growth. Dark ACF obtained the ability to grow invasively and was converted to colon cancer. We found that β-catenin and MMP-7 expression was not changed in classic ACF, which suggested that oncogene activation and expression does not exist in classic ACF. Therefore, the chance of conversion from classic ACF to tumors is relatively small.</p>" ]
[ "<title>Conclusion</title>", "<p>In summary, we identified two types of ACF: dark ACF and classic ACF. Dark ACF is similar to tumor with regard to its morphological and pathological changes, the increased number of crypts and the expression oncogenes. However, all these features identified in dark ACF and tumors were not observed in classic ACF. These results suggested that not all ACF are early indications of colon cancers and only a specific type of ACF (dark ACF) may be used for diagnosis and evaluation of anticancer abilities of chemicals and food additives. Furthermore, our results also showed that the key proteins in the Wnt signal transduction pathway were abnormally expressed suggesting that the activation of Wnt pathway exists in the stage of ACF during the cancer development. Future studies will be focused on the pathological changes and activation of signal transduction pathways during earlier stages of ACF.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>To evaluate the relationship between Aberrant Crypt Foci (ACF) and tumorigenesis, we observed the sequential development from ACF to tumor in the colon of azoxymethane-exposed wistar rats.</p>", "<title>Methods</title>", "<p>Sixty wistar rats were sacrificed at different time points after exposure to azoxymethane and the colons were stained with methylene blue for stereomicroscopic analysis.</p>", "<title>Results</title>", "<p>We found two types of early lesions: classic ACF and dark ACF. Dark ACF were characterized by dark blue staining, mildly enlarged or small compressed crypts that are not elevated from the surrounding epithelium. Large dark ACF and nascent tumors displayed the same surface morphology. Furthermore, dark ACF grew significantly faster than classic ACF and showed dysplasia without hyperplasia. In contrast, classic ACF showed hyperplasia without dysplasia. Dark ACF has significant higher expression rate of β-catenin (100%) and MMP-7 (81.82%) compared with the expression of β-catenin and MMP-7 in classic ACF (4.84% and 7.87%, respectively).</p>", "<title>Conclusion</title>", "<p>Our data indicated that dark ACF is closely related to tumorigenesis while classic ACF is not. Furthermore, Wnt signal pathway was activated during the early period of dark ACF.</p>" ]
[ "<title>Abbreviations</title>", "<p>ACF: aberrant crypt foci; AOM: azoxymethane.</p>", "<title>Authors' contributions</title>", "<p>LQ: Principal investigator and Experimental Designer. JB: Department head and Experimental Designer. CL, TS: Technician.</p>" ]
[ "<title>Acknowledgements</title>", "<p>We thank Prof. Zhang Hongquan and Dr. Wang Xinyin for their excellent technical assistance.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Morphological observations of classic ACF, dark ACF and tumors</bold>. Morphological observations of classic ACF, dark ACF and tumors. Lesions were stained with methylene blue and examined with the stereomicroscope (A, D, ×20); black arrowheads represent classic ACF and white arrowheads represent dark ACF. Histological observation (B, C, E) are derived from the same lesions as seen in A and D. Classic ACF showed hyperplasia without dysplasia (B, ×100); dark ACF showed moderate dysplasia (C, ×100); small tumor showed severe dysplasia (E, ×100).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Immunohistochemical analyses for the expression of β-catenin and mmp-7 in classic ACF, dark ACF and histological sections of lesions</bold>. Immunohistochemical analyses for the expression of β-catenin and mmp-7 in classic ACF, dark ACF and histological sections of lesions. The expression of β-catenin (A) and mmp-7 (B) is not altered in classic ACF. The expression of β-catenin (C) and mmp-7 (D) is altered in dark ACF. Magnification: ×100.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Histopathologic examination of classic ACF, dark ACF, and tumor</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">Hyperplasia without dysplasia</td><td align=\"center\" colspan=\"3\">Mild dysplasia</td><td align=\"center\" colspan=\"3\">Morderate dysplasia</td><td align=\"center\" colspan=\"3\">Severe dysplasia</td></tr><tr><td colspan=\"1\"><hr/></td><td/><td colspan=\"9\"><hr/></td></tr><tr><td/><td/><td align=\"left\">W8-14</td><td align=\"left\">W16-21</td><td align=\"left\">W22-25</td><td align=\"left\">W8-14</td><td align=\"left\">W16-21</td><td align=\"left\">W22-25</td><td align=\"left\">W8-14</td><td align=\"left\">W16-21</td><td align=\"left\">W22-25</td></tr></thead><tbody><tr><td align=\"left\">Classic ACF</td><td align=\"left\">165</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td></tr><tr><td align=\"left\">Dark ACF</td><td align=\"left\">0</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">2</td><td align=\"left\">7</td><td align=\"left\">2</td><td align=\"left\">0</td><td align=\"left\">4</td><td align=\"left\">6</td></tr><tr><td align=\"left\">tumor</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">3</td><td align=\"left\">0</td><td align=\"left\">1</td><td align=\"left\">6</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Crypt multiplicity of classic ACF, dark ACF, and tumors (mean ± SD)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"3\">crypt multiplicity (No. of crypts/lesion/group)</td><td/><td/></tr><tr><td colspan=\"4\"><hr/></td><td/><td/></tr><tr><td/><td align=\"left\">W8-14</td><td align=\"left\">W16-21</td><td align=\"left\">W22-25</td><td align=\"left\">F</td><td align=\"left\">P</td></tr></thead><tbody><tr><td align=\"left\">Classic ACF</td><td align=\"left\">3.93 ± 0.89</td><td align=\"left\">4.73 ± 1.12</td><td align=\"left\">4.52 ± 1.03</td><td align=\"left\">3.17</td><td align=\"left\">0.278</td></tr><tr><td align=\"left\">Dark ACF</td><td align=\"left\">6.00 ± 3.00</td><td align=\"left\">10.73 ± 4.03</td><td align=\"left\">18.38 ± 3.54</td><td align=\"left\">15.40</td><td align=\"left\">0.000</td></tr><tr><td align=\"left\">t</td><td align=\"left\">3.330</td><td align=\"left\">9.883</td><td align=\"left\">11.204</td><td/><td/></tr><tr><td align=\"left\">P</td><td align=\"left\">0.002</td><td align=\"left\">0.000</td><td align=\"left\">0.000</td><td/><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Immunohistochemical analysis for the expression of β-catenin, mmp-7 in classic ACF, dark ACF and tumor</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"4\">β-catenin</td><td align=\"center\" colspan=\"2\">mmp-7</td></tr></thead><tbody><tr><td/><td align=\"left\">No. of normal samples/total No. of samples</td><td align=\"left\">No. of samples with reduced expression in membrane/total No. of samples</td><td align=\"left\">No. of samples with overexpression in cytoplasm/total No. of samples</td><td align=\"left\">No. of samples with accumulated expression in nucleus/total No. of samples</td><td align=\"left\">No. of samples with mmp-7 expression/total No. of samples</td><td align=\"left\">No. of samples without mmp-7 expression/total No. of samples</td></tr><tr><td colspan=\"7\"><hr/></td></tr><tr><td align=\"left\">Classic ACF</td><td align=\"left\">157/165</td><td align=\"left\">4/165</td><td align=\"left\">4/165</td><td align=\"left\">0/165</td><td align=\"left\">13/165</td><td align=\"left\">152/165</td></tr><tr><td align=\"left\">Dark ACF</td><td align=\"left\">0/22</td><td align=\"left\">6/22</td><td align=\"left\">8/22</td><td align=\"left\">8/22</td><td align=\"left\">18/22</td><td align=\"left\">4/22</td></tr><tr><td align=\"left\">Tumor</td><td align=\"left\">0/10</td><td align=\"left\">0/10</td><td align=\"left\">3/10</td><td align=\"left\">7/10</td><td align=\"left\">10/10</td><td align=\"left\">0/10</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Expression of β-catenin in dark ACF at different time point</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\" colspan=\"3\">β-catenin expression</td></tr></thead><tbody><tr><td/><td align=\"left\">No. of samples with reduced expression in membrane/total No. of samples</td><td align=\"left\">No. of samples with overexpression in cytoplasm/total No. of samples</td><td align=\"left\">No. of samples with accumulated expression in nucleus/total No. of samples</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\">Wk8-14</td><td align=\"left\">3/3</td><td align=\"left\">0/3</td><td align=\"left\">0/3</td></tr><tr><td align=\"left\">Wk16-21</td><td align=\"left\">3/11</td><td align=\"left\">5/11</td><td align=\"left\">3/11</td></tr><tr><td align=\"left\">Wk22-25</td><td align=\"left\">0/8</td><td align=\"left\">3/8</td><td align=\"left\">5/8</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><p>Note: No. rats (n) used at each time point after AOM treatment: Wk8, 10, 12, 14, 16, 18, 20, 21,22, 23, 24, 25 = 5, respectively.</p></table-wrap-foot>", "<table-wrap-foot><p>Note: The rate of ectopic expression of β-catenin in classic ACF is significantly lower than that in tumors (<italic>P </italic>= 0.0023). The rate of mmp-7 expression in classic ACF is significantly lower than that in tumors (<italic>P </italic>= 0.000). The rate of β-catenin ectopic expression and positive expression of mmp-7 in dark ACF is not significantly different with those in tumor (<italic>P </italic>= 0.143 and <italic>P </italic>= 0.283, respectively). The rate of ectopic expression of β-catenin in dark ACF is significantly higher than that in classic ACF (<italic>P </italic>= 0.000). The rate of mmp-7 expression in dark ACF is significantly higher than that in classic ACF (<italic>P </italic>= 0.000).</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1756-9966-27-26-1\"/>", "<graphic xlink:href=\"1756-9966-27-26-2\"/>" ]
[]
[{"surname": ["Bird", "Good"], "given-names": ["PR", "CK"], "article-title": ["The significance of aberrant cryptfoci in understanding the pathogenesis of colon cancer"], "source": ["Toxicol Lett"], "year": ["2000"], "volume": ["113"], "fpage": ["295"], "lpage": ["402"]}, {"surname": ["Gregorieff", "Clevers"], "given-names": ["Alex", "Hans"], "suffix": ["1"], "article-title": ["Wnt signaling in the intestinal epithelium: from endoderm to cancer"], "source": ["GENES & DEVE"], "year": ["2005"], "volume": ["19"], "fpage": ["877"], "lpage": ["90"]}, {"surname": ["Giles", "van Es", "Clevers"], "given-names": ["RH", "JH", "H"], "article-title": ["Caught up in aWnt storm: Wnt signaling in cancer"], "source": ["Biochim Biophys Acta"], "year": ["2003"], "volume": ["1653"], "issue": ["Suppl 1"], "fpage": ["12"], "lpage": ["24"]}, {"surname": ["jie", "chu"], "given-names": ["DUAN Guang", "YAN Xiao"], "article-title": ["The signif icance of \u03b2-catenin and matrix metalloproteinase-7 expression in colorectal adenoma and carcinoma"], "source": ["Chin J Pathol"], "year": ["2004"], "volume": ["33"], "issue": ["Suppl 6"], "fpage": ["518"], "lpage": ["522"]}]
{ "acronym": [], "definition": [] }
24
CC BY
no
2022-01-12 14:47:25
J Exp Clin Cancer Res. 2008 Aug 5; 27(1):26
oa_package/6a/38/PMC2529269.tar.gz
PMC2529270
18652658
[ "<title>Background</title>", "<p>Hypertension is the leading cause of morbidity and mortality worldwide [##REF##12493255##1##]. The concomitant manifestation of type 2 diabetes mellitus leads to a substantial further increase in risk [##UREF##0##2##,##REF##12788299##3##]. While about 50% of patients in German primary care were hypertensive in a recent cross-sectional survey, 12% of all patients had a co-manifestation of hypertension and diabetes [##UREF##1##4##].</p>", "<p>Not only hypertensive patients with diabetes, but also hypertensive patients without diabetes tend to be resistant to insulin stimulated glucose uptake and are hyperinsulinaemic compared with normotensive controls [##REF##2405235##5##]. About 20% of patients with hypertension will develop type 2 diabetes in a three year period [##REF##16980380##6##] and new onset diabetes in treated hypertensive patients is not trivial as recent studies suggest [##REF##17101936##7##,##REF##14988289##8##].</p>", "<p>The risk for subsequent cardiovascular (CV) disease in patients with pre-diabetes is not different from those who had both hypertension and diabetes already at baseline [##REF##15037557##9##]. The adjusted relative risk of events was about 3-times higher in both previous and new onset diabetes compared to patients with hypertension but without diabetes [##REF##15037557##9##].</p>", "<title>Antihypertensive drugs and new-onset diabetes</title>", "<p>The roles of antihypertensive agents and in particular those that inhibit the RAS in the acceleration or deceleration of diabetes manifestation have been discussed controversial and study results on this question are not consistent.</p>", "<p>The RAS itself plays a pivotal role in the development of diabetes. Over-activity appears to be linked to reduced insulin and glucose delivery to the peripheral skeletal muscle and impaired glucose transport and response to insulin signalling pathways, thus increasing insulin resistance [##REF##15716683##10##]. Activation of a local pancreatic RAS, in particular within the islets, may represent an independent mechanism for the progression of islet cell damage in diabetes. In fact, impaired pancreatic islet function may predominate quantitatively over peripheral insulin resistance in impaired glucose tolerance [##REF##12879253##11##].</p>", "<p>Drugs that interrupt the RAS like angiotensin converting enzyme inhibitors (ACEi) and angiotensin receptors blockers (ARBs) are likely to be beneficial in the prevention of diabetes [##REF##15716683##10##,##REF##16397519##12##]. A series of recent large-scale prospective randomised studies of 3–6 year duration such as CAPP, INSIGHT, LIFE or ALLHAT, reported a remarkably consistent reduction in the incidence of type 2 diabetes in hypertensive patients reported with either ACEi-based or ARB-based therapy (reviewed by Jandeleit-Dahm in [##REF##15716683##10##]). The comparator groups were based on thiazide-diuretics, β-blockers, the calcium channel blocker amlodipine or placebo, respectively.</p>", "<p>In a large meta-analyis, Abuissa et al. calculated the average risk reduction in 6 of these trials using ACEi and 7 trials using ARBs. The reduction of new onset-diabetes was 24% for ACEi, 23% for ARBs and 23% for the combined data-set [##UREF##2##13##]. Furthermore a recent network meta-analysis of randomized controlled trials showed that while patients taking betablockers and diuretics show an increased incidence of diabetes, it is reduced in patients using ACEi or ARBs (Figure ##FIG##0##1##) [##REF##17482972##14##,##REF##17240286##15##].</p>", "<p>Endpoint studies to elucidate the role of antihypertensive agents on new onset diabetes related morbidity and mortality are however scarce. The VALUE trial with valsartan was the only trial to include new-onset diabetes as a pre-specified endpoint [##REF##15207952##16##]. Patients were normoglycemic, those with abnormal glucose values were excluded. While 16.4% of patients in the amlodipine arm (up to 10 mg) developed diabetes over a mean follow-up of 4.2 years, 13.1% developed such in the valsartan arm (up to 160 mg); p &lt; 0.0001. The ASCOT-BPLA study, which was a randomised controlled trial of the prevention of CHD and other vascular events by BP and cholesterol lowering in a factorial study design, was prematurely stopped in December 2004 [##REF##16154016##17##,##REF##16154017##18##]. The study was designed to resolve whether newer antihypertensive strategies that use calcium channel blockers (CCBs) and ACEi are superior to older treatments using betablockers and diuretics. As a key finding there was a substantial excess of new diabetes (increase of 30%) in the beta blocker/diuretic arm [##REF##16154016##17##]. DREAM investigated the effect of ramipril (up to 15 mg) compared to placebo [##REF##16980380##6##]. In this randomised controlled trial ramipril significantly increased regression to normoglycemia in patients with impaired glucose tolerance. It did however not influence the risk of a combined endpoint consisting of new-onset diabetes or death over a 3 year observational period. Interpretation of DREAM is limited by a number of details: 1) hypertension was not an inclusion criterion (mean blood pressure at baseline 136/83 mmHg), 2) comparison was made to placebo instead of diuretics or betablockers (which would be reasonable based on the analysis of Elliott [##REF##17240286##15##]) and 3) betablockers were allowed in both the ramipril and placebo groups.</p>", "<title>Rationale for ADaPT</title>", "<p>Thus, despite the strong evidence for a reduction of new-onset diabetes from several RCTs and meta-analyses, there is an ongoing controversy about the clinical significance, the comparability of agents within one drug class, or the generalisability of these findings into clinical practice [##REF##14767003##19##, ####REF##12584373##20##, ##REF##15983290##21####15983290##21##]. The \"<italic>ACE inhibitor-based versus diuretic-based antihypertensive primary treatment in patients with prediabetes</italic>\" (ADaPT) study addresses this issue. On the basis of the existing body of evidence, it appeared likely that patients with impaired fasting glucose (IGF) according to the screening on pre-diabetes (PreDiSc Score) will benefit from tight blood pressure control and further effects from RAS-inhibition by the ACEi ramipril in terms of manifestation of type 2 diabetes. The long-term outcomes of this treatment regimen will be compared to a regimen based on diuretics and/or β-blockers.</p>" ]
[ "<title>Materials and methods</title>", "<title>Design</title>", "<p>The ADaPT investigation is a comparative Post Marketing Surveillance according to §67(6) German Drug Law, performed by the German Hypertension League. It was designed as an open, prospective, non-randomised parallel group observational investigation in 150 general practices (general physicians and internists) throughout Germany.</p>", "<title>Patient population</title>", "<title>Inclusion criteria</title>", "<p>Patients eligible for this study were at high risk for the development of type 2 diabetes according to the modified <italic>PreDiSc </italic>Score [##REF##15924589##22##]: They had to be ≥ 45 year old (amended, original protocol ≥ 55 years), have hypertension (systolic blood pressure ≥ 140 and/or diastolic blood pressure ≥ 90 mmHg), impaired fasting glucose (IFG) defined as glucose level 110–125 mg/dl in venous plasma or 100–109 mg/dl in capillary whole blood, and a glycosylated haemoglobin A1c (HbA1c) of 6–6.5% determined within the last six months.</p>", "<title>Exclusion criteria</title>", "<p>Patients were excluded if they received any antidiabetic drug treatment, had overt type 2 diabetes, fasting blood glucose level ≥ 126 mg/dl in venous plasma or ≥ 110 mg/dl in capillary blood, or casual plasma glucose concentrations ≥ 200 mg/dl, congestive heart failure, chronic renal insufficiency, history of myocardial infarction, stroke, drug or alcohol abuse, or contraindications against one of the drugs applied.</p>", "<title>Definitions</title>", "<p>For definitions of normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and overt diabetes (DM) see Table ##TAB##0##1##. <italic>PreDiSc </italic>Score: The score indicates the presence of pre-diabetes with a diagnostic sensivity of 78% and a specificity of 37% using the following parameters: blood pressure (BP) ≥ 140/90 mmHg, capillary fasting blood glucose ≥ 100 mg/dl (STIX) and age ≥ 55 years. Sensivity can be increased to 79% and specificity to 74% by additional determination of the HbA1c value (≥ 6%) [##REF##15924589##22##].</p>", "<title>Antihypertensive treatment</title>", "<p>Patients in <bold>Group 1 </bold>receive Ramipril either as monotherapy (Delix<sup>®</sup>, Sanofi-Aventis, Berlin) or in combination with Felodipin (Delmuno<sup>®</sup>, Sanofi-Aventis, Berlin), patients in <bold>Group 2 </bold>received any other diuretic-based or β-blocker-based therapy without using ACEi or ARBs. Ramipril was chosen as the antihypertensive drug in one group, as long-standing experience from several clinical studies including large endpoint studies have accumulated with this agent [##REF##12076194##23##, ####REF##7774515##24##, ##REF##8879894##25####8879894##25##]. The Heart Outcomes Prevention Evaluation (HOPE) Study showed that ramipril is effective in preventing major cardiovascular events in high-risk patients without hypertension or those whose hypertension was sufficiently controlled with other treatments [##REF##10639539##26##].</p>", "<p>Generally, treatment regimens in this study can be chosen in accordance with the recommendations of the German Hypertension League and the European Society of Hypertension [##UREF##3##27##,##REF##17563527##28##] For initial treatment monotherapy or a low-dose combination regimen is suggested. If the response is inadequate, possible options include increasing the dose, changing the drug or introduction of further combination drugs [##UREF##4##29##]. All drugs are administered within the approved labelling.</p>", "<p>Advice about the investigation has been obtained by the institutional review board of the Charité Berlin, Germany – Campus Benjamin-Franklin. Written informed consent was obtained from every patient in writing. The planned follow-up period is four years.</p>", "<title>Endpoints</title>", "<p>The primary evaluation criterion of this observational study is the first manifestation of type 2 diabetes according to the current guidelines of the German Diabetes Society (see table ##TAB##0##1##). [##UREF##5##30##] Further evaluation criteria are the deterioration of pre-diabetes indicated by an increase of HbA1c of at least 10% compared to baseline value within 4 years, the initiation of antidiabetic glucose lowering medication, an increase of fasting glucose levels, change of HbA1c compared to baseline, BP reduction, achievement of the target BP &lt;130/80 mmHg after 12 months and at the 4 year follow-up, time needed to reach target BP, major cardiovascular (CV) events (first manifestation of symptomatic coronary heart disease (CHD) and/or peripheral arterial occlusive disease and/or cerebrovascular events), type and frequency of Adverse Events (AE) or Serious Adverse Events (SAE), and total mortality. In the diuretic-based therapy group, time to switch to ACE-based or ARB-based therapy will also be analysed.</p>", "<title>Investigational plan</title>", "<p>Table ##TAB##1##2## summarizes the investigational plan. Patients are seen at 7 scheduled visits. Vital signs (BP, heart rate) will be complemented by height and weight measurements (determinations of body mass index) and waist and hip circumference measurement at after 3, 6 and 12 months and thereafter at yearly intervals. Further, lab examinations of glucose, lipids, inflammatory (high sensitive C-reactive protein) and renal parameters (with cystatin C to assess renal function [##UREF##5##30##,##REF##15901858##31##]) will be done in the same intervals. Ambulatory BP monitoring is facultative and will be performed in a subset of patients. AEs will be recorded and their severity, course and relation to the medication assessed by the treating physician. Prior as well as concomitant diseases and concomitant medication will also be assessed.</p>", "<title>Statistical Assumptions and sample size calculation</title>", "<p>Sample size calculation for the primary endpoint was made under the assumption – based on the results of the ALLHAT study – that during the 4-year observational period 10.0% of patients in the ramipril-based antihypertensive regimen and 14.3% in the diuretic-based regimen will develop overt diabetes mellitus [##REF##15983290##21##]. The detectable risk increase is compatible with 80% power and significance of 0.05 is 49% (OR 1,489). Based on this assumption, a sample size of <italic>n </italic>= 2001 was required. In terms of randomisation, addressing the literature evidence in favour of ACE inhibitor treatment, an imbalanced ratio of 2:1 for the number of patients in both treatment groups was chosen, resulting in a target inclusion number of 1334 patients in group 1 and 667 patients in group 2, respectively.</p>", "<title>Statistical analyses</title>", "<p>The following parameters will be analysed: patient demographics including patient history, capillary und venous fasting glucose, HbA1c, BP, BMI, waist/hip ratio, concomitant medications, percentage of patients with prediabetes according to PreDiSc parameters [##REF##15924589##22##], total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, urinary albumin, and high-sensitive C-reactive protein.</p>", "<p>Patients treated with ramipril (and ramipril-based combination therapy) and patients who received various other antihypertensive drugs (with the exception of ACEi or ARBs) will be compared. Statistical analyses of the data will be performed as exploratory analyses. Descriptive statistics for continuous target data per treatment group and per total contain the following: number of patients, means ± standard deviation, median, minimum and maximum. The absolute and relative frequency in percentages will be determined. Per treatment group 95% confidence intervals for the means of continuous target data as well as for the relative frequency of categorical target data will be calculated using appropriate methods.</p>", "<p>For comparison of the treatment groups with respect to the incidence of specific events (e.g. patients with first manifestation of diabetes mellitus type 2 or proportion of patients with deterioration of pre-diabetes), the chi-square or the log-rank tests will be used to compare the \"survival curves\". An interim analysis is scheduled after the first and second year of the observation. However, the overall significance levels will not be adjusted.</p>", "<title>Baseline Characteristics</title>", "<p>Enrolment in AdaPT started in August 2004. The last patient out of a total of 2,015 patients was included in March 2006 (table ##TAB##2##3##). 1,353 patients were enrolled into the ACEi-based group and 662 patients got a diuretic-based therapy. While age was similar between both groups the ACEi-based group had more male patients (51.4 vs. 42.6%). BMI (29.9 ± 5.0 vs. 29.8 ± 4.8 kg/m<sup>2</sup>) and waist-to-hip-ratio (0.95 ± 0.1 vs. 0.93 ± 0.1) were almost identical in both study groups. Dyslipidemia (56.5%), hypertension (57.8%) and overweight (43.3%) and obesity (42.6%) were the frequent baseline characteristics of patients in both groups. Baseline characteristics of AdaPT are displayed in table ##TAB##2##3## and compared to other recent studies on the incidence of diabetes being treated with RAS blocking agents vs. conventional drugs.</p>" ]
[]
[ "<title>Discussion</title>", "<p>Although there are several trials with a RAS based pharmacotherapy that report a reduction in the development of diabetes compared to diuretics and betablockers, these analyses were mostly post-hoc and not predefined. The only trials with a pre-defined new-onset diabetes endpoint were ASCOT-BPLA [##REF##16154016##17##,##REF##16154017##18##], VALUE [##REF##15207952##16##] and DREAM [##REF##16980380##6##] (new-onset diabetes as part of the primary endpoint). While there was a significant reduction of new-onset diabetes in ASCOT-BLPA (HR 0.70 [95%CI 0.63–0.78]) and VALUE (HR 0.77 [95%CI 0.69–0.86]) there was none in DREAM (HR 0.91 [95%CI 0.80–1.03]). Study duration was 5.5 (median), 4.2 (mean) and 3.0 (median) years. Within this setting the trial with the longest study duration had the most pronounced effect on diabetes development (see table ##TAB##3##4##).</p>", "<p>Inclusion and exclusion criteria were also substantially different between ASCOT-BPLA, VALUE and DREAM. ASCOT-BPLA and VALUE included patients with treated or newly diagnosed hypertension being at least 40 years (ASCOT-BLPA) or 50 years old (VALUE). Neither impaired fasting plasma glucose nor glucose tolerance was an inclusion criterion. DREAM on the other hand included patients 30 years and above with either impaired fasting plasma glucose or glucose tolerance. Diagnosis of hypertension was not required.</p>", "<title>What is the additional value of AdaPT ?</title>", "<p>The primary goal of AdaPT is to compare the effects of two antihypertensive combination therapies, an ACE inhibitor based treatment with a diuretic- (or betablocker)-based treatment on the incidence of new-onset of type 2 diabetes. To provide adequate power to discriminate a potential differential effect of these therapies, the trial is being conducted in pre-diabetic patients with hypertension and metabolic disorders in which there is a high probability for the development of diabetes.</p>", "<p>AdaPT is conducted as an observational study in daily practice in Germany allowing for the widest possible applicability of the results obtained. This is important because there is considerable heterogeneity in patient management in daily practice and patients with pre-diabetes in clinical trials usually differ substantially from those in clinical trials. Taking the <italic>PreDiSc </italic>Score as a screening algorithm, the study allows physicians to effectively screen for a high risk for the development of diabetes. This would, in case of positive results, serve as an easy screening tool for high risk patients in the future.</p>", "<p>Compared to the previously mentioned trials AdaPT included patients at a higher risk for the development of diabetes based on the selection criteria age, presence of hypertension, an impaired fasting glucose and the missing exclusion of prior cardiovascular disease. As opposed to DREAM (Placebo control) AdaPT includes patients treated with betablockers or diuretics as a control group. This further enhances the likelihood of a differential effect based on the previously mentioned data by Abuissa [##REF##16139131##32##], Elliott [##REF##17240286##15##] and Lam [##REF##17482972##14##] (see also Figure ##FIG##0##1##). Together with the 4 year follow-up it appears likely that AdaPT may document a reduced incidence of diabetes.</p>", "<title>The PreDiSc Score</title>", "<p>The fact that a score (PreDiSc) is applied in the ADaPT investigation to prospectively identify eligible patients represents a novel approach, since it shifts the conventional focus from individual risk factors (hypertension, dyslipidaemia) to a more comprehensive view that considers absolute patient risk [##REF##12788299##3##,##REF##10710556##33##]. To our knowledge, such an approach has only been pursued in retrospective post-hoc investigations, for example in a current analysis of the LIFE study [##UREF##6##34##].</p>", "<p>The oral glucose tolerance test (OGTT) is the standard screening test in high risk populations (identified by medical history), but the fasting plasma glucose test is more convenient under daily practice conditions. [##UREF##7##35##] Determining fasting plasma glucose lacks sensitivity however and may miss a number of patients with diabetes. Another possible variable to determine glycaemic control is HbA1c but is less suitable for a general screening. [##REF##9539993##36##,##UREF##8##37##]</p>", "<p>Against this background, the Pre-Diabetes Score (PreDiSc) Study established a set of easy-to-determine clinical and/or laboratory parameters with close correlation to the outcomes of an OGTT [##REF##15924589##22##]. Indeed, using the HbA1c alone yielded low sensitivity (58%) as did fasting glucose alone (62%). However, a combination of age ≥ 55 years, systolic BP of ≥ 140 mmHg, fasting glucose ≥ 110 – 126 mg/dl, elevated HbA1c ≥ 6% and abdominal obesity (waist circumference &gt; 88 cm in women and 102 cm in men) was associated with high sensitivity of IGT (i.e. identification of individuals with pre-diabetes: 79%) as well as high specificity (i.e. exclusion of individuals without pre-diabetes: 74%) [##REF##15924589##22##]. The low acceptance of the OGTT and the non satisfying sensitivity and/or specificity of the sole HbA1c determination speak against these parameters as inclusion criteria for studies in daily practice. In contrast, the satisfactory predictive value of the clinically easy to determine dataset evaluated in PreDiSc was the rationale to use this score as screening procedure in a prospective study. Hypertensive patients fulfilling the PreDiSc criteria have a very high likelihood to progress to overt diabetes. Compared to the original PreDiSc score, on the basis of practical experiences in the initiation phase of the study two amendments to the ADaPT protocol became necessary: first, HbA<sub>1c </sub>was to be measured in a central laboratory instead of the originally foreseen local laboratories (due to wide variation in locally determined values). Second, the age criterion was reduced to 45 years or older in order to accelerate the inclusion rate.</p>", "<title>Strength and limitations</title>", "<p>Certain limitations of the investigation have to be taken into account: first, it is controlled, but not randomised. This means that the comparability of both cohorts can be assessed retrospectively in terms of known factors that may influence the outcomes (e.g. age, gender, comorbidity), but not in terms of unknown bias. Second, the majority of patients will require combination therapy of two, three or even more antihypertensive drugs of various classes. At clinical practice conditions, a substantial proportion of patients may receive \"unallowed\" medications in the course of the investigation (i.e., from the regimen of the other arm). On the other hand, the real practice conditions in this investigation convey substantial benefits that will extend the knowledge from randomised studies. Patients will be less selected than typical study patients. The dosing regimens and the combinations will reflect current use (e.g. lower doses as compared to the US for some drugs, inclusion of ARBs, etc.) and thus the results can be easily extrapolated to day-to-day clinical use.</p>" ]
[ "<title>Conclusion</title>", "<p>Although there are some published RCTs on the development of diabetes in patients on antihypertensive drugs, the results are inconclusive and require further investigation. The ADaPT study will provide important clinical data in a group of patients being at high risk to develop diabetes for which clear guideline recommendations regarding choice of antihypertensives are still missing.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Recent clinical trials reported conflicting results on the reduction of new-onset diabetes using RAS blocking agents. Therefore the role of these agents in preventing diabetes is still not well defined. Ramipril is an ACE inhibitor (ACEi), that has been shown to reduce cardiovascular events in high risk patients and post-hoc analyses of the HOPE trial have provided evidence for its beneficial action in the prevention of diabetes.</p>", "<title>Methods</title>", "<p>The ADaPT investigation (\"ACE inhibitor-based versus diuretic-based antihypertensive primary treatment in patients with pre-diabetes\") is a 4-year open, prospective, parallel group phase IV study. It compares an antihypertensive treatment regimen based on ramipril versus a treatment based on diuretics or betablockers. The primary evaluation criterion is the first manifestation of type 2 diabetes. The study is conducted in primary care to allow the broadest possible application of its results. The present article provides an outline of the rationale, the design and baseline characteristics of AdaPT and compares these to previous studies including ASCOT-BLPA, VALUE and DREAM.</p>", "<title>Results</title>", "<p>Until March 2006 a total of 2,015 patients in 150 general practices (general physicians and internists) throughout Germany were enrolled. The average age of patients enrolled was 67.1 ± 10.3 years, with 47% being male and a BMI of 29.9 ± 5.0 kg/m<sup>2</sup>. Dyslipidemia was present in 56.5%. 37.8% reported a family history of diabetes, 57.8% were previously diagnosed with hypertension (usually long standing). The HbA1c value at baseline was 5.6 %. Compared to the DREAM study patients were older, had more frequently hypertension and patients with cardiovascular disease were not excluded.</p>", "<title>Conclusion</title>", "<p>Comparing the ADaPT design and baseline data to previous randomized controlled trial it can be acknowledged that AdaPT included patients with a high risk for diabetes development. Results are expected to be available in 2010. Data will be highly valuable for clinical practice due to the observational study design.</p>" ]
[ "<title>Conflict of interest</title>", "<p>Dr. Paar declares to be an employee of Sanofi-Aventis Germany. All other authors have attended advisory boards and have held lectures for a number of pharmaceutical companies including Sanofi-Aventis.</p>", "<title>Authors' contributions</title>", "<p>All authors have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. PB has drafted the manuscript. The other authors revised the manuscript for important intellectual content. All authors have given final approval of the version to be published.</p>" ]
[ "<title>Acknowledgements</title>", "<p>The AdaPT investigation is a project of the Germany Hypertension League. It is supported by an unrestricted educational grant by Sanofi-Aventis Germany, Berlin. Committee chair: Prof. Dr. Zidek, Berlin. Committee members: Prof. Dr. Schrader, Cloppenburg, Prof. Dr. Matthaei, Quakenbrück, Prof. Dr. Hasslacher, Heidelberg, Prof. Dr. Hoyer, Marburg, PD Dr. Paar, Berlin (without voting right).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Diabetes incidence – results of full Bayesian network meta-analysis of 22 trials with 143153 patients [##REF##17482972##14##], modified from [##REF##17240286##15##].</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Values of plasma glucose (venous blood) for the diagnosis of diabetes mellitus* and other categories of hyperglycemia according to DDG criteria [##UREF##5##30##]</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td/><td align=\"center\" colspan=\"3\"><bold>mg/dl</bold></td><td align=\"center\" colspan=\"3\"><bold>mmol/l</bold></td></tr></thead><tbody><tr><td/><td/><td align=\"left\"><bold>Fasting</bold></td><td/><td align=\"left\"><bold>2 h OGTT</bold></td><td align=\"left\"><bold>Fasting</bold></td><td/><td align=\"left\"><bold>2 h OGTT</bold></td></tr><tr><td colspan=\"8\"><hr/></td></tr><tr><td align=\"left\"><bold>NGT</bold></td><td align=\"left\"><bold>Normal glucose tolerance</bold></td><td align=\"left\">&lt; 100</td><td/><td align=\"left\">&lt; 140</td><td align=\"left\">&lt; 5.6</td><td/><td align=\"left\">&lt; 7.8</td></tr><tr><td align=\"left\"><bold>IFG</bold></td><td align=\"left\"><bold>Impaired fasting glucose</bold></td><td align=\"left\">100–125</td><td/><td align=\"left\">-</td><td align=\"left\">5.6–6.9</td><td/><td align=\"left\">-</td></tr><tr><td align=\"left\"><bold>IGT</bold></td><td align=\"left\"><bold>Impaired glucose tolerance</bold></td><td align=\"left\">&lt; 126</td><td align=\"left\">and</td><td align=\"left\">140–199</td><td align=\"left\">&lt; 7.0</td><td align=\"left\">and</td><td align=\"left\">7.8–11.0</td></tr><tr><td align=\"left\"><bold>DM</bold></td><td align=\"left\"><bold>Diabetes mellitus</bold></td><td align=\"left\">≥ 126</td><td align=\"left\">and/or</td><td align=\"left\">≥ 200</td><td align=\"left\">≥ 7.0</td><td align=\"left\">and/or</td><td align=\"left\">≥ 11.1</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>ADaPT study plan</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Documentation/Investigation</bold></td><td align=\"center\"><bold>Baseline</bold></td><td align=\"center\"><bold>12 wk</bold></td><td align=\"center\"><bold>6 mo</bold></td><td align=\"center\"><bold>1 yr</bold></td><td align=\"center\"><bold>2 yr</bold></td><td align=\"center\"><bold>3 yr</bold></td><td align=\"center\"><bold>4 yr</bold></td></tr></thead><tbody><tr><td align=\"left\">Clinical examination and medical history</td><td align=\"center\">x</td><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\">Information on data protection</td><td align=\"center\">x</td><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\">Blood pressure, heart rate</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td></tr><tr><td align=\"left\">Physical examination: height, weight, waist and hip circumference</td><td align=\"center\">x</td><td/><td/><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td></tr><tr><td align=\"left\">Laboratory screening (central laboratory): Blood glucose, HbA1c</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td></tr><tr><td align=\"left\">Laboratory values: hsCRP, total cholesterol, LDL-C, HDL-C, TG, cystatin C, potassium, albumin, OGTT</td><td align=\"center\">x</td><td/><td/><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td></tr><tr><td align=\"left\">ABPM (optional)</td><td align=\"center\">x</td><td/><td/><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td></tr><tr><td align=\"left\">AE, SAE</td><td/><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td><td align=\"center\">x</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Baseline characteristics of the enrolled patients in AdaPT-study – compared to other trials</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\"><bold>VALUE </bold>[##REF##15207952##16##]</td><td align=\"center\" colspan=\"2\"><bold>ASCOT-BPLA </bold>[##REF##16154016##17##]</td><td align=\"center\" colspan=\"2\"><bold>DREAM </bold>[##REF##16980380##6##,##REF##15322749##38##]</td><td align=\"center\" colspan=\"2\"><bold>AdaPT</bold></td></tr></thead><tbody><tr><td/><td align=\"center\"><bold>Valsartan</bold></td><td align=\"center\"><bold>Amlodipine</bold></td><td align=\"center\"><bold>Amlodipine</bold></td><td align=\"center\"><bold>Atenolol</bold></td><td align=\"center\"><bold>Ramipril</bold></td><td align=\"center\"><bold>Placebo</bold></td><td align=\"center\"><bold>ACEi</bold></td><td align=\"center\"><bold>Diuretic</bold></td></tr><tr><td/><td align=\"center\"><bold>(n = 7649)</bold></td><td align=\"center\"><bold>(n = 7596)</bold></td><td align=\"center\"><bold>(n = 9639)</bold></td><td align=\"center\"><bold>(n = 9618)</bold></td><td align=\"center\"><bold>(n = 2.623)</bold></td><td align=\"center\"><bold>(n = 2.646)</bold></td><td align=\"center\"><bold>(n = 1.353)</bold></td><td align=\"center\"><bold>(n = 662)</bold></td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"left\"><bold>Age (mean, years)</bold></td><td align=\"center\">67.2 ± 8.2</td><td align=\"center\">67.3 ± 8.1</td><td align=\"center\">63.0 ± 8.5</td><td align=\"center\">63.0 ± 8.5</td><td align=\"center\">54.7 ± 10.9</td><td align=\"center\">54.7 ± 10.9</td><td align=\"center\">67.3 ± 10.4</td><td align=\"center\">66.5 ± 10.2</td></tr><tr><td align=\"left\"><bold>Male gender (%)</bold></td><td align=\"center\">57.6</td><td align=\"center\">57.5</td><td align=\"center\">77</td><td align=\"center\">77</td><td align=\"center\">40.3</td><td align=\"center\">41.3</td><td align=\"center\">51.4</td><td align=\"center\">42.6</td></tr><tr><td align=\"left\"><bold>BMI (mean, kg/m<sup>2</sup>)</bold></td><td align=\"center\">28.6 ± 5.1</td><td align=\"center\">28.7 ± 5.0</td><td align=\"center\">28.7 ± 4.6</td><td align=\"center\">28.7 ± 4.5</td><td align=\"center\">30.9 ± 5.6</td><td align=\"center\">30.9 ± 5.7</td><td align=\"center\">29.9 ± 5.0</td><td align=\"center\">29.8 ± 4.8</td></tr><tr><td align=\"left\"> 25–29.9 kg/m<sup>2 </sup>(%)</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">43.7</td><td align=\"center\">42.6</td></tr><tr><td align=\"left\"> ≥30 kg/m<sup>2 </sup>(%)</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">42.4</td><td align=\"center\">43.2</td></tr><tr><td align=\"left\"><bold>Waist-Hip-ratio</bold></td><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Men</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">0.96 ± 0.07</td><td align=\"center\">0.96 ± 0.07</td><td align=\"center\">0.99 ± 0.08</td><td align=\"center\">0.98 ± 0.07</td></tr><tr><td align=\"left\"> Women</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">0.86 ± 0.08</td><td align=\"center\">0.87 ± 0.08</td><td align=\"center\">0.90 ± 0.09</td><td align=\"center\">0.90 ± 0.08</td></tr><tr><td align=\"left\"><bold>History of Hypertension (%)</bold></td><td align=\"center\">92.7*</td><td align=\"center\">92.0*</td><td align=\"center\">81*</td><td align=\"center\">81*</td><td align=\"center\">34.4</td><td align=\"center\">43.5</td><td align=\"center\">56.8</td><td align=\"center\">59.8</td></tr><tr><td align=\"left\"><bold>Heart rate (bpm)</bold></td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">74.0 ± 9.4</td><td align=\"center\">73.0 ± 9.8</td></tr><tr><td align=\"left\"><bold>Office systolic/diastolic BP (mmHg)</bold></td><td align=\"center\">154.5 ± 19.0/87.4 ± 10.9</td><td align=\"center\">154.8 ± 19.0/87.6 ± 10.7</td><td align=\"center\">164.1 ± 18.1/94.8 ± 10.4</td><td align=\"center\">163.9 ± 18.0/94.5 ± 10.4</td><td align=\"center\">136.1 ± 18.6/83.4 ± 10.8</td><td align=\"center\">136.0 ± 18.1/83.4 ± 10.8</td><td align=\"center\">147.4 ± 15.9/87.3 ± 9.3</td><td align=\"center\">144.6 ± 15.3/86.5 ± 9.4</td></tr><tr><td align=\"left\"><bold>HbA1c (mean %)</bold></td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">5.6 ± 0.6</td><td align=\"center\">5.6 ± 0.7</td></tr><tr><td align=\"left\"><bold>Risk factors</bold></td><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Smoker (%)</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">33</td><td align=\"center\">32</td><td align=\"center\">44.1</td><td align=\"center\">45.0</td><td align=\"center\">15.6</td><td align=\"center\">13.6</td></tr><tr><td align=\"left\"> Dyslipidemia (%)</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">35.6</td><td align=\"center\">35.4</td><td align=\"center\">56.4</td><td align=\"center\">56.6</td></tr><tr><td align=\"left\"> Hyperuricemia (%)</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">21.7</td><td align=\"center\">23.0</td></tr><tr><td align=\"left\"> MAU (%)</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">6.2</td><td align=\"center\">5.4</td></tr><tr><td align=\"left\"> CHD (%)</td><td align=\"center\">45.6</td><td align=\"center\">46.0</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\" colspan=\"2\">exclusion</td><td align=\"center\">13.9</td><td align=\"center\">14.2</td></tr><tr><td align=\"left\"> Stroke/TIA (%)</td><td align=\"center\">19.8</td><td align=\"center\">19.8</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\" colspan=\"2\">exclusion</td><td align=\"center\" colspan=\"2\">exclusion</td></tr><tr><td align=\"left\"><bold>Drug therapy</bold></td><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Aspirin or antiplatelet agents (%)</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">19</td><td align=\"center\">19</td><td align=\"center\">14.3</td><td align=\"center\">14.3</td><td align=\"center\">27.7</td><td align=\"center\">21.9</td></tr><tr><td align=\"left\"> Thiazide diuretics (%)</td><td align=\"center\">35.9</td><td align=\"center\">35.1</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">9.5</td><td align=\"center\">10.0</td><td align=\"center\">0.4</td><td align=\"center\">0</td></tr><tr><td align=\"left\"> Nonthiazide diuretics (%)</td><td/><td/><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">5.9</td><td align=\"center\">5.6</td><td align=\"center\">0</td><td align=\"center\">0</td></tr><tr><td align=\"left\"> ACEi</td><td align=\"center\">41.3</td><td align=\"center\">41.4</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td/><td/><td align=\"center\">0</td><td align=\"center\">0.6</td></tr><tr><td align=\"left\"> ARBs (%)</td><td align=\"center\">10.7</td><td align=\"center\">10.6</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">5.6</td><td align=\"center\">5.3</td><td align=\"center\">0.9</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\"> Betablockers (%)</td><td align=\"center\">32.7</td><td align=\"center\">33.7</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">17.2</td><td align=\"center\">17.5</td><td align=\"center\">0.3</td><td align=\"center\">0</td></tr><tr><td align=\"left\"> CCBs (%)</td><td align=\"center\">41.7</td><td align=\"center\">40.2</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">12.8</td><td align=\"center\">12.9</td><td align=\"center\">18.6</td><td align=\"center\">15.3</td></tr><tr><td align=\"left\"> Alphablockers (%)</td><td align=\"center\">7.1</td><td align=\"center\">6.5</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">1.9</td><td align=\"center\">2.2</td><td align=\"center\">2.1</td><td align=\"center\">2.0</td></tr><tr><td align=\"left\"> Statins (%)</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td align=\"center\">11**</td><td align=\"center\">10**</td><td align=\"center\">12.4</td><td align=\"center\">13.5</td><td align=\"center\">19.4</td><td align=\"center\">17.4</td></tr><tr><td align=\"left\"> Fibrates (%)</td><td align=\"center\">n.a.</td><td align=\"center\">n.a.</td><td/><td/><td align=\"center\">2.1</td><td align=\"center\">2.3</td><td align=\"center\">0.1</td><td align=\"center\">0</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Design characteristics of AdaPT in comparison to other trials</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\"><bold>VALUE </bold>[##REF##15207952##16##]</td><td align=\"center\" colspan=\"2\"><bold>ASCOT-BPLA </bold>[##REF##16154016##17##]</td><td align=\"center\" colspan=\"2\"><bold>DREAM </bold>[##REF##16980380##6##,##REF##15322749##38##]</td><td align=\"center\" colspan=\"2\"><bold>AdaPT</bold></td></tr></thead><tbody><tr><td/><td align=\"center\"><bold>Valsartan</bold></td><td align=\"center\"><bold>Amlodipine</bold></td><td align=\"center\"><bold>Amlodipine</bold></td><td align=\"center\"><bold>Atenolol</bold></td><td align=\"center\"><bold>Ramipril</bold></td><td align=\"center\"><bold>Placebo</bold></td><td align=\"center\"><bold>ACEi</bold></td><td align=\"center\"><bold>Diuretic</bold></td></tr><tr><td/><td align=\"center\"><bold>(n = 7649)</bold></td><td align=\"center\"><bold>(n = 7596)</bold></td><td align=\"center\"><bold>(n = 9639)</bold></td><td align=\"center\"><bold>(n = 9618)</bold></td><td align=\"center\"><bold>(n = 2.623)</bold></td><td align=\"center\"><bold>(n = 2.646)</bold></td><td align=\"center\"><bold>(n = 1.353)</bold></td><td align=\"center\"><bold>(n = 662)</bold></td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"left\"><bold>Study design</bold></td><td align=\"center\" colspan=\"2\">RCT</td><td align=\"center\" colspan=\"2\">RCT</td><td align=\"center\" colspan=\"2\">RCT</td><td align=\"center\" colspan=\"2\">Observational study</td></tr><tr><td align=\"left\"><bold>Endpoint</bold></td><td align=\"center\" colspan=\"2\">New onset diabetes (secondary objective)</td><td align=\"center\" colspan=\"2\">New onset diabetes (tertiary objective)</td><td align=\"center\" colspan=\"2\">New onset diabetes or death (primary endpoint)</td><td align=\"center\" colspan=\"2\">New onset diabetes (primary evaluation criterion)</td></tr><tr><td align=\"left\"><bold>Inclusion criteria</bold></td><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Age (years)</td><td align=\"center\" colspan=\"2\">≥ 50</td><td align=\"center\" colspan=\"2\">40–79</td><td align=\"center\" colspan=\"2\">≥ 30</td><td align=\"center\" colspan=\"2\">≥ 45</td></tr><tr><td align=\"left\"> Hypertension</td><td align=\"center\" colspan=\"2\">160–210/&lt;115 mmHg</td><td align=\"center\" colspan=\"2\">≥ 160/100 mmHg</td><td align=\"center\" colspan=\"2\">n.a.</td><td align=\"center\" colspan=\"2\">≥ 140/90 mmHg</td></tr><tr><td align=\"left\"> Fasting plasma glucose</td><td align=\"center\" colspan=\"2\">normal</td><td align=\"center\" colspan=\"2\">Normal</td><td align=\"center\" colspan=\"2\">110–125 mg/dl [6.1–7.0 mmol/l]</td><td align=\"center\" colspan=\"2\">110–125 mg/dl [6.1–7.0 mmol/l]</td></tr><tr><td align=\"left\"> Impaired glucose tolerance</td><td align=\"center\" colspan=\"2\">normal</td><td align=\"center\" colspan=\"2\">Normal</td><td align=\"center\" colspan=\"2\">140–199 mg/dl [7.8–11.0 mmol/l]</td><td align=\"center\" colspan=\"2\">no inclusion criterion</td></tr><tr><td align=\"left\"><bold>Exclusion criteria</bold></td><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Diabetes</td><td align=\"center\" colspan=\"2\">exclusion (for diabetes endpoint)</td><td align=\"center\" colspan=\"2\">exclusion (for diabetes endpoint)</td><td align=\"center\" colspan=\"2\">exclusion</td><td align=\"center\" colspan=\"2\">exclusion</td></tr><tr><td align=\"left\"> Cardiovascular disease</td><td align=\"center\" colspan=\"2\">possible</td><td align=\"center\" colspan=\"2\">Possible</td><td align=\"center\" colspan=\"2\">exclusion</td><td align=\"center\" colspan=\"2\">possible</td></tr><tr><td align=\"left\"><bold>Follow-up (years)</bold></td><td align=\"center\" colspan=\"2\">4.2 (mean)</td><td align=\"center\" colspan=\"2\">5.5 (median)</td><td align=\"center\" colspan=\"2\">3.0 (median)</td><td align=\"center\" colspan=\"2\">4 (planned)</td></tr><tr><td align=\"left\"><bold>HR new-onset diabetes</bold></td><td align=\"center\" colspan=\"2\">0.77 [95%CI 0.69–0.86]</td><td align=\"center\" colspan=\"2\">0.70 [95%CI 0.63–0.78]</td><td align=\"center\" colspan=\"2\">0.91 [95%CI 0.80–1.03]</td><td align=\"center\" colspan=\"2\">n.a.</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>IGT classification according to 2 h OGG is only appropriate if the NGT value is below the threshold value for diabetes mellitus.</p></table-wrap-foot>", "<table-wrap-foot><p>Wk = weeks; mo = months; yr = years; hsCRP = high sensitive C-reactive protein; ABPM = ambulatory blood pressure monitoring; AE = Adverse Events; SAE = Serious Adverse Events</p></table-wrap-foot>", "<table-wrap-foot><p>* previously treated for hypertension; n.a. = not available; BMI = body mass index; MAU = microalbuminuria; CHD = coronary heart disease; TIA = transitory ischemic attack; ACEi = ACE inhibitors; ARBs = Angiotensin receptor blockers; CCBs = calcium channel blockers</p><p>** statins and fibrates combined.</p></table-wrap-foot>", "<table-wrap-foot><p>n.a. = not available</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1475-2840-7-22-1\"/>" ]
[]
[{"collab": ["American Diabetes Association"], "article-title": ["Treatment of hypertension in adults with diabetes. Position statement"], "source": ["Diabetes Care"], "year": ["2003"], "volume": ["26"], "fpage": ["80S"], "lpage": ["82"], "pub-id": ["10.2337/diacare.26.7.2194"]}, {"surname": ["Lehnert", "Wittchen", "Pittrow", "Bramlage", "Kirch", "B\u00f6hler", "H\u00f6fler", "Ritz"], "given-names": ["H", "HU", "D", "P", "W", "S", "M", "E"], "article-title": ["Pr\u00e4valenz und Pharmakoepidemiologie des Diabetes mellitus in Deutschland"], "source": ["Deutsche Med Wochenschr"], "year": ["2005"], "volume": ["130"], "fpage": ["323"], "lpage": ["328"], "pub-id": ["10.1055/s-2005-863050"]}, {"surname": ["Abuissa", "O'Keefe", "Bell", "Jones", "Marso"], "given-names": ["H", "J", "D", "P", "S"], "article-title": ["ACE inhibitors or angiotensin receptor blockers for prevention of type 2 diabetes"], "source": ["J Am Coll Cardiol"], "year": ["2005"], "volume": ["45"], "fpage": ["Abstract 1058"], "lpage": ["1117"]}, {"surname": ["Deutsche"], "given-names": ["Hochdruckliga"], "article-title": ["Leitlinien zur Diagnostik und Behandlung der arteriellen Hypertonie"], "source": ["Nieren- und Hochdruckkrankheiten"], "year": ["2005"], "volume": ["34"], "fpage": ["481"], "lpage": ["498"]}, {"collab": ["Deutsche Liga zur Bek\u00e4mpfung des hohen Blutdruckes e.V., [German Hypertension League]"], "article-title": ["[Guidelines for the prevention, detection, diagnotics and therapy of hypertension]"], "source": ["Bonn"], "year": ["2003"]}, {"surname": ["Kerner", "Br\u00fcckel", "B\u00f6hm"], "given-names": ["W", "J", "B"], "article-title": ["Definition, Klassifikation und Diagnostik des Diabetes mellitus. Deutsche Diabetes Gesellschaft DDG, November 2004. Herausgeber: W. A. Scherbaum, W. Kiess. Aktualisierung der 1. Auflage vom Juli 2001"]}, {"surname": ["Kjeldsen", "Hille", "Lyle", "Julius", "Dahl\u00f6f", "Devereux"], "given-names": ["S", "D", "P", "S", "B", "R"], "article-title": ["Risk Predictors for Cardiovascular Outcomes in Patients With Hypertension and Left Ventricular Hypertrophy: The LIFE Study. Abstract 7C.1. 15. European Meeting on Hypertension. Milan, Italy, 20. June 2005"]}, {"collab": ["American Diabetes Association"], "article-title": ["Standards of medical care for patients with diabetes mellitus"], "source": ["Diabetes Care"], "year": ["2002"], "volume": ["26"], "fpage": ["33"], "lpage": ["50"]}, {"collab": ["American Diabetes Association"], "article-title": ["Standards of Medical Care in Diabetes"], "source": ["Diabetes Care"], "year": ["2005"], "volume": ["28"], "fpage": ["4"], "lpage": ["36"], "pub-id": ["10.2337/diacare.28.suppl_1.S4"]}]
{ "acronym": [], "definition": [] }
38
CC BY
no
2022-01-12 14:47:25
Cardiovasc Diabetol. 2008 Jul 24; 7:22
oa_package/0b/af/PMC2529270.tar.gz
PMC2529271
18680588
[]
[]
[]
[ "<title>Discussion</title>", "<p>Intussusception is an uncommon cause of intestinal obstruction and more than 95% of cases occur in the paediatric age group [##REF##14632903##1##]. Intussusception in adults is a rare pathology its incidence is around 2–3 per 1000,000 per year [##REF##11308002##2##]. Due to this rare nature of the disease there are no large scale/multi-centre studies or meta-analyses published to investigate the management of adult intussusception. The aetiology, presentation and management of intussusception in adults is different from children. In children intussusception is usually idiopathic or secondary to a viral illness. However in adults in more than 90% of cases a lead point can be identified causing the intussusception [##REF##10494696##3##,##REF##15759123##4##]. This is usually a polyp or a tumour and in majority of these cases the colonic tumours are malignant [##REF##5028582##5##,##REF##9296505##6##].</p>", "<p>The clinical presentation is very non-specific which makes this a difficult condition to diagnose. Abdominal pain, nausea, diarrhoea and bleeding per rectum are the common symptoms. Rarely this can present with acute intestinal obstruction. The classical triad of abdominal pain, sausage shaped palpable mass and passage of red current jelly stools seen in children is rarely observed in adults [##REF##9296505##6##,##REF##10195723##7##]. The use of investigations including a barium enema, ultrasound scan, and computed tomography can be helpful to establish the diagnosis [##REF##11308002##2##,##REF##10494696##3##,##REF##10195723##7##, ####REF##9915523##8##, ##REF##11004336##9####11004336##9##]. CT scan has been reported to have a diagnostic accuracy of around 80% [##REF##11893645##10##]. The classical finding on a CT scan is a target lesion or target sign which represents the outer intussuscepiens and the inner intussusceptum (Figure ##FIG##1##2##, ##FIG##2##3##). The dense intussuscepted mass comprising of swollen bowel and mesentery within the lumen of the bowel is responsible for the characteristic target lesion seen on the CT scan [##REF##10195723##7##,##REF##1879648##11##,##REF##9074370##12##]. Ultrasound scan is a less invasive and reproducible investigation. The classical features include a donut sign in transverse view and pseudo-kidney sign on longitudinal view [##REF##8470658##13##]. The examination is of limited value in the presence of significant amount of air in the intestine. A few studies have reported the use of colonoscopy in preoperative diagnosis particularly in the cases presenting with symptoms of large bowel obstruction [##REF##3721141##14##]. However the examination is technically challenging and the diagnosis is difficult to make.</p>", "<p>Benign lesions account for almost 25% cases of intussusception in adults. The commonest benign lesion is a lipoma in the colon. These are solitary submucosal lesions with 75% occurring in the right colon. Small lipomas are asymptomatic. Other benign lesions include adenomatous polyps and Peutz-Jeghers polyps. However in more than two thirds of cases there is a malignant tumour in the colon or small bowel resulting in intussusception [##REF##2662787##15##, ####REF##1879648##16##, ##REF##16990978##17####16990978##17##].</p>", "<p>Operative intervention is required in all cases of adult intussusception and unlike children conservative treatment does not work [##REF##11308002##2##,##REF##9296505##6##]. This usually involves segmental colonic resection. The optimal treatment for adult intussusception is slightly controversial. The type of procedure depends upon the location of intussusception, pre-operative diagnosis and condition of the intestine at the time of laparotomy. A few authors have described intra-operative reduction of intussusception before resection [##REF##5028582##5##]. However most authors do not recommend this due to a higher incidence of malignancy in these cases [##REF##11308002##2##,##REF##9296505##6##, ####REF##10195723##7##, ##REF##9915523##8####9915523##8##,##REF##937658##18##,##REF##7469558##19##] and hence the risk of tumour embolisation and seedling.</p>", "<p>In most cases of adult colonic intussusception, primary resection without reduction should be performed particularly in those more than 60 years of age due to a higher risk of malignancy. In cases of small bowel intussusception reduction before resection should be carried out only if there is a pre-operative diagnosis of benign etiology, the bowel is viable or it entails resecting massive lengths of small bowel with the risk of short gut syndrome [##REF##9074370##12##,##REF##12488701##20##].</p>" ]
[ "<title>Conclusion</title>", "<p>Intussusception is a rare cause of acute abdomen in adults. A high index of suspicion and appropriate investigations (USS, Barium enema and CT scan) can result in prompt diagnosis. Unlike children 75% of cases are due to a malignant tumour in the small bowel or colon. The extent of resection and operative technique depend upon the age of the patient, results of investigations (benign or malignant) and the length of the bowel involved.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>Colonic Intussusception although common in children, is a rare cause of acute intestinal obstruction in adults. The etiology, clinical presentation and management of this condition is different in adults as compared to children. Pre-operative diagnosis is usually difficult due to the non specific and intermittent nature of the symptoms. CT scan can be a helpful adjunct in establishing the diagnosis. We present a case report of adult ileocolic intussusception with classical radiological signs and operative findings. A brief literature review is also presented with emphasis on the controversy of reduction of the intussusception before resection.</p>" ]
[ "<title>Case report</title>", "<p>A 74 year old male presented with a four month history of colicky right upper quadrant pain, altered bowel habits and weight loss. He did not have any rectal bleeding. On examination there were signs of anaemia and a palpable mass in the right upper quadrant. Base line blood tests including FBC were normal. An abdominal USS showed a 5 × 7 cm ill defined bowel related mass in the right upper quadrant. The liver was normal.</p>", "<p>A subsequent barium enema showed an intra-luminal filling defect measuring 8 × 10 cm in the proximal transverse colon with no flow of barium proximally (Figure ##FIG##0##1##). CT scan of the abdomen showed significantly thickened bowel loops with fat density within a proximal segment and a target lesion, consistent with a neoplasm or a lipoma causing Intussusception (Figure ##FIG##1##2##).</p>", "<p>A provisional diagnosis of colonic carcinoma was made although the tumour markers including CEA and CA 19.9 were normal. The patient underwent a laparotomy where an ileocolic intussusception was found at the level of hepatic flexure (Figure ##FIG##2##3##). A right hemicolectomy was carried out with a hand sewn end to end ileocolic anastomosis. The specimen was opened to reveal a protruding polypoidal mass in the caecum (Figure ##FIG##3##4##). The patient made an uneventful post operative recovery and was discharged seven days later. The histology of the specimen showed this to be a benign submucosal lipoma of the caecum protruding into the caecal lumen.</p>", "<title>Authors' contributions</title>", "<p>MNK carried out the literature review and drafted the paper. AA designed the paper, literature review and reviewed the manuscript. PS literature review, provided the figures and reviewed the manuscript. All authors have read and approved the final manuscript.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>" ]
[]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Barium enema showing a classical \"claw sign\".</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>CT scan of abdomen.</bold> Red arrow demonstrates a \"target lesion\" diagnostic of intussusception.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Operative picture demonstrating ileocolic intussusception.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>Resected specimen opened up to show the presence of intussusception.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"1749-7922-3-26-1\"/>", "<graphic xlink:href=\"1749-7922-3-26-2\"/>", "<graphic xlink:href=\"1749-7922-3-26-3\"/>", "<graphic xlink:href=\"1749-7922-3-26-4\"/>" ]
[]
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{ "acronym": [], "definition": [] }
20
CC BY
no
2022-01-12 14:47:25
World J Emerg Surg. 2008 Aug 4; 3:26
oa_package/f2/da/PMC2529271.tar.gz
PMC2529272
18715501
[ "<title>Background</title>", "<p>Proper homeostasis is achieved <italic>during </italic>an immune response by controlling the appropriate size and activity of the effector T cell pool to maximize immunity and minimize immunopathology. <italic>After </italic>an immune response, homeostasis depends on the efficient contraction of the expanded T effector pool. Both processes require the selective death of effector T cells [##REF##15489190##1##, ####REF##16791883##2##, ##REF##8325332##3##, ##REF##10358758##4##, ##REF##8642275##5####8642275##5##]. When resting T cells become activated and proliferate under the influence of growth cytokines, they display heightened sensitivity to apoptosis [##REF##8325332##3##,##REF##8642275##5##,##REF##1944559##6##]. The mechanisms by which apoptosis is provoked have been thought to differ depending on the level of antigen in the T cell milieu. In one simple schema, T cell apoptosis proceeds through either an \"intrinsic\" (Bcl-2 superfamily/mitochondrial-dependent) program when antigen levels are low, or an \"extrinsic\" (Fas/CD95/APO-1/death receptor-mediated) pathway under conditions of high or repeated antigen stimulation [##REF##15489190##1##,##REF##10358758##4##,##REF##8642275##5##,##REF##18275825##7##]. In the first case, antigen clearance at the conclusion of an immune response results in diminished growth and survival cytokines (such as interleukin-2 (IL-2)), thus activating the mitochondrial death program. Cytokine withdrawal apoptosis (CWA) dramatically reduces the expanded T effector population to re-establish homeostasis, but permits a small population to persist as memory T cells. CWA is principally regulated by the pro- and anti-apoptotic members of the Bcl-2 family. In particular, the pro-apoptotic \"BH3-only\" proteins Bim and Puma have been implicated in CWA, as revealed by expanded memory T cells in knockout mice [##REF##10576740##8##, ####REF##17178918##9##, ##REF##18287039##10####18287039##10##]. These \"BH3 only\" members of the Bcl-2 superfamily cause caspase activation and apoptosis by binding pro-survival congeners and releasing the proapoptotic proteins Bax and Bak [##REF##18196961##11##]. Moreover, we recently discovered that a gain-of-function mutation in N-RAS, which suppresses Bim expression via constitutive extracellular signal-related kinase (ERK) activation, could cause a novel form of ALPS in humans [##REF##17517660##12##]. Indeed, Bim expression is tightly controlled by several transcriptional and post-translational mechanisms that underscore its role in central and peripheral T cell tolerance [##REF##11973609##13##].</p>", "<p>On the other hand, the extrinsic apoptosis pathway involves restimulation of activated T cells with high doses of antigen during the immune response; a pathway often referred to as \"activation-induced cell death (AICD)\" [##REF##15489190##1##,##REF##18275825##7##,##REF##12752672##14##]. However, it is important not to obfuscate the critical functional distinction between \"activation\" – the process entrained to the antigen receptor that causes resting cells to cycle, expand, and acquire effector function – and the death mechanism induced by TCR restimulation of those effector T cells that counterposes their expansion. So the term \"TCR restimulation\" or \"TCR-induced\" apoptosis will be used herein. The key immunoregulatory consideration is why restimulation by same antigen that produced the immune response, can kill the participating T cells in a highly specific way. At first glance, this event would seem to debilitate the immune response since the antigen, and presumably its pathogenic source, are still present. However, it is best understood as a negative feedback mechanism that constrains effector T cell proliferation to avoid immunopathology, previously termed \"propriocidal\" regulation [##REF##8325332##3##,##REF##10358758##4##,##REF##1944559##6##]. Propriocidal or TCR-induced death increases proportionately with high or persistent levels of antigen in IL-2. TCR-induced death has hitherto been primarily equated with the Fas death receptor. Indeed, the upregulation of Fas ligand (FasL) on the surface of restimulated T cells engages Fas on effector T cells in <italic>cis </italic>(\"suicide\") or in <italic>trans </italic>(\"fratricide\") leading to apoptosis [##REF##7528780##15##, ####REF##7530336##16##, ##REF##7530335##17##, ##REF##7530337##18####7530337##18##]. Moreover, debilitating mutations in Fas or FasL result in defective lymphocyte homeostasis and autoimmunity first characterized in mice (<italic>lpr </italic>and <italic>gld</italic>, respectively) and later in humans with ALPS type Ia or Ib [##REF##16551252##19##,##REF##15640690##20##].</p>", "<p>The Bim vs. Fas paradigm recently restated for intrinsic vs. extrinsic T cell apoptosis is appealing in its simplicity but illusory. For instance, other BH3-only proteins such as PUMA are likely instrumental in CWA [##REF##17178918##9##,##REF##18287039##10##]. Also, the evidence suggests that Fas may not be the sole mediator of TCR-induced death and that TNF or nonapoptotic pathways may be involved [##REF##7566090##21##,##REF##12444127##22##]. Data from conditional knockout mice in which Fas is ablated or blocked in distinct hematopoietic compartments indicate that Fas-mediated apoptosis may also counter autoimmunity by ensuring the removal of antigen presenting cells, including B cells and dendritic cells rather than T cells [##REF##16497935##23##,##REF##17509906##24##]. Although autoreactive T cells accumulate in T cell-specific Fas knockout mice, surprisingly, loss of Fas confers no selective survival advantage for T cells exposed to repeated antigen challenge [##REF##17509906##24##]. Also, Fas engagement can intersect with the intrinsic pathway through a caspase-8 activating cleavage of Bid – a Bcl-2 superfamily member that can trigger mitochondrial apoptosis. Based on these insights, we asked whether death effector pathways other than Fas, including intrinsic signals routed through mitochondrial activation, were important for TCR-induced death of human T cells.</p>", "<p>In re-examining human T cells in which FAS signaling is blocked or genetically impaired, we found that TCR-induced apoptosis can proceed through rapid induction of BIM expression in the absence of FAS signals, which contributes to mitochondrial permeabilization and cell death in the presence of IL-2. Knockdown of BIM expression partially rescued cells from TCR-induced death, particularly for CD8<sup>+ </sup>human T cells. Moreover, we show that TCR-induced apoptosis is normal for ALPS Ia patients displaying elevated BIM expression, but impaired in an ALPS type IV patient in which BIM expression is repressed. Collectively, these data indicate that FAS and BIM can cooperate as independent effector molecules in TCR-induced apoptosis. Our results show BIM plays a key role in T cell contraction even when cytokines are abundant, indicating that FAS- and BIM-mediated T cell apoptosis are not mutually exclusive pathways as recently reinforced in the literature [##REF##18275825##7##].</p>" ]
[ "<title>Methods</title>", "<title>Cells and Treatments</title>", "<p>Patients were enrolled and blood samples were obtained with informed consent under protocols approved by the National Institutes of Health (NIH). Peripheral blood lymphocytes (PBL) from normal donors were isolated by Ficoll density gradient centrifugation, and T cells were activated by either 5 μg/ml ConA or 1 μg/ml OKT3 mAb (Ortho Biotech, Raritan, NJ) plus 25 U/ml rhIL-2 (Peprotech, Rocky Hill, NJ), washed 3× in PBS, then cultured in 100 U/ml rhIL-2 for at least 7 days before apoptosis assays were performed. Activated T cell subsets were separated using CD4 or CD8 Microbeads and MACS magnetic bead cell separation (Miltenyi Biotec, Auburn, CA). In some experiments, inhibitors to caspase 8 (IETD-fmk) or caspase 9 (LEHD) (BioVision, Palo Alto, CA) were added at 20 μM. Caspase 9 enzymatic activity was measured using a Caspase 9 Colorimetric Assay Kit (BioVision) according to the manufacturer's instructions.</p>", "<title>Flow Cytometry</title>", "<p>Apoptosis assays were performed as previously described [##REF##17517660##12##]. Briefly, activated T cells were resuspended in fresh media + IL-2 and stimulated for 24 h with soluble OKT3 mAb, agonistic anti-Fas mAb APO1.3 (Alexis, San Diego, CA) plus 200 ng/ml Protein A, or 2 μM staurosporine. In some experiments, 1 μg/ml of an antagonistic Fas blocking Ab (clone SM1/23, Alexis) was added to cells 30 minutes prior to OKT3 restimulation. The level of apoptosis was determined by staining with 1 μg/ml propidium iodide and flow cytometry analysis using constant time acquisition as previously described. Mitochondrial permeability was measured by staining with 40 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC6) (EMD Biosciences, San Diego, CA) for 15 min at 37°C before flow cytometry analysis. For surface staining, cells were stained with 5 μg anti-CD4-fluorescin isothiocyanate (FITC), anti-CD8-phycoerythrin (PE), or anti-CD95-PE (BD Biosciences).</p>", "<title>Electron Microscopy</title>", "<p>Treated cells (5 × 10<sup>6</sup>) were pelleted and overlaid with 2% glutaraldehyde in 0.1 M cacodylate buffer fixative for 2 h at room temperature (RT). Sample preparation and electron microscopy was performed at the Image Analysis Laboratory of the National Cancer Institute (Frederick, MD).</p>", "<title>Mircoarray Analysis</title>", "<p>RNA was isolated from two normal donor activated T cells at 0 or 6 h after OKT3 restimulation using Trizol (Invitrogen) and RNeasy mini-columns (Qiagen, Valencia, CA). Purified RNA was amplified using the Ovation Aminoallyl Amplification System (NuGEN, San Carlos, CA), labeled with Cy5 using the Cy5 Reactive Dye Pack (GE Healthcare, Piscataway, NJ), and cleaned up using Vivaspin columns (VivaScience AG, Hanover, Germany). Amplified RNA (2 μg) was hybridized to Hsbb 23K human spotted arrays (NIAID Mircoarray Research Facility) versus Cy3-labeled reference RNA pooled from six normal donor cycling T cells. Data was analyzed using GenePix and mAdb software.</p>", "<title>Immunoblotting</title>", "<p>Cells were lysed in 1% NP-40 lysis buffer for 15 min on ice, then cleared by centrifugation. Protein concentration was determined by BCA assay (Pierce, Rockford, IL), and 20–30 μg total protein was separated by SDS-PAGE. Blots were probed with the following antibodies (Abs): anti-BIM (Stressgen, Ann Arbor, MI); anti-BAX, anti-cytochrome c (clone 7H8.2C12), anti-BCL-xL, anti-BCL-2, anti-MCL-1 (BD Pharmingen); anti-PUMA (Alexis); anti-β-actin (clone AC-15, Sigma). Bound Abs were detected using appropriate horseradish peroxidase-conjugated secondary Abs (Southern Biotech, Birmingham, AL) and ECL (Pierce).</p>", "<title>siRNA Transfections</title>", "<p>Activated human PBL were transfected with 200 pmol of either specific small interfering RNA oligoribonucleotides (siRNA) or a non-specific (NS) control oligo (Invitrogen, Carlsbad, CA) using the Amaxa Nucleofection system (Amaxa, Koln, Germany). Assessment of knockdown efficiency and all subsequent assays were performed 4 days (human) post-transfection. siRNA sequences are available from Invitrogen (Stealth Select).</p>" ]
[ "<title>Results and discussion</title>", "<title>TCR restimulation induces apoptosis signals independent of FAS</title>", "<p>To examine TCR-induced death in human T cells, activated peripheral blood lymphocytes (PBL) from normal donors were restimulated with the anti-CD3 mAb OKT3 after cycling in IL-2 for 7–14 days. The majority of these cells are CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells, with the latter generally more abundant in culture. Data was obtained for numerous human donors. We found that apoptosis was readily induced in restimulated T cells, marked by chromatin condensation and shrinkage (Figure ##FIG##0##1A##). This was followed by loss of membrane integrity due to secondary necrosis. Apoptosis was verified by PI exclusion; however, we noted that blocking FAS with an antagonistic Ab (SM1/23) provided only partial protection against TCR-induced death (Figure ##FIG##0##1B##). Flow cytometric analysis of restimulated T cells also confirmed cell shrinkage and loss of mitochondrial membrane potential, as indicated by decreased DiOC<sub>6 </sub>staining following 12 h of OKT3 treatment, signifying apoptosis (Figure ##FIG##0##1C##). Again, blocking Fas with an antagonistic Ab (SM1/23) only partially rescued this drop in mitochondrial membrane potential and cell viability. Remarkably, T cells from an ALPS Ia patient with a FAS death domain mutation also showed only a modest loss of mitochondrial membrane potential and viability (Figure ##FIG##0##1C##), suggesting a mitochondria-dependent apoptotic signal could proceed despite compromised FAS function. Similarly, cytochrome c released from mitochondria in response to OKT3 restimulation was only modestly decreased by FAS blockade (Figure ##FIG##0##1D##). We also tested caspase 9 activation, which occurs downstream of cytochrome c release and \"apoptosome\" formation. As expected, caspase 9 activation was only partially reduced in restimulated cells in the presence of FAS blocking Ab, but completely abrogated in the presence of the caspase 9 specific inhibitor LEHD-fmk (Figure ##FIG##0##1E##). In contrast, the SM1/23 Ab effectively blocked APO1.3 anti-Fas induced apoptosis, indicating that the cells were competent for FAS-mediated death (Additional File ##SUPPL##0##1##). Taken together, our data confirms that TCR-induced death relies in part on intrinsic mitochondrial signals triggered independently of FAS-FASL interactions.</p>", "<title>Role for BIM induction in TCR-induced death</title>", "<p>Initial studies of AICD indicated that <italic>de novo </italic>transcription was required for the execution of apoptosis in response to T cell restimulation[##REF##7530335##17##]. Since our data pointed toward a mitochondrial component, we surveyed expression of several pro- and anti-apoptotic BCL-2 family members using microarrays following TCR restimulation of activated human PBL for 6 h. As a positive control, we detected significant induction of FASL expression. Notably, we detected an even greater increase (&gt; 5 fold) in BIM transcription in response to OKT3 stimulation (Figure ##FIG##1##2A##). Only BCL-xL was also increased with restimulation, whereas other BCL-2 family members remained largely unchanged or slightly decreased. The expression of all three BIM protein isoforms (extra long (EL), long (L), and short (S)) also increased substantially over time with OKT3 restimulation, whether Fas blockade was applied or not (Figure ##FIG##1##2B##). Although BCL-xL protein levels also increased, the ratio of BIM:BCL-xL expression rose substantially over time, suggesting heightened Bim expression represents a \"tipping point\" for overcoming the anti-apoptotic function of BCL-xL and related proteins in driving mitochondrial depolarization. PUMAβ levels also showed a minor increase (Figure ##FIG##1##2B##). Remarkably, the quick induction of BIM upon restimulation occurred in the presence of IL-2, which is required for TCR-induced death[##REF##1944559##6##]. IL-2 signaling alone can destabilize BIM mRNA or promotes BIM protein degradation via Raf/ERK or phosphoinositide kinase 3 (PI-3K) signaling pathways [##REF##10102273##25##, ####REF##12646560##26##, ##REF##17218274##27####17218274##27##]. However, our results suggest the TCR restimulation overrides this signal to allow for rapid BIM upregulation. These data are consistent with previous observations indicating BIM expression can be induced upon TCR triggering in human CTL clones, depending on the agonistic peptide used [##REF##17174744##28##,##REF##14970329##29##]. However, these studies did not establish whether loss of BIM expression had functional consequences for TCR-induced apoptosis sensitivity, or how this related to FAS-FASL signaling.</p>", "<p>To definitively test whether BIM contributes to the TCR-induced apoptosis signal, we silenced BIM expression by RNA interference (RNAi) in activated PBL and restimulated them with OKT3 with or without FAS blockade. Knockdown of BIM expression significantly reduced the sensitivity of activated PBL to TCR-induced death (Figure ##FIG##1##2C##). Control immunoblots showed that BIM expression was silenced effectively in cells that received BIM-specific siRNA both before and after restimulation (Figure ##FIG##1##2D##, Additional File ##SUPPL##1##2##). As noted above, FAS blockade also partially rescued cells from death in these experiments, and had an additive protective effect when BIM expression was reduced (Figure ##FIG##1##2C##). The protective effects of BIM suppression and Fas blockade were noted in multiple human donors (Figure ##FIG##1##2E##). Knockdown of FAS associated death domain (FADD) rescued cells from TCR-induced death to a similar extent, further illustrating that death receptor signaling is only part of the apoptotic signal triggered by TCR restimulation (Additional File ##SUPPL##2##3##). In addition, knockdown of PUMA also provided some protection from TCR-induced death (Additional File ##SUPPL##3##4##), although this effect was variable in different donors tested. Collectively, our data definitively shows that intrinsic apoptosis mediators, particularly BIM, are required for optimal apoptosis triggered by TCR re-engagement separate from extrinsic FAS-induced apoptotic signals.</p>", "<title>Relative contribution of BIM in CD4<sup>+ </sup>versus CD8<sup>+ </sup>TCR-induced death</title>", "<p>We next tested whether BIM induction played a role in TCR-induced death of both CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells. Purified CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells sorted from activated PBL were transfected with NS or BIM-specific siRNA and tested for sensitivity to OKT3-induced death. Whereas Fas blockade alone substantially rescued the apoptosis of purified CD4<sup>+ </sup>T cells, knockdown of Bim expression had little effect (Figure ##FIG##2##3A##). Conversely, CD8<sup>+ </sup>T cells relied on both FAS and BIM for TCR-induced apoptosis signaling. Although BIM expression was consistently higher in CD4<sup>+ </sup>T cells compared to CD8<sup>+ </sup>T cells from multiple donors (Figure ##FIG##2##3B##), BIM induction from steady state levels was as good or better in CD8<sup>+ </sup>T cells upon restimulation (Additional Files ##SUPPL##1##2## &amp;##SUPPL##4##5##). We cannot rule out that residual BIM expression in CD4<sup>+ </sup>T cells following BIM siRNA transfection contributed to the Fas-independent of apoptosis observed. However, other experiments revealed that BIM knockdown using the same siRNA provided greater protection from IL-2 withdrawal apoptosis in CD4<sup>+ </sup>T cells (Additional File ##SUPPL##5##6##), suggesting BIM levels could be sufficiently depleted to hinder BIM-dependent death. Collectively, the data suggests that human CD8<sup>+ </sup>T cells rely on BIM more extensively for TCR-induced deletion than CD4<sup>+ </sup>T cells, which are largely dependent on FAS signaling. This idea agrees with landmark studies that implicated FAS in TCR-induced apoptosis, which focused primarily on CD4<sup>+ </sup>T cell lines or clones from humans or mice [##REF##7528780##15##, ####REF##7530336##16##, ##REF##7530335##17##, ##REF##7530337##18####7530337##18##]. Moreover, our data potentially explain new studies suggesting BIM drives Ag-specific CD8<sup>+ </sup>T cell deletion in establishing peripheral tolerance in both mice and humans [##REF##18398508##30##,##REF##18390708##31##].</p>", "<title>Bim and Fas cooperate in TCR-induced apoptosis of murine T cells</title>", "<p>In light of our findings in human T cells, we re-examined TCR-induced death in murine T cells. Surprisingly, we observed that activated splenic T cells from Fas-deficient <italic>lpr </italic>mice showed only minor resistance to anti-CD3-induced death induced by restimulation, whereas <italic>bim </italic>knockout mice showed no difference in sensitivity compared to WT cells (Figure ##FIG##3##4A##). We also tested for Bim induction in restimulated WT and <italic>lpr </italic>T cells in the presence of IL-2. Consistent with data in human T cells, activated mouse T cells (WT or <italic>lpr</italic>) showed a clear increase in BimEL expression after 6 hours of restimulation (Figure ##FIG##3##4B##). We also detected a change in the migration of BimEL and BimL isoforms, suggesting post-translational modifications may affect of bim function in mice, perhaps via phosphorylation.</p>", "<p>Next, we reasoned that differences in apoptosis sensitivity caused by loss of Fas or Bim may differ in CD4<sup>+ </sup>and CD8<sup>+ </sup>T cell cultures, as noted in for human T cells. Therefore, we assayed TCR-induced apoptosis sensitivity in purified CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells from WT, <italic>lpr</italic>, and <italic>bim</italic><sup>-/- </sup>mice. As expected from previous reports, CD4<sup>+ </sup><italic>lpr </italic>cells showed a profound defect in restimulation-induced death (Figure ##FIG##3##4C##). This concurred with our results in human CD4<sup>+ </sup>T cells using Fas blocking Ab (Figure ##FIG##2##3A##), indicating Fas is necessary for CD4<sup>+ </sup>T cell restimulation apoptosis. In contrast, there were no differences in CD8<sup>+ </sup>T cell death between restimulated WT and <italic>lpr </italic>cells, explaining the cumulatively minor rescue of TCR-induced death in total splenic T cells when Fas is absent. Furthermore, genetic ablation of <italic>bim </italic>had little protective effect for activated CD4<sup>+ </sup>T cells upon TCR restimulation, and no discernible effect on apoptosis in CD8<sup>+ </sup>T cells (Figure ##FIG##3##4D##).</p>", "<p>We hypothesized that loss of Bim from development, through germline gene ablation, may permit T cells to \"compensate\" accordingly via enhanced expression or function of pro-apoptotic molecules. Therefore, we acutely silenced Bim using RNAi in activated WT and <italic>lpr </italic>T cells. Knockdown of Bim significantly protected activated WT and <italic>lpr </italic>T cells from apoptosis induced by 100 ng/ml anti-CD3 stimulation (Figure ##FIG##3##4E##, left panel), demonstrating that Bim can play a prominent role in this apoptosis pathway. This effect was also noted in purified CD4<sup>+ </sup>and CD8<sup>+ </sup>T cell populations (data not shown), even though loss of Fas alone reduced sensitivity only in CD4<sup>+ </sup>T cells again as expected. Control blots showed that Bim siRNA effectively suppressed Bim protein expression in both WT and <italic>lpr </italic>T cells (Figure ##FIG##3##4E##, right panel). This protective effect was less pronounced at higher doses of anti-CD3 stimulation (data not shown), suggesting stronger restimulation may override Bim siRNA effects and/or trigger alternative death effector pathways. Thus, our data suggests that Fas or Bim may partially compensate for the loss of one or the other from development in murine T cells during development.</p>", "<title>Relative BIM expression correlates with sensitivity to TCR-induced death in ALPS patients</title>", "<p>Based on our aforementioned results, we revisited TCR restimulation-induced apoptosis in PBL derived from several ALPS patients. Similar to controls, PBL cultures from ALPS Ia patients were primarily comprised of CD8<sup>+ </sup>T cells (data not shown). Surprisingly, we found that PBL from several ALPS Ia patients displayed normal or slightly more death in response to OKT3 titration compared to normal controls, despite impaired apoptosis upon direct Fas crosslinking. (Figure ##FIG##4##5A##, Additional File ##SUPPL##6##7##). Similarly, T cells derived from an ALPS Ib patient harboring a dominant interfering mutation in <italic>FASL </italic>[##REF##17605793##32##] were also killed effectively upon TCR restimulation (Figure ##FIG##4##5B##). Consistent with defective FASL function, TCR-induced apoptosis was unaffected by Fas blockade. These results exposed a glaring contradiction in the concept that FAS mediates most or all TCR-induced death.</p>", "<p>We next assessed the relative expression of BIM before and after restimulation of PBL in ALPS Ia patients. In general, we noted higher BIM protein expression in restimulated ALPS Ia T cells relative to controls (Figure ##FIG##4##5C##). In 4/6 ALPS Ia patients, steady-state BIM expression was also elevated relative to controls. Using spot densitometry, we estimated that ALPS Ia T cells had between 30–80% more BIM protein than normal controls both before and after TCR ligation (Figure ##FIG##4##5C##, bottom panel). BIM siRNA treatment did not result in a significantly greater rescue of TCR-induced death in ALPS Ia cells compared to normal controls (data not shown), perhaps due to incomplete depletion of BIM or compensation by other mediators (e.g. PUMA). Nevertheless, elevated BIM levels in cycling T cells with defective FAS function may suggest that these T cells are \"primed\" for apoptotic deletion through a compensatory increase in BIM expression.</p>", "<p>Finally, we tested TCR-induced apoptosis in T cells derived from an ALPS Type IV patient (P58) with a gain-of-function, germline NRAS mutation that constitutively activates ERK and suppresses BIM expression. We recently demonstrated that P58 T cells are resistant to apoptosis induced by IL-2 withdrawal due to BIM suppression[##REF##17517660##12##]. Remarkably, P58 T cells displayed partial resistance to TCR-induced death when compared to normal donor and ALPS Ia cells, despite comparable expression of FAS on the cell surface (Figure ##FIG##4##5D##, Additional File ##SUPPL##7##8##). Moreover, BIM expression was attenuated in P58 T cells and could not be rescued by TCR restimulation (Figure ##FIG##4##5E##), providing stronger evidence that BIM serves a physiologically relevant role in the restimulation apoptosis pathway, especially for CD8<sup>+ </sup>T cell homeostasis. Moreover, our data implies that relative BIM expression may represent an important determinant of TCR-induced apoptosis sensitivity, independently of FAS. However, we concede that NRAS/ERK dysregulation in P58 could alter TCR-induced death through BIM-independent mechanisms as well. Indeed, pharmacological ERK inhibitors actually provided a small but reproducible rescue of TCR-induced death in both normal and P58 T cells.</p>", "<p>The physiological function of Bim was originally revealed from characterization of Bim-deficient mice, from which T cells were profoundly resistant to lymphokine withdrawal death[##REF##10576740##8##]. The pro-apoptotic function of Bim also enforces immune tolerance through thymocyte negative selection, CD8<sup>+ </sup>T cell cross tolerance, and the regulation of antigen presenting cells including B cells and dendritic cells[##REF##16497935##23##,##REF##11859372##33##, ####REF##12370256##34##, ##REF##14517273##35####14517273##35##]. Here we demonstrate that BIM also plays a significant role in TCR-induced death of activated human T cells, working in tandem with FAS signaling as a separate signal to kill T cells. This provides a new mechanism besides the cleavage of BID for an extrinsic signal to activate the intrinsic mitochondrial death program. This paradigm may be distinct from Bim-dependent \"activated T cell death\" described by Hildeman et al. in mice challenged with a single dose of superantigen [##REF##12121658##36##], which may be interpreted as predominantly cytokine withdrawal apoptosis, not restimulation-induced death with repeated Ag dosing. On the other hand, the marked accumulation and persistence of Bim-deficient murine CD8<sup>+ </sup>T cells in chronic viral infection models could be connected to failed deletion in response to repeated TCR stimulation [##REF##16912311##37##,##REF##14623954##38##].</p>", "<p>Our results show that direct signals from the TCR program T cells to die through Bim, which is fundamentally different from the secretion of death cytokines such as FasL that engage external death receptors. This has some interesting implications. First, it may be advantageous in conditions where Fas may not be effective. For example, Bim has a greater influence in CD8<sup>+ </sup>T cells that can utilize FasL:Fas as a calcium-independent cytolytic mechanism against infected target cells and therefore may be inured to its lethal effects. Second, the direct molecular connection inside the cell may make the Bim pathway more efficient. Careful investigation of the temporal effects of killing after TCR engagement may reveal differences between Fas and Bim effectiveness. Third, as Bim expression is extensively regulated post-translationally, the fact that translation inhibitors only partially block TCR-induced death could indicate there is a direct death pathway entrained to TCR restimulation that does not require new protein synthesis [##REF##7530335##17##]. Finally, pro-apoptotic mediators like Bim or Puma acting at the convergence of TCR and CWA may help to restrain these pathways at a focal point for tight control of those T cells escape death and emerge as memory T cells.</p>", "<p>Recently, three groups reported that loss of both Bim and Fas in mice results in massive lymphadenopathy/splenomegaly, early onset of SLE-like autoimmune manifestations, and even greater accumulation of antigen-specific CD8<sup>+ </sup>T cells upon chronic viral infection [##REF##18275830##39##, ####REF##18275831##40##, ##REF##18275832##41####18275832##41##]. These experiments reprised earlier work that obtained very similar results when transgenic Bcl-2 overexpressing mice were crossed onto an <italic>lpr </italic>background [##REF##7594564##42##,##REF##8557033##43##]. However, their general conclusions still emphasized the traditional model, reiterated in an accompanying review, that Fas and Bim control T cell homeostasis through two distinct pathways: restimulation-driven versus IL-2 withdrawal-induced apoptosis, respectively[##REF##8642275##5##,##REF##18275825##7##,##REF##16551252##19##]. Our study illustrates that death of activated T cells via Fas or Bim are not mutually exclusive pathways, as both can operate in IL-2 dependent TCR-induced apoptosis. During infections this combination of potent extrinsic and intrinsic signals may act to ensure rapid and efficient killing of hyper-responsive or cross-reactive autoimmune T cells upon repeated antigen encounter, thus preventing immunotoxicity and maintaining peripheral tolerance. Another intriguing possibility relates to the potential of Bim and Fas to partially compensate for one another in driving TCR-induced apoptosis. This applies to situations where either gene function is lost from development, such as in <italic>lpr </italic>or <italic>bim</italic><sup>-/- </sup>mice, and may explain why only acute knockdown of Bim resulted in significant reduction of TCR-induced apoptosis in murine T cells <italic>in vitro</italic>. The idea that Bim participates in ensuring T cell homeostasis both during and after effector T cell responses may also explain why Bcl-2 Tg <italic>lpr </italic>mice described years ago have strikingly worse lymphocyte accumulation compared to either Bcl-2 Tg or <italic>lpr </italic>mice alone[##REF##8557033##43##]. Our results provide a new interpretation of the mouse studies by revealing that the infection-induced derangement of T cell homeostasis caused by Bim-deficiency could be accounted for by an impairment of both intrinsic and extrinsic apoptosis. It is also notable that ALPS patients show wide variability in conventional CD3<sup>+ </sup>T cell numbers, with a substantial fraction showing no increases. By contrast, the fraction and absolute number of \"double negative\" (CD4<sup>-</sup>CD8<sup>-</sup>) α/β T cells are invariably elevated [##REF##10189330##44##]. This may reflect that alternative effectors such as BIM could preserve equipoise in the conventional T cell compartment.</p>", "<p>In humans, our data are consistent with previous studies suggesting that TCR-induced death involves multiple effector molecules, and clearly includes components other than FAS or BIM that remain to be elucidated[##REF##12444127##22##]. We have previously noted a role for tumor necrosis factor-alpha (TNF-α) in this process for murine CD8<sup>+ </sup>T cells[##REF##7566090##21##]; however, blockade of this pathway in human T cells had little demonstrable effect (data not shown), which requires further exploration. A recent paper from Mateo et al. implicated perforin and cytotoxic granules in the execution of TCR-induced death, particularly for ALPS Ia patient cells[##REF##17724145##45##]. Other inputs implicated in control of AICD sensitivity, including NF-κB regulation through HPK-1 or generation of reactive oxygen species (ROS), likely relate more to the regulation of FasL or Bim expression[##REF##17712048##46##,##REF##17339328##47##]. However, we are studying patients with impaired TCR-induced apoptosis despite normal induction of FASL and BIM, and normal apoptosis triggered by FAS ligation or IL-2 deprivation (A.L. Snow, unpublished data). Insights gleaned from such patients may further advance our understanding of the biochemical complexity and physiological relevance of apoptosis in different immune cell compartments. Nevertheless, our current findings further elucidate FAS-independent signals for the restimulation-induced death of activated T cells via BIM induction.</p>" ]
[ "<title>Results and discussion</title>", "<title>TCR restimulation induces apoptosis signals independent of FAS</title>", "<p>To examine TCR-induced death in human T cells, activated peripheral blood lymphocytes (PBL) from normal donors were restimulated with the anti-CD3 mAb OKT3 after cycling in IL-2 for 7–14 days. The majority of these cells are CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells, with the latter generally more abundant in culture. Data was obtained for numerous human donors. We found that apoptosis was readily induced in restimulated T cells, marked by chromatin condensation and shrinkage (Figure ##FIG##0##1A##). This was followed by loss of membrane integrity due to secondary necrosis. Apoptosis was verified by PI exclusion; however, we noted that blocking FAS with an antagonistic Ab (SM1/23) provided only partial protection against TCR-induced death (Figure ##FIG##0##1B##). Flow cytometric analysis of restimulated T cells also confirmed cell shrinkage and loss of mitochondrial membrane potential, as indicated by decreased DiOC<sub>6 </sub>staining following 12 h of OKT3 treatment, signifying apoptosis (Figure ##FIG##0##1C##). Again, blocking Fas with an antagonistic Ab (SM1/23) only partially rescued this drop in mitochondrial membrane potential and cell viability. Remarkably, T cells from an ALPS Ia patient with a FAS death domain mutation also showed only a modest loss of mitochondrial membrane potential and viability (Figure ##FIG##0##1C##), suggesting a mitochondria-dependent apoptotic signal could proceed despite compromised FAS function. Similarly, cytochrome c released from mitochondria in response to OKT3 restimulation was only modestly decreased by FAS blockade (Figure ##FIG##0##1D##). We also tested caspase 9 activation, which occurs downstream of cytochrome c release and \"apoptosome\" formation. As expected, caspase 9 activation was only partially reduced in restimulated cells in the presence of FAS blocking Ab, but completely abrogated in the presence of the caspase 9 specific inhibitor LEHD-fmk (Figure ##FIG##0##1E##). In contrast, the SM1/23 Ab effectively blocked APO1.3 anti-Fas induced apoptosis, indicating that the cells were competent for FAS-mediated death (Additional File ##SUPPL##0##1##). Taken together, our data confirms that TCR-induced death relies in part on intrinsic mitochondrial signals triggered independently of FAS-FASL interactions.</p>", "<title>Role for BIM induction in TCR-induced death</title>", "<p>Initial studies of AICD indicated that <italic>de novo </italic>transcription was required for the execution of apoptosis in response to T cell restimulation[##REF##7530335##17##]. Since our data pointed toward a mitochondrial component, we surveyed expression of several pro- and anti-apoptotic BCL-2 family members using microarrays following TCR restimulation of activated human PBL for 6 h. As a positive control, we detected significant induction of FASL expression. Notably, we detected an even greater increase (&gt; 5 fold) in BIM transcription in response to OKT3 stimulation (Figure ##FIG##1##2A##). Only BCL-xL was also increased with restimulation, whereas other BCL-2 family members remained largely unchanged or slightly decreased. The expression of all three BIM protein isoforms (extra long (EL), long (L), and short (S)) also increased substantially over time with OKT3 restimulation, whether Fas blockade was applied or not (Figure ##FIG##1##2B##). Although BCL-xL protein levels also increased, the ratio of BIM:BCL-xL expression rose substantially over time, suggesting heightened Bim expression represents a \"tipping point\" for overcoming the anti-apoptotic function of BCL-xL and related proteins in driving mitochondrial depolarization. PUMAβ levels also showed a minor increase (Figure ##FIG##1##2B##). Remarkably, the quick induction of BIM upon restimulation occurred in the presence of IL-2, which is required for TCR-induced death[##REF##1944559##6##]. IL-2 signaling alone can destabilize BIM mRNA or promotes BIM protein degradation via Raf/ERK or phosphoinositide kinase 3 (PI-3K) signaling pathways [##REF##10102273##25##, ####REF##12646560##26##, ##REF##17218274##27####17218274##27##]. However, our results suggest the TCR restimulation overrides this signal to allow for rapid BIM upregulation. These data are consistent with previous observations indicating BIM expression can be induced upon TCR triggering in human CTL clones, depending on the agonistic peptide used [##REF##17174744##28##,##REF##14970329##29##]. However, these studies did not establish whether loss of BIM expression had functional consequences for TCR-induced apoptosis sensitivity, or how this related to FAS-FASL signaling.</p>", "<p>To definitively test whether BIM contributes to the TCR-induced apoptosis signal, we silenced BIM expression by RNA interference (RNAi) in activated PBL and restimulated them with OKT3 with or without FAS blockade. Knockdown of BIM expression significantly reduced the sensitivity of activated PBL to TCR-induced death (Figure ##FIG##1##2C##). Control immunoblots showed that BIM expression was silenced effectively in cells that received BIM-specific siRNA both before and after restimulation (Figure ##FIG##1##2D##, Additional File ##SUPPL##1##2##). As noted above, FAS blockade also partially rescued cells from death in these experiments, and had an additive protective effect when BIM expression was reduced (Figure ##FIG##1##2C##). The protective effects of BIM suppression and Fas blockade were noted in multiple human donors (Figure ##FIG##1##2E##). Knockdown of FAS associated death domain (FADD) rescued cells from TCR-induced death to a similar extent, further illustrating that death receptor signaling is only part of the apoptotic signal triggered by TCR restimulation (Additional File ##SUPPL##2##3##). In addition, knockdown of PUMA also provided some protection from TCR-induced death (Additional File ##SUPPL##3##4##), although this effect was variable in different donors tested. Collectively, our data definitively shows that intrinsic apoptosis mediators, particularly BIM, are required for optimal apoptosis triggered by TCR re-engagement separate from extrinsic FAS-induced apoptotic signals.</p>", "<title>Relative contribution of BIM in CD4<sup>+ </sup>versus CD8<sup>+ </sup>TCR-induced death</title>", "<p>We next tested whether BIM induction played a role in TCR-induced death of both CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells. Purified CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells sorted from activated PBL were transfected with NS or BIM-specific siRNA and tested for sensitivity to OKT3-induced death. Whereas Fas blockade alone substantially rescued the apoptosis of purified CD4<sup>+ </sup>T cells, knockdown of Bim expression had little effect (Figure ##FIG##2##3A##). Conversely, CD8<sup>+ </sup>T cells relied on both FAS and BIM for TCR-induced apoptosis signaling. Although BIM expression was consistently higher in CD4<sup>+ </sup>T cells compared to CD8<sup>+ </sup>T cells from multiple donors (Figure ##FIG##2##3B##), BIM induction from steady state levels was as good or better in CD8<sup>+ </sup>T cells upon restimulation (Additional Files ##SUPPL##1##2## &amp;##SUPPL##4##5##). We cannot rule out that residual BIM expression in CD4<sup>+ </sup>T cells following BIM siRNA transfection contributed to the Fas-independent of apoptosis observed. However, other experiments revealed that BIM knockdown using the same siRNA provided greater protection from IL-2 withdrawal apoptosis in CD4<sup>+ </sup>T cells (Additional File ##SUPPL##5##6##), suggesting BIM levels could be sufficiently depleted to hinder BIM-dependent death. Collectively, the data suggests that human CD8<sup>+ </sup>T cells rely on BIM more extensively for TCR-induced deletion than CD4<sup>+ </sup>T cells, which are largely dependent on FAS signaling. This idea agrees with landmark studies that implicated FAS in TCR-induced apoptosis, which focused primarily on CD4<sup>+ </sup>T cell lines or clones from humans or mice [##REF##7528780##15##, ####REF##7530336##16##, ##REF##7530335##17##, ##REF##7530337##18####7530337##18##]. Moreover, our data potentially explain new studies suggesting BIM drives Ag-specific CD8<sup>+ </sup>T cell deletion in establishing peripheral tolerance in both mice and humans [##REF##18398508##30##,##REF##18390708##31##].</p>", "<title>Bim and Fas cooperate in TCR-induced apoptosis of murine T cells</title>", "<p>In light of our findings in human T cells, we re-examined TCR-induced death in murine T cells. Surprisingly, we observed that activated splenic T cells from Fas-deficient <italic>lpr </italic>mice showed only minor resistance to anti-CD3-induced death induced by restimulation, whereas <italic>bim </italic>knockout mice showed no difference in sensitivity compared to WT cells (Figure ##FIG##3##4A##). We also tested for Bim induction in restimulated WT and <italic>lpr </italic>T cells in the presence of IL-2. Consistent with data in human T cells, activated mouse T cells (WT or <italic>lpr</italic>) showed a clear increase in BimEL expression after 6 hours of restimulation (Figure ##FIG##3##4B##). We also detected a change in the migration of BimEL and BimL isoforms, suggesting post-translational modifications may affect of bim function in mice, perhaps via phosphorylation.</p>", "<p>Next, we reasoned that differences in apoptosis sensitivity caused by loss of Fas or Bim may differ in CD4<sup>+ </sup>and CD8<sup>+ </sup>T cell cultures, as noted in for human T cells. Therefore, we assayed TCR-induced apoptosis sensitivity in purified CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells from WT, <italic>lpr</italic>, and <italic>bim</italic><sup>-/- </sup>mice. As expected from previous reports, CD4<sup>+ </sup><italic>lpr </italic>cells showed a profound defect in restimulation-induced death (Figure ##FIG##3##4C##). This concurred with our results in human CD4<sup>+ </sup>T cells using Fas blocking Ab (Figure ##FIG##2##3A##), indicating Fas is necessary for CD4<sup>+ </sup>T cell restimulation apoptosis. In contrast, there were no differences in CD8<sup>+ </sup>T cell death between restimulated WT and <italic>lpr </italic>cells, explaining the cumulatively minor rescue of TCR-induced death in total splenic T cells when Fas is absent. Furthermore, genetic ablation of <italic>bim </italic>had little protective effect for activated CD4<sup>+ </sup>T cells upon TCR restimulation, and no discernible effect on apoptosis in CD8<sup>+ </sup>T cells (Figure ##FIG##3##4D##).</p>", "<p>We hypothesized that loss of Bim from development, through germline gene ablation, may permit T cells to \"compensate\" accordingly via enhanced expression or function of pro-apoptotic molecules. Therefore, we acutely silenced Bim using RNAi in activated WT and <italic>lpr </italic>T cells. Knockdown of Bim significantly protected activated WT and <italic>lpr </italic>T cells from apoptosis induced by 100 ng/ml anti-CD3 stimulation (Figure ##FIG##3##4E##, left panel), demonstrating that Bim can play a prominent role in this apoptosis pathway. This effect was also noted in purified CD4<sup>+ </sup>and CD8<sup>+ </sup>T cell populations (data not shown), even though loss of Fas alone reduced sensitivity only in CD4<sup>+ </sup>T cells again as expected. Control blots showed that Bim siRNA effectively suppressed Bim protein expression in both WT and <italic>lpr </italic>T cells (Figure ##FIG##3##4E##, right panel). This protective effect was less pronounced at higher doses of anti-CD3 stimulation (data not shown), suggesting stronger restimulation may override Bim siRNA effects and/or trigger alternative death effector pathways. Thus, our data suggests that Fas or Bim may partially compensate for the loss of one or the other from development in murine T cells during development.</p>", "<title>Relative BIM expression correlates with sensitivity to TCR-induced death in ALPS patients</title>", "<p>Based on our aforementioned results, we revisited TCR restimulation-induced apoptosis in PBL derived from several ALPS patients. Similar to controls, PBL cultures from ALPS Ia patients were primarily comprised of CD8<sup>+ </sup>T cells (data not shown). Surprisingly, we found that PBL from several ALPS Ia patients displayed normal or slightly more death in response to OKT3 titration compared to normal controls, despite impaired apoptosis upon direct Fas crosslinking. (Figure ##FIG##4##5A##, Additional File ##SUPPL##6##7##). Similarly, T cells derived from an ALPS Ib patient harboring a dominant interfering mutation in <italic>FASL </italic>[##REF##17605793##32##] were also killed effectively upon TCR restimulation (Figure ##FIG##4##5B##). Consistent with defective FASL function, TCR-induced apoptosis was unaffected by Fas blockade. These results exposed a glaring contradiction in the concept that FAS mediates most or all TCR-induced death.</p>", "<p>We next assessed the relative expression of BIM before and after restimulation of PBL in ALPS Ia patients. In general, we noted higher BIM protein expression in restimulated ALPS Ia T cells relative to controls (Figure ##FIG##4##5C##). In 4/6 ALPS Ia patients, steady-state BIM expression was also elevated relative to controls. Using spot densitometry, we estimated that ALPS Ia T cells had between 30–80% more BIM protein than normal controls both before and after TCR ligation (Figure ##FIG##4##5C##, bottom panel). BIM siRNA treatment did not result in a significantly greater rescue of TCR-induced death in ALPS Ia cells compared to normal controls (data not shown), perhaps due to incomplete depletion of BIM or compensation by other mediators (e.g. PUMA). Nevertheless, elevated BIM levels in cycling T cells with defective FAS function may suggest that these T cells are \"primed\" for apoptotic deletion through a compensatory increase in BIM expression.</p>", "<p>Finally, we tested TCR-induced apoptosis in T cells derived from an ALPS Type IV patient (P58) with a gain-of-function, germline NRAS mutation that constitutively activates ERK and suppresses BIM expression. We recently demonstrated that P58 T cells are resistant to apoptosis induced by IL-2 withdrawal due to BIM suppression[##REF##17517660##12##]. Remarkably, P58 T cells displayed partial resistance to TCR-induced death when compared to normal donor and ALPS Ia cells, despite comparable expression of FAS on the cell surface (Figure ##FIG##4##5D##, Additional File ##SUPPL##7##8##). Moreover, BIM expression was attenuated in P58 T cells and could not be rescued by TCR restimulation (Figure ##FIG##4##5E##), providing stronger evidence that BIM serves a physiologically relevant role in the restimulation apoptosis pathway, especially for CD8<sup>+ </sup>T cell homeostasis. Moreover, our data implies that relative BIM expression may represent an important determinant of TCR-induced apoptosis sensitivity, independently of FAS. However, we concede that NRAS/ERK dysregulation in P58 could alter TCR-induced death through BIM-independent mechanisms as well. Indeed, pharmacological ERK inhibitors actually provided a small but reproducible rescue of TCR-induced death in both normal and P58 T cells.</p>", "<p>The physiological function of Bim was originally revealed from characterization of Bim-deficient mice, from which T cells were profoundly resistant to lymphokine withdrawal death[##REF##10576740##8##]. The pro-apoptotic function of Bim also enforces immune tolerance through thymocyte negative selection, CD8<sup>+ </sup>T cell cross tolerance, and the regulation of antigen presenting cells including B cells and dendritic cells[##REF##16497935##23##,##REF##11859372##33##, ####REF##12370256##34##, ##REF##14517273##35####14517273##35##]. Here we demonstrate that BIM also plays a significant role in TCR-induced death of activated human T cells, working in tandem with FAS signaling as a separate signal to kill T cells. This provides a new mechanism besides the cleavage of BID for an extrinsic signal to activate the intrinsic mitochondrial death program. This paradigm may be distinct from Bim-dependent \"activated T cell death\" described by Hildeman et al. in mice challenged with a single dose of superantigen [##REF##12121658##36##], which may be interpreted as predominantly cytokine withdrawal apoptosis, not restimulation-induced death with repeated Ag dosing. On the other hand, the marked accumulation and persistence of Bim-deficient murine CD8<sup>+ </sup>T cells in chronic viral infection models could be connected to failed deletion in response to repeated TCR stimulation [##REF##16912311##37##,##REF##14623954##38##].</p>", "<p>Our results show that direct signals from the TCR program T cells to die through Bim, which is fundamentally different from the secretion of death cytokines such as FasL that engage external death receptors. This has some interesting implications. First, it may be advantageous in conditions where Fas may not be effective. For example, Bim has a greater influence in CD8<sup>+ </sup>T cells that can utilize FasL:Fas as a calcium-independent cytolytic mechanism against infected target cells and therefore may be inured to its lethal effects. Second, the direct molecular connection inside the cell may make the Bim pathway more efficient. Careful investigation of the temporal effects of killing after TCR engagement may reveal differences between Fas and Bim effectiveness. Third, as Bim expression is extensively regulated post-translationally, the fact that translation inhibitors only partially block TCR-induced death could indicate there is a direct death pathway entrained to TCR restimulation that does not require new protein synthesis [##REF##7530335##17##]. Finally, pro-apoptotic mediators like Bim or Puma acting at the convergence of TCR and CWA may help to restrain these pathways at a focal point for tight control of those T cells escape death and emerge as memory T cells.</p>", "<p>Recently, three groups reported that loss of both Bim and Fas in mice results in massive lymphadenopathy/splenomegaly, early onset of SLE-like autoimmune manifestations, and even greater accumulation of antigen-specific CD8<sup>+ </sup>T cells upon chronic viral infection [##REF##18275830##39##, ####REF##18275831##40##, ##REF##18275832##41####18275832##41##]. These experiments reprised earlier work that obtained very similar results when transgenic Bcl-2 overexpressing mice were crossed onto an <italic>lpr </italic>background [##REF##7594564##42##,##REF##8557033##43##]. However, their general conclusions still emphasized the traditional model, reiterated in an accompanying review, that Fas and Bim control T cell homeostasis through two distinct pathways: restimulation-driven versus IL-2 withdrawal-induced apoptosis, respectively[##REF##8642275##5##,##REF##18275825##7##,##REF##16551252##19##]. Our study illustrates that death of activated T cells via Fas or Bim are not mutually exclusive pathways, as both can operate in IL-2 dependent TCR-induced apoptosis. During infections this combination of potent extrinsic and intrinsic signals may act to ensure rapid and efficient killing of hyper-responsive or cross-reactive autoimmune T cells upon repeated antigen encounter, thus preventing immunotoxicity and maintaining peripheral tolerance. Another intriguing possibility relates to the potential of Bim and Fas to partially compensate for one another in driving TCR-induced apoptosis. This applies to situations where either gene function is lost from development, such as in <italic>lpr </italic>or <italic>bim</italic><sup>-/- </sup>mice, and may explain why only acute knockdown of Bim resulted in significant reduction of TCR-induced apoptosis in murine T cells <italic>in vitro</italic>. The idea that Bim participates in ensuring T cell homeostasis both during and after effector T cell responses may also explain why Bcl-2 Tg <italic>lpr </italic>mice described years ago have strikingly worse lymphocyte accumulation compared to either Bcl-2 Tg or <italic>lpr </italic>mice alone[##REF##8557033##43##]. Our results provide a new interpretation of the mouse studies by revealing that the infection-induced derangement of T cell homeostasis caused by Bim-deficiency could be accounted for by an impairment of both intrinsic and extrinsic apoptosis. It is also notable that ALPS patients show wide variability in conventional CD3<sup>+ </sup>T cell numbers, with a substantial fraction showing no increases. By contrast, the fraction and absolute number of \"double negative\" (CD4<sup>-</sup>CD8<sup>-</sup>) α/β T cells are invariably elevated [##REF##10189330##44##]. This may reflect that alternative effectors such as BIM could preserve equipoise in the conventional T cell compartment.</p>", "<p>In humans, our data are consistent with previous studies suggesting that TCR-induced death involves multiple effector molecules, and clearly includes components other than FAS or BIM that remain to be elucidated[##REF##12444127##22##]. We have previously noted a role for tumor necrosis factor-alpha (TNF-α) in this process for murine CD8<sup>+ </sup>T cells[##REF##7566090##21##]; however, blockade of this pathway in human T cells had little demonstrable effect (data not shown), which requires further exploration. A recent paper from Mateo et al. implicated perforin and cytotoxic granules in the execution of TCR-induced death, particularly for ALPS Ia patient cells[##REF##17724145##45##]. Other inputs implicated in control of AICD sensitivity, including NF-κB regulation through HPK-1 or generation of reactive oxygen species (ROS), likely relate more to the regulation of FasL or Bim expression[##REF##17712048##46##,##REF##17339328##47##]. However, we are studying patients with impaired TCR-induced apoptosis despite normal induction of FASL and BIM, and normal apoptosis triggered by FAS ligation or IL-2 deprivation (A.L. Snow, unpublished data). Insights gleaned from such patients may further advance our understanding of the biochemical complexity and physiological relevance of apoptosis in different immune cell compartments. Nevertheless, our current findings further elucidate FAS-independent signals for the restimulation-induced death of activated T cells via BIM induction.</p>" ]
[ "<title>Conclusion</title>", "<p>Although Fas-FasL signaling is often considered synonymous with TCR restimulation-induced death, the data provided herein show it has a quantitatively lesser role than previously acknowledged, and support a critical role for BIM induction in the execution of antigen-driven \"extrinsic\" apoptosis. Increased BIM expression following TCR restimulation, even with a surfeit of IL-2, works in parallel to FAS signaling in driving mitochondrial depolarization, caspase 9 activation, and eventual apoptosis. Like FAS blockade, suppression of BIM induction via RNAi or increased NRAS activity in an ALPS variant patient results in partial resistance to TCR-induced death. These data build upon previous work from Sandalova and colleagues by demonstrating that BIM is indispensable for maximum sensitivity to restimulation-induced apoptosis of human T cells. More importantly, our findings revise previous models in showing that FAS and BIM both participate in eliminating activated T cells through this IL-2 dependent, propriocidal death pathway.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Upon repeated or chronic antigen stimulation, activated T cells undergo a T cell receptor (TCR)-triggered propriocidal cell death important for governing the intensity of immune responses. This is thought to be chiefly mediated by an extrinsic signal through the Fas-FasL pathway. However, we observed that TCR restimulation still potently induced apoptosis when this interaction was blocked, or genetically impaired in T cells derived from autoimmune lymphoproliferative syndrome (ALPS) patients, prompting us to examine Fas-independent, intrinsic signals.</p>", "<title>Results</title>", "<p>Upon TCR restimulation, we specifically noted a marked increase in the expression of BIM, a pro-apoptotic Bcl-2 family protein known to mediate lymphocyte apoptosis induced by cytokine withdrawal. In fact, T cells from an ALPS type IV patient in which BIM expression is suppressed were more resistant to restimulation-induced death. Strikingly, knockdown of BIM expression rescued normal T cells from TCR-induced death to as great an extent as Fas disruption.</p>", "<title>Conclusion</title>", "<p>Our data implicates BIM as a critical mediator of apoptosis induced by restimulation as well as growth cytokine withdrawal. These findings suggest an important role for BIM in eliminating activated T cells even when IL-2 is abundant, working in conjunction with Fas to eliminate chronically stimulated T cells and maintain immune homeostasis.</p>", "<title>Reviewers</title>", "<p>This article was reviewed by Dr. Wendy Davidson (nominated by Dr. David Scott), Dr. Mark Williams (nominated by Dr. Neil Greenspan), and Dr. Laurence C. Eisenlohr.</p>" ]
[ "<title>Abbreviations</title>", "<p>ALPS: Autoimmune lymphoproliferative syndrome; FasL: Fas ligand; PBL: Peripheral blood lymphocytes; PI: Propidium iodide.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>ALS and JBO designed and performed all experiments. ALS wrote the manuscript. LZ helped with activation and analysis of ALPS Type Ib cells. JKD. cared for ALPS patients and facilitated sample collection and distribution. MJL and TAF helped direct the research and contributed to final revisions.</p>", "<title>Reviewers' comments</title>", "<p>Reviewer #1, Snow et al.:</p>", "<p>Dr. Wendy Davidson, University of Maryland School of Medicine</p>", "<p><bold>Editor: </bold>Dr. David Scott, University of Maryland School of Medicine</p>", "<p>This manuscript examines the role of BIM in TCR restimulation-induced death in human T cells from healthy individuals and patients with ALPS. The authors show that restimulation of normal unfractionated T cell blasts results in significant upregulation of BIM expression and that reactivation-induced cell death (AICD) is reduced when FAS/FASL interactions are blocked with antagonist Ab or BIM expression is decreased by siRNA. Blockade of both pathways appeared to have additive effects suggesting that both BIM- and FAS-mediated death pathways contribute to AICD. Further analysis of T cell subsets showed that although CD4<sup>+ </sup>T cell blasts express higher levels of BIM than CD8<sup>+ </sup>T cell blasts prior to and after restimulation, BIM-mediated death appears to be restricted to CD8<sup>+ </sup>T cells. Combined, the data on subsets suggest that AICD in CD4<sup>+ </sup>T cells is predominantly FAS-mediated whereas CD8<sup>+ </sup>T cells are susceptible to death induced by FAS and BIM. Additional studies with unfractionated T cell blasts from ALPS patients deficient in FAS or FASL indicated sensitivity to FAS-independent AICD, increased basal levels of BIM and increased expression of BIM post restimulation. However, attempts to block AICD using BIM siRNA were unsuccessful. As an alternative indirect approach to determine the contribution of BIM to AICD in the context of ALPS, the investigators used T cells from an ALPS type IV patient with a germline NRAS mutation resulting in constitutive activation of ERK and suppression of BIM expression. Unfractionated T cell blasts from this patient were efficiently killed by anti-FAS mAb but were relatively insensitive to TCR-induced death, suggesting that BIM might contribute to AICD.</p>", "<p>The findings with normal human CD8<sup>+ </sup>T cells are novel and consistent with recent studies in mice indicating the importance of both the BIM and FAS cell death pathways in regulating CD8<sup>+ </sup>T cell homeostasis. The data relating to the role of BIM in T cell death in ALPS patients are less compelling because of the difficulties in efficiently downregulating BIM in T cell blasts from ALPS type I patients. Further, in the ALPS type IV patient the authors assume, but do not prove, that the only way that dysregulated expression of NRAS protects T cells from restimulation-induced death is through down regulation of BIM.</p>", "<p>Critique:</p>", "<p>Major points:</p>", "<p>1. The key data shown in Figs. ##FIG##1##2C## and ##FIG##2##3A## appear to be from a single experiment utilizing cells from one individual. The investigators should indicate how reproducible the studies were and whether there was significant variation in susceptibility to BIM-mediated killing among T cells from different individuals.</p>", "<title>Author Response</title>", "<p><italic>The reviewer poses an important question. Although some variation was noted in cells from different donors, the experiments described were very reproducible in multiple replicate experiments, utilizing several human donors. In order to clarify this point, Figure </italic>##FIG##1##2E##<italic> has been added to summarize the extent of TCR-induced apoptosis inhibition, relative to NS siRNA-transfected cells, for 6 different human donors tested.</italic></p>", "<p>2. In Fig. ##FIG##1##2D##, the authors show that BIM expression is significantly reduced in T cell blasts after transfection with BIM siRNA. The authors do not specify whether the cell lysates came from blasts 4 days post transfection or from restimulated transfected blasts. Similar data shown in Additional File ##SUPPL##1##2## indicate that western blots were performed prior to restimulation. It is imperative that the authors demonstrate that restimulation-induced upregulation of BIM is prevented in the BIM siRNA transfected blasts. Otherwise, the data cannot be interpreted.</p>", "<title>Author Response</title>", "<p><italic>We agree with the reviewer's critique, as blots shown represent cells lysed 4 days post-transfection of siRNA without restimulation. To address this point, the new Additional File </italic>##SUPPL##1##2##<italic> now demonstrates that Bim siRNA effectively suppresses BIM expression/induction in both resting and restimulated T cells.</italic></p>", "<p>3. Data in Fig. ##FIG##2##3A## suggest that BIM either does not contribute to restimulation-induced death in CD4<sup>+ </sup>T cell blasts or that the BIM death pathway is far less efficient than the FAS death pathway in inducing apoptosis in this population. Studies with Bim KO mice may help to distinguish between these two possibilities, assuming that murine CD4<sup>+ </sup>T cells reliably model human CD4<sup>+ </sup>T cells in this regard. Decreased sensitivity of BIM knockdown CD4<sup>+ </sup>blasts to IL-2 withdrawal-induced death would provide additional evidence that BIM levels are sufficiently depleted to impair BIM-dependent death.</p>", "<title>Author Response</title>", "<p><italic>Following the reviewer's suggestion, we have added a new figure detailing several experiments performed with mouse T cells from Bim KO as well as lpr mice (new Figure </italic>##FIG##3##4##<italic>). These data support the conclusion that CD4<sup>+ </sup>T cells are much less dependent on Bim compared to Fas for TCR-induced death, although we found that CD8<sup>+ </sup>T cells were equally sensitive to the blockade of either pathway. On the other hand, acute knockdown of Bim expression in mouse T cells rescued a substantial proportion of WT T cells, and almost all lpr T cells, from TCR-induced apoptosis. We hypothesize that Bim KO cells maintain sensitivity to TCR-induced death through a compensatory effector molecule (Fas or otherwise), such that the acute knockdown in WT cells does not allow sufficient time or the physiological context for a hypothetical compensatory mechanism to have a significant effect.</italic></p>", "<p><italic>We also provide new data (Additional File </italic>##SUPPL##5##6##<italic>) showing that our RNAi approach provides sufficient depletion of BIM to impair BIM-dependent, IL-2 withdrawal apoptosis in CD4<sup>+ </sup>T cells. In fact, the extent of this protection was more pronounced for CD4<sup>+ </sup>T cells compared to CD8<sup>+ </sup>T cells. This contrast suggests the more prominent role of BIM in human CD8<sup>+ </sup>T cell deletion, supported by our data in Figure </italic>##FIG##2##3##<italic>, is specific to TCR restimulation-induced death.</italic></p>", "<p>In Fig. ##FIG##1##2C##, the data suggest that the FAS and BIM death pathways are equally effective in killing blasts enriched for CD8<sup>+ </sup>cells. However, in Fig. ##FIG##2##3A## purified CD8<sup>+ </sup>blasts appear to be more sensitive to FAS-mediated death. This difference deserves comment. The author's should also indicate whether the greater sensitivity of CD8<sup>+ </sup>blasts to FAS-mediated death is a consistent finding in healthy humans. As discussed above for the unfractionated blast populations, data showing that siRNA blocks the restimulation-induced increase in BIM expression in the purified CD4<sup>+ </sup>and CD8<sup>+ </sup>are essential.</p>", "<title>Author Response</title>", "<p><italic>In general, we cannot definitively say that purified CD8<sup>+ </sup>T cells are more sensitive to the Fas-FasL component of TCR-induced death than unfractionated PBL. The data shown in Figure </italic>##FIG##2##3A##<italic> are representative of several experiments, although some variability in the extent of rescue by the Fas antagonist antibody SM1/23 was noted in both purified CD8<sup>+ </sup>T cells and PBL. As noted in the text, the majority of T cells found in normal donor PBL (roughly 55–80%) were CD8<sup>+ </sup>T cells following a week of cycling in IL-2. As mentioned above, Additional File </italic>##SUPPL##1##2##<italic> demonstrates that Bim-specific siRNA blocks the restimulation-induced increase in BIM expression in purified T cell subsets.</italic></p>", "<p>4. Although T cells from ALPS patients with defects in FAS signaling are clearly susceptible to AICD and exhibit increased expression of BIM after restimulation, no conclusions can be drawn regarding the contribution of BIM in FAS-independent apoptosis in CD4<sup>+ </sup>or CD8<sup>+ </sup>T cells since the authors were unable to block BIM expression. While the ALPS type IV patient, P58, with reduced BIM levels and decreased sensitivity to AICD provides additional preliminary evidence for a role for BIM in T cell homeostasis, the authors should address the possibility that the enhanced cell survival associated with NRAS dysregulation may result from effects other than, or in addition to, BIM downregulation.</p>", "<p>In Fig. ##FIG##3##4D##, the investigators show that P58 blasts are highly sensitive to anti-FAS-induced apoptosis but do not establish whether FAS-mediated AICD is intact. Ideally, the AICD experiments should be repeated with isolated P58 CD4<sup>+ </sup>and CD8<sup>+ </sup>blasts in the presence or absence of antagonist anti-FAS Ab to determine the level of FAS-mediated apoptosis in each population. If P58 CD4<sup>+ </sup>blasts are sensitive to AICD and death is significantly inhibited by FAS blockade, the investigators will have additional evidence that BIM contributes minimally to AICD in CD4<sup>+ </sup>T cells.</p>", "<title>Author Response</title>", "<p>We appreciate the authors' interpretation. Accordingly, we now provide additional commentary in the Discussion to point out that the gain-of-function NRAS mutation in P58 T cells could be altering TCR-induced death signaling through additional unidentified BIM-independent pathways. Although purified CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells were not tested, TCR-induced death in P58 PBL could be partially rescued by Fas blockade to a similar extent as normal donor T cells, suggesting FasL function is intact. We also concur that enhanced BIM expression in ALPS Ia patients is suggestive, but not definitive, evidence for BIM-dependent AICD short of better knockdown experiments.</p>", "<p>Minor Points:</p>", "<p>1. In their discussion (page 14, line 10), the authors overstate the differences between their interpretation of how FAS and BIM may work to regulate T cell homeostasis and the conclusions of three other groups studying mice deficient in Bim and functional Fas.</p>", "<title>Authors Response</title>", "<p>We respectfully disagree with the author's opinion in this case, as we feel it is imperative to highlight the novelty of our findings in relation to the three cited articles pertaining to Bim-deficient lpr mice. The \"preview\" article published alongside those articles in Immunity (see references in the manuscript) maintains that Fas controls antigen-stimulated or \"active\" death, and cytokine withdrawal death governed by Bim and its interactions with other Bcl-2 family members controls antigen depletion or \"passive\" death in a mutually exclusive manner. Actually our data indicate that both of these contentions are oversimplified. Ample data indicates that Fas is not the only mediator of TCR-stimulated death of activated cells illustrated in all of the experiments in this paper by the fact that substantial residual death evident even with combined Fas/Bim blockade. Moreover, our data demonstrate that Fas and Bim can both contribute to apoptosis caused by TCR restimulation of activated T cells. This provides a different molecular schema for interpreting the synergistic increase in lymphadenopathy and autoimmunity they reported resulting from combined genetic lesions of Fas and Bim. It is clear from those papers and the overview written by Green that the interpretation is confined to a model that has existed in the field for over a decade, viz Lenardo, J. Exp. Med. 183: 1071, 1996. Our new findings reveal a functional connection between the TCR-induced pathway and Bim that was previously hinted at in the literature (Sandalova et al., Hum Immunol. 2006 Dec;67(12):958–65) and not considered in the 2008 Immunity papers. This sheds new light on the described phenotype of these mouse experiments, particularly with regard to excessive CD8<sup>+ </sup>T cell lymphoproliferation, that was not considered.</p>", "<p>2. Since the difference in susceptibility of CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells to BIM-mediated AICD is a novel observation, the authors should discuss this data more extensively.</p>", "<p>3. Information on the source of the SM1/23 mAb and a reference for the cell death assay need to be included in the Methods section.</p>", "<p>4. In Fig. ##FIG##3##4B##, the axis label for OKT3 concentration should read μg/ml. On page 7 there is are typos in the spelling of microarray. Data is plural.</p>", "<title>Author Response</title>", "<p>We have elaborated on our discussion of differences in BIM-driven TCR-induced death of CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells as recommended by the reviewer. We have also added two very recent references that demonstrate a prominent role for BIM in Ag-specific peripheral deletion of CD8<sup>+ </sup>T cells in mice and humans, in agreement with our in vitro data. The information requested is now provided in the text, and typos have been corrected.</p>", "<p>Reviewer #2, Snow et al.:</p>", "<p>Dr. Mark Williams, University of Maryland School of Medicine</p>", "<p>Editor: Dr. Neil Greenspan, Case Western Reserve University</p>", "<p>Recent papers suggest that the role of Bim in classic TCR-induced AICD is not well defined. This paper addresses this question.</p>", "<p>The data in Figure ##FIG##1##2## suggest that Bim plays a role in AICD in the T cells from normal controls, but the data from the ALPS patients in Figure ##FIG##3##4## do not allow such an interpretation. The P58 cells could be resistant to AICD for many other reasons besides a lack of Bim. For example FasL levels are not examined or TCR signals needed to sensitize to AICD may be deficient. The Bim siRNA data in ALPS patients also should be shown (page 13).</p>", "<title>Authors Response</title>", "<p><italic>We concede that because we were unable to knockdown BIM expression adequately in ALPS Ia patient cells, data shown in Figure </italic>##FIG##4##5C##<italic> do not conclusively show that BIM plays a more prominent role in TCR-induced death of ALPS Ia patient cells. However, elevated BIM expression noted in several ALPS Ia patients strongly suggests that other apoptosis effector molecules, including BIM, may be compensating for the loss of functional Fas in maintaining sensitivity to TCR-induced death. The striking sensitivity of ALPS cells to TCR-induced death is a surprising effect that we believe has been unequivocally demonstrated. Indeed, a similar interpretation may be derived from our new data in Bim KO and lpr T cells (see new Figure </italic>##FIG##3##4##<italic>). Please note our response to Reviewer 1 with regard to interpreting data from P58 cells.</italic></p>", "<p>Statistical analysis is needed for all graphs (the word \"significantly\" is used throughout the text) especially in Figure ##FIG##2##3## to establish that Bim knockdown gives additional protection above Fas blockade.</p>", "<title>Authors Response</title>", "<p>We point out that statistical analysis (paired T tests) was applied to all of the graphs shown throughout the paper, as noted in the Figure Legends (p values are included). This statistical analysis is now mentioned in the Methods section for clarification. The word \"significantly\" was applied in the manuscript with these statistical analyses in mind, referring only to differences with p &lt; 0.05.</p>", "<p>Overall, the data support a role for Bim in AICD, albeit not as strongly as the authors suggest. More studies in which Bim expression is manipulated are necessary to support such a conclusion.</p>", "<title>Authors Response</title>", "<p><italic>Currently, direct silencing of BIM using siRNA is our most effective tool for manipulating BIM expression in primary human T cells. Unfortunately, ectopic expression of BIM in primary cells is more problematic, as cell viability is poor due to both BIM overexpression and increased cell loss during electroporation of an expression plasmid. We hope that the inclusion of new data in Figure </italic>##FIG##1##2E##<italic> and Additional File </italic>##SUPPL##1##2##<italic> strengthens our conclusion that BIM siRNA effectively suppresses BIM expression during restimulation, translating to AICD resistance in multiple human donors tested. Furthermore, the effect of acute Bim knockdown in WT and lpr mouse T cells resembles our data in human T cells and supports our general hypothesis further.</italic></p>", "<p>Reviewer #3, Snow et al.:</p>", "<p>Editor: Laurence C. Eisenlohr, Thomas Jefferson University</p>", "<p>Comments:</p>", "<p>1. In reference to the term \"propriocidal cell death\": I'm familiar with \"proprioception\" but not \"propriocidal\".</p>", "<title>Author Response</title>", "<p>We employ the term \"propriocidal\", based on the Latin prefix (\"proprio\" = \"of his own accord\"), to refer to TCR-induced apoptosis as a self-regulatory form of T cell death. This has been introduced in the literature on this topic in the past: Nature 353: 858–861, 1991, Eur. J. Immunol., 23: 1552–1560, 1993. Although this term may be somewhat recondite, it is interesting that the Editor draws the intended analogy. Just as the proprioceptive system of inhibitory neurons provides negative feedback to control motor movements, we theorize that the negative feedback of propriocidal T cell death can govern the intensity of an immune response to prevailing conditions of antigen and IL-2. To avoid confusion we will clarify this term in the Introduction and Discussion.</p>", "<p>2. In reference to first sentence of \"Conclusions\" section of Abstract: This sentence is a speed bump for me. This is the first mention of IL-2 but the implication is that you see BIM upregulation under both conditions – restimulation and IL-2 withdrawal. Is the IL-2/BIM connection already published?</p>", "<title>Author Response</title>", "<p>As explained in the Introduction, the connection between IL-2 withdrawal and BIM upregulation is well established in the literature. However, we emphasize our novel finding that robust BIM upregulation in the presence of IL-2 upon TCR restimulation suggests a different signal emanating from the TCR can override BIM destabilization normally conferred by IL-2R signaling.</p>", "<p>3. In reference to Introduction (pg 4, paragraph 2): This sentence seems a little off point. If I understand correctly, it is illustrating how Fas might play a role beyond T cells in controlling autoimmunity, not how TCR-induced death can be mediated via a non-Fas mechanism.</p>", "<title>Author Response</title>", "<p>We agree with the reviewer's opinion, and have modified the sentence in question to emphasize that these data suggest Fas is less important for killing activated T cells in the context of repeated antigen restimulation than previously presumed. The next sentence provides further detail on this point.</p>", "<p>4. You need to confirm that the SM1/23 antibody is saturating. Have you tried a dose-response where it flattens below 100%? Does this adequately address the issue of saturating antibody, as PBL were used in both cases.</p>", "<title>Author Response</title>", "<p>We have confirmed that use of SM1/23 Fas blocking Ab is saturating at the 1 μg/ml dose used throughout the study, both in primary human T cells as well as Jurkat T cell lines. Adding more than 1 μg/ml did not provide additional protection.</p>", "<p>5. In reference to sentence on pg 10 (\"IL-2 signaling <italic>normally </italic>destabilizes BIM mRNA or promotes BIM protein degradation via Raf/ERK or phosphoinositide kinase 3 (PI-3K) signaling pathways.\") Is this the right word or would \"alone\" be better?</p>", "<title>Author Response</title>", "<p>Per the reviewer's suggestion, we have modified the sentence to read \"IL-2 signaling alone can destabilize BIM mRNA...\"</p>", "<p>6. In reference to Bim knockdown in CD8<sup>+ </sup>T cells (Figure ##FIG##2##3B##, Additional File ##SUPPL##2##3##): Could you titrate your siRNA so that you get a similar level of residual Bim in CD8s and see if you get the same compromise in apoptosis rescue?</p>", "<title>Author Response</title>", "<p>We appreciate the reviewer's suggestion, and will try this approach in subsequent studies. On the other hand, our experience indicates that adding more siRNA does not improve BIM knockdown and may lead to off-target effects, which is why we did not pursue this further in the context of the current study.</p>", "<p>7. Insert \"T cell\" on pg 13 to clarify the term \"cross-tolerance\".</p>", "<title>Author Response</title>", "<p>The term is added as suggested, thank you.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>The authors wish to thank the patients for their participation in NIH ALPS studies, T. Myers and Q. Su in the NIAID Research Technologies Branch for help with microarray hybridization and analysis, K. Nagashima and J. de la Cruz at SAIC-Frederick for electron microscopy analysis, and members of the Lenardo laboratory for helpful suggestions. This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. A.L.S. was supported by a Pharmacology Research Associate Training (PRAT) Program Fellowship, National Institute of General Medical Sciences.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>TCR re-stimulation signals mitochondrial-dependent apoptosis independent of FAS</bold>. (A) Electron micrographs (upper panels, 2500× magnification or lower panels, 10000×) of activated human PBL either not restimulated (NRS) or restimulated with OKT3 mAb for 18 h. Arrows indicate apoptotic cells. (B) Activated human T cells were untreated (NRS) or restimulated with OKT3 for 24 h in the presence of FAS blocking Ab (SM1/23) or isotype control Ab. Cells were stained with PI and analyzed by flow cytometry; gates indicate % viable cells. (C) Activated human T cells from a normal donor or ALPS 1a patient were untreated (NRS) or restimulated with OKT3 for 12 h in the presence of FAS blocking Ab (SM1/23) or isotype control Ab. Cells were stained with DiOC6 and analyzed by flow cytometry (right panels). Viable gates are shown at left, and the percentage of DiOC6 low cells are indicated in the histograms on the right. (D) Cytosolic extracts from activated human PBL were immunoblotted for the presence of cytochrome c following stimulation with OKT3 or staurosporine (STS) for the indicated timepoints, in the presence or absence of SM1/23 Ab. (E) Lysates prepared as described in (D) were incubated with the caspase 9 specific substrate LEHD-pNA for 2 h, and caspase 9 enzymatic activity was quantitated as OD at 405 nm minus background (OD405 at 5 min).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Induction of BIM expression contributes to TCR-induced apoptosis</bold>. (A) Mircoarray analysis of designated Bcl-2 family members was performed using RNA purified from activated human PBL either untreated (0 h) or stimulated with OKT3 for 6 h. Relative expression values normalized to reference RNA from normal human PBL are shown at left, fold change following TCR restimulation is quantitated at right. (B) Activated human PBL were stimulated with OKT3 for the indicated times, and whole cell lysates were prepared and immunoblotted for the proteins indicated on the right. All three isoforms of BIM (extra-long (EL), long (L), short (S)) were detected. Spot densitometry analysis of the ratio of BIM-EL to BCL-xL (normalized to β-actin loading control) is plotted below. (C) Activated human PBL were transfected with nonspecific (NS) or Bim-specific siRNA, rested 4 days, and then restimulated for 24 h with increasing doses of OKT3 in the presence or absence of SM1/23. Percent cell loss was calculated in triplicate by PI exclusion. Differences in apoptosis sensitivity (relative to NS alone) were statistically significant for each dose of OKT3 (p &lt; 0.04), except for SM1/23 treated NS cells at 1 μg/ml. (D) Lysates from cells transfected in (C) were immunoblotted for BIM as in (B). β-actin serves as a loading control. (E) Average extent of TCR-induced apoptosis inhibition (relative to NS siRNA alone) is shown for each condition described in (D) for 6 different normal donor PBL tested.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Bim is important for TCR-induced apoptosis of CD8<sup>+ </sup>T cells</bold>. (A) CD4<sup>+ </sup>or CD8<sup>+ </sup>T cells purified from activated human PBL were transfected with NS or Bim-specific siRNA, rested 4 days, then restimulated with increasing doses of OKT3 in the presence or absence of SM1/23. Percent cell loss was calculated in triplicate by PI exclusion. Differences in apoptosis sensitivity were statistically significant for SM1/23 treated CD4<sup>+ </sup>cells (NS and Bim) compared to NS cells alone (p &lt; 0.007), except for SM1/23 treated NS cells at 1 μg/ml OKT3 (p &lt; 0.07). Differences in apoptosis sensitivity for CD8<sup>+ </sup>T cells (relative to NS alone) were all statistically significant (p &lt; 0.05). (B) Lysates from cells transfected in (A) were immunoblotted for BIM. β-actin serves as a loading control.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Fas and Bim cooperate in driving TCR-induced apoptosis of murine T cells</bold>. (A) Activated splenic T cells from wild-type (WT), <italic>lpr</italic>, or <italic>bim</italic><sup>-/- </sup>mice were restimulated with platebound anti-CD3 for 24 h. Percent cell loss was calculated in triplicate by PI exclusion. (B) Lysates from splenic T cells from the indicated genetic backgrounds left untreated or restimulated with platebound anti-CD3 for 6 h were immunoblotted for Bim isoform expression. β-actin serves as a loading control; asterisk indicates non-specific band. (C, D) CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells were purified from activated WT, <italic>lpr</italic>, or <italic>bim</italic><sup>-/- </sup>splenocytes and restimulated with platebound anti-CD3 for 24 h. Percent cell loss was calculated in triplicate by PI exclusion. (E) Splenic T cells from WT or <italic>lpr </italic>mice were stimulated for 48 h with platebound anti-CD3/anti-CD28, washed, and transfected with NS or Bim-specific siRNA. Three days post-transfection, cells were restimulated with 100 ng/ml platebound anti-CD3; percent cell loss was calculated in triplicate by PI exclusion. Differences in apoptosis sensitivity (relative to NS-treated WT cells) were statistically significant (p &lt; 0.04). Lysates made from cells three days post-transfection were assessed for Bim knockdown by immunoblotting, right.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Suppression of Bim expression in ALPS type IV patient causes resistance to TCR-induced death</bold>. (A) Activated human PBL from normal control donors (NC1, NC2), or 6 ALPS type Ia patients were restimulated with increasing doses of OKT3 for 24 h. Percent cell loss was calculated in triplicate by PI exclusion. (B) Activated human PBL from an ALPS type Ib patient or a normal control donor (NC) were treated as in (A). Percent cell loss was calculated in triplicate by PI exclusion. (C) Activated human PBL from normal control donors (NC1, NC2), or 6 ALPS type Ia patients restimulated with 100 ng/ml OKT3 for 0 (-) or 8 h (+), lysed and immunoblotted for BIM. β-actin serves as a loading control. Spot densitometry analysis of the ratio of BIM (EL isoform) to β-actin (normalized to NC1 untreated, dashed line) is plotted below. (D) Activated human PBL from normal control donors (NC1, NC2), an ALPS type Ia patient, and an ALPS Type IV patient (P58) were restimulated with OKT3 for 24 h. Percent cell loss was calculated in triplicate by PI exclusion. Differences in apoptosis sensitivity (relative to NC1 or NC2) were statistically significant (p &lt; 0.01). (E) Activated human PBL as in (D) were restimulated with 100 ng/ml OKT3 for 0 (-) or 8 h (+), lysed and immunoblotted for BIM. β-actin serves as a loading control.</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional File 1</title><p><bold>Blockade of Fas-induced apoptosis in SM1/23 treated PBL</bold>. Activated PBL were pre-treated with 1 ug/ml SM1/23 Ab prior to addition of increasing amounts of APO1.3 Ab. Percent cell loss was calculated 24 h later by PI exclusion in triplicate.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional File 2</title><p><bold>Bim siRNA effectively suppresses restimulation-induced BIM expression in activated T cells</bold>. Activated human PBL, purified CD4+ and CD8+ T cells were transfected with nonspecific (NS) or Bim-specific siRNA and rested for 4 days. Lysates were made from cells left untreated (0 h) or restimulated for 8 h with 100 ng/ml OKT3. Knockdown of protein expression was confirmed by immunoblotting.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional File 3</title><p><bold>Knockdown of FADD or Bim expression results in partial resistance to TCR-induced death</bold>. Activated human PBL were transfected with nonspecific (NS), FADD-specific, or Bim-specific siRNA, rested for 4 days, and then restimulated for 24 h with increasing doses of OKT3 in the presence or absence of Fas blocking Ab (SM1/23). Percent cell loss was calculated in triplicate by PI exclusion (left panel). Knockdown of protein expression was confirmed by immunoblotting in whole lysates 4 days post-transfection (right panel).</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S4\"><caption><title>Additional File 4</title><p><bold>Knockdown of PUMA results in partial resistance to TCR-induced death</bold>. Activated human PBL were transfected with nonspecific (NS), Puma-specific, or Bim-specific siRNA, rested for 4 days, and then restimulated for 24 h with increasing doses of OKT3 in the presence or absence of Fas blocking Ab (SM1/23). Percent cell loss was calculated in triplicate by PI exclusion (left panel). Knockdown of protein expression was confirmed by immunoblotting in whole lysates 4 days post-transfection (right panel).</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S5\"><caption><title>Additional File 5</title><p><bold>BIM induction in CD4<sup>+ </sup>vs CD8<sup>+ </sup>human T cells</bold>. CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells were purified from activated human PBL and restimulated with OKT3 for the indicated times. Whole cell lysates were prepared, separated by SDS-PAGE, and immunoblotted for BIM isoforms or BCL-2 expression. β-actin serves as a loading control.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S6\"><caption><title>Additional File 6</title><p><bold>BIM siRNA impairs IL-2 withdrawal apoptosis in both CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells</bold>. Purified CD4<sup>+ </sup>and CD8<sup>+ </sup>T cells were transfected with nonspecific (NS) or Bim-specific siRNA and rested 24 hrs in IL-2. IL-2 was removed by thorough washing, and percent cell loss was calculated 72 and 96 hrs later by PI exclusion (left panel). The percent of apoptosis inhibition afforded by BIM siRNA (relative to NS) is graphed in the right panel.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S7\"><caption><title>Additional File 7</title><p><bold>Impaired Fas-induced apoptosis in ALPS Ia patients</bold>. Activated human PBL from normal control donors (NC1, NC2), or 6 ALPS type Ia patients were treated with increasing doses of APO1.3 mAb for 24 h. Percent cell loss was calculated in triplicate by PI exclusion.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S8\"><caption><title>Additional File 8</title><p><bold>ALPS Type IV patient T cells express functional FAS</bold>. (A) Activated PBL from a normal control donor (NC, open histogram), an ALPS type Ia patient (gray), or ALPS type IV (P58, black) were stained with FITC-conjugated anti-CD95 or isotype control Ab (dashed line) and analyzed by flow cytometry. (B) Activated PBL from a normal donor (NC), an ALPS type IV patient (P58) and an ALPS type Ia patient were treated with 20 or 200 ng/ml APO1.3 mAb plus 200 ng/ml Protein A. Percent cell loss was calculated in triplicate by PI exclusion.</p></caption></supplementary-material>" ]
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{ "acronym": [], "definition": [] }
47
CC BY
no
2022-01-12 14:47:25
Biol Direct. 2008 Aug 20; 3:34
oa_package/00/a5/PMC2529272.tar.gz
PMC2529273
18554397
[ "<title>Background</title>", "<p>GABA<sub>A </sub>receptors (GABA<sub>A</sub>-R) are pentameric membrane proteins that belong to the superfamily of cys-loop ligand-gated ion channels (LGIC), which operate as GABA-gated Cl<sup>-</sup>-selective channels. GABA<sub>A</sub>-R mediate most of the fast inhibitory neurotransmission in the CNS [##REF##8578436##1##, ####REF##7568326##2##, ##UREF##0##3####0##3##]. Initially, two subunits of the GABA<sub>A</sub>-R named α and β were purified [##UREF##1##4##,##REF##6304068##5##] and subsequently their cDNAs were isolated [##REF##6202691##6##]. Twenty related GABA<sub>A</sub>-R subunits have been so far identified in mammals (α<sub>1–6</sub>, β<sub>1–4</sub>, γ<sub>1–3</sub>, δ, ε, π, θ, and ρ<sub>1–3 </sub>[##REF##3037384##7##,##REF##9647870##8##]), yielding a high degree of potential diversity. If all of these subunits could randomly co-assemble, more than one hundred thousand GABA<sub>A</sub>-R subtypes with distinct subunit composition and arrangement would be formed [##REF##10449790##9##]. The composition of the most abundant GABA<sub>A</sub>-R type in the CNS is αβγ, and immunohistochemistry studies suggest that receptors containing α<sub>1</sub>, β<sub>2/3 </sub>and γ<sub>2 </sub>subunits are the most widespread GABA<sub>A</sub>-R subtype in adult mammalian brain and represent about 50% of the total receptor pool [##REF##7568326##2##,##REF##1661244##10##].</p>", "<p>Typical αβγ GABA<sub>A</sub>-Rs harbor two agonist (GABA) binding sites located at the two α/β subunit interfaces [##REF##7568326##2##,##REF##9824848##11##]. The function of GABA<sub>A</sub>-Rs can be modulated by various compounds acting at different allosteric sites located on GABA<sub>A</sub>-Rs. The benzodiazepine (BZD) site, which is located at an α/γ interface [##REF##10366619##12##,##REF##11438573##13##], is the most frequently targeted site for therapeutic agents, and ligands that enhance GABA<sub>A</sub>-R function through positive modulation at this site possess anxiolytic, sedative, myorelaxant, anesthetic and amnestic properties [##REF##7568326##2##,##UREF##0##3##,##REF##1661244##10##,##UREF##2##14##]. Based on pharmacological studies in transgenic mice, it has been proposed that GABA<sub>A</sub>-Rs can be classified into the following pharmacological classes according to the effects of BZ site ligands: α<sub>1</sub>-containing receptors (GABA<sub>A1</sub>) that mediate sedative effects; α<sub>2</sub>-containing receptors (GABA<sub>A2</sub>) that mediate anxiolytic effects; α<sub>3</sub>-containing receptors (GABA<sub>A3</sub>) that mediate myorelaxation; and α<sub>5</sub>-containing receptors (GABA<sub>A5</sub>) that are involved in learning and memory processes [##REF##3037384##7##,##UREF##3##15##,##REF##11021797##16##]. This classification is consistent with the sedative/hypnotic profile of GABA<sub>A1</sub>-preferring compounds such as zolpidem and zaleplon [##REF##14744255##17##], but pharmacological studies in wild-type animals and in man have raised questions regarding the attribution of anxiolytic effects to GABA<sub>A2 </sub>receptors. In particular, a number of compounds have been identified that exhibit an anxioselective profile <italic>in vivo </italic>despite lacking the expected GABA<sub>A2 </sub>selectivity. A series of compounds with mixed preference for α<sub>2</sub>/α<sub>3</sub>-containing receptors has been reported to produce robust anxiolysis in animals without noticeable sedation, including one compound that exhibits selectivity for α<sub>3</sub>-containing receptors [##REF##15252823##18##, ####REF##15655523##19##, ##REF##16279764##20##, ##REF##16392789##21####16392789##21##]. Other compounds, such as ocinaplon [##REF##16610803##22##] and DOV 51,892 [##REF##15870187##23##], are anxiolytic in humans and animals without undesired side effects such as sedation and myorelaxation, but do not exhibit strong selectivity among GABA<sub>A</sub>-Rs sensitive to benzodiazepines (that is, those receptors containing α<sub>1–3 </sub>and/or α<sub>5</sub>-subunits)</p>", "<p>One hypothesis that could explain the anxioselective profile of ocinaplon is the presence of one or more biotransformation products that exhibit selectivity at GABA<sub>A2 </sub>receptors. To test this hypothesis, we characterized the pharmacological properties of the major biotransformation product of ocinaplon in dogs, rats and man, DOV 315,090 (Fig. ##FIG##0##1##), using <italic>in vitro </italic>radioligand binding and two-electrode voltage-clamp electrophysiology. We now report that like its parent compound, DOV 315,090 acts as a positive modulator at GABA receptors, and like its parent, does not exhibit marked selectivity among α<sub>1–3 </sub>and α<sub>5 </sub>containing receptors. Thus, while DOV 315,090 may contribute to the pharmacological actions of ocinaplon, the anxioselective profile of ocinaplon cannot be explained on the basis of enhanced subunit selectivity on the part of DOV 315,090.</p>" ]
[ "<title>Methods</title>", "<title>Radioligand Binding Assays</title>", "<p>HEK293 cells (CRL 1573, American type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco's modified Eagle's medium (D-MEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% MEM Non-Essential Amino Acids Solution (Invitrogen, Carlsbad, CA, USA). cDNAs encoding rat GABA<sub>A</sub>-R subunits were in the following vectors: α<sub>1 </sub>and α<sub>5 </sub>in pRc/CMV, α<sub>2</sub>, α<sub>3</sub>, γ<sub>2S </sub>and γ<sub>3 </sub>in pcDNA3 and β<sub>2 </sub>in pcDNA1. The cells were transiently transfected (5 μg of each cDNA per 100 mm dish) using FuGene™ (Roche Diagnostics Corporation) at a 3:1 FuGene:DNA ratio. Transfection efficiency was 50–80% as measured by co-transfection with green fluorescent protein cDNA (2.5 μg/100 mm dish). Forty-eight hours after transfection, cells were washed with ice-cold PBS, harvested and homogenized. Cell homogenates were centrifuged (100,000 <italic>g</italic>, 25 min) and washed three times by homogenization in ice-cold PBS buffer followed by centrifugation at 100,000 <italic>g </italic>for 25 min. The final pellets were stored at -20°C until needed.</p>", "<p>For competition binding, 100 μg of membrane protein was incubated in 500 μl of PBS buffer with 0.5 nM [<sup>3</sup>H]Ro15–1788 (78.6 Ci/mmol, PerkinElmer Life Sciences) in the presence of diazepam (1 nM – 10 μM, Sigma-Aldrich), ocinaplon (0.1 – 250 μM, DOV Pharmaceuticals) or DOV 315,090 (0.1 – 50 μM, DOV Pharmaceuticals) for 1 h at 0°C. The samples were then diluted with 5 ml of ice-cold buffer and filtered under vacuum through glass-fiber filters (GF/B Whatman). Filters were washed 3 times with 5 ml of buffer and the radioactivity was quantitated by liquid scintillation counting in 5 ml of Ecolite scintillation fluid (ICN). Non-specific binding determined in the presence of 100 μM Ro 15–1788 (Sigma-Aldrich) was subtracted from total binding to calculate specific binding. Data were analyzed by non-linear regression (Prism, Graph-Pad software).</p>", "<title>Recording of GABA-Gated Currents from GABA<sub>A </sub>Receptors Expressed in Xenopus Oocytes</title>", "<p>cRNAs encoding GABA<sub>A</sub>-R α<sub>1</sub>, α<sub>2</sub>, α<sub>3</sub>, α<sub>5</sub>, β<sub>2 </sub>and γ<sub>2S </sub>subunits were injected into oocytes from <italic>Xenopus laevis</italic>. Forty-eight hours later, measurements of the effects of diazepam, ocinaplon and DOV 315,090 on GABA-gated Cl<sup>- </sup>currents from oocytes expressing GABA<sub>A</sub>-Rs were performed using a Warner TEVC amplifier (Warner Instruments, Inc., Foster City, CA) (Park-Chung et al., 1999). GABA (Sigma) was prepared as a 1 M stock solution in ND96. Microelectrodes of 1–3 MΩ when filled with 3 M KCl were used to record from oocytes in a recording chamber continuously perfused with ND-96 buffer solution. During data acquisition, oocytes were clamped at a holding potential of -70 mV. Drugs were applied by perfusion at a rate of approximately 50 μl sec<sup>-1 </sup>for 20 s followed by a 120 s wash. At the end of each experiment 3 μM of diazepam was applied as a potentiation control. All experiments were performed at room temperature (22–24°C).</p>", "<p>GABA concentration-response data was obtained for each subunit combination, and the GABA EC<sub>10 </sub>was determined by nonlinear regression using the logistic equation. This concentration of GABA was used for modulation studies. Peak current measurements were normalized and expressed as a fraction of the peak control current measurements. Control responses to an EC<sub>10 </sub>concentration of GABA were re-determined after every 2 – 4 applications of modulator + GABA. Percent potentiation is defined as [I<sub>(GABA + Drug)</sub>/I<sub>GABA</sub>)-1] × 100, where I<sub>(GABA + Drug) </sub>is the current response in the presence of diazepam, and I<sub>GABA </sub>is the control GABA current. Potentiation data from each oocyte was fitted to the equation Potentiation = E<sub>max </sub>× [Drug]/([Drug + EC<sub>50</sub>) by non-linear regression (Prism, Graph-Pad software). Due to a decline in the response at high diazepam concentrations, concentrations of diazepam above 3 μM were excluded from the fit. Some oocytes expressing α1β1γ2 receptors appeared to exhibit a biphasic modulatory response to diazepam, suggesting the possible presence of an additional component of modulation with a sub-nM EC<sub>50</sub>. For 6 of 8 oocytes, the fit was significantly improved by adding a second, higher-potency component of modulation, but the affinity of this second component was not well resolved in fitting due to its small amplitude. Given the lack of consistency of this possible high affinity effect, we have omitted it in fitting our concentration-effect curves. The choice of fitting to a monophasic or biphasic equation had only a small effect on the EC<sub>50 </sub>for the major component of modulation. For diazepam, the mean EC<sub>50 </sub>of the major component was increased from 35 nM to 42 nM when a two-component fit was used for those oocytes in which it produced a significant improvement in the sum of squares.</p>" ]
[ "<title>Results</title>", "<title>Biotransformation of ocinaplon into DOV 315,090 <italic>in vivo</italic></title>", "<p>As shown in Figure ##FIG##1##2##, DOV 315,090 appears rapidly in plasma following i.v. or oral administration of a behaviorally active dose of ocinaplon (5 mg/kg) to rats. At 1 h, corresponding to the time at which the anticonflict effect of ocinaplon was evaluated [##REF##16610803##22##], the plasma concentration of DOV 315,090 is ~38% of the concentration of parent compound.</p>", "<title>Comparison of the binding properties of diazepam, ocinaplon and DOV 315,090</title>", "<p>Figure ##FIG##2##3## and Table ##TAB##0##1## document the binding properties of diazepam, ocinaplon and DOV 315,090 in HEK293 cells expressing different GABA<sub>A</sub>-R subunit combinations. Examination of binding constants shows that ocinaplon and DOV 315,090 have lower affinity than diazepam at all of the receptor subunit combinations tested. The binding profile of DOV 315,090 is similar to that of ocinaplon, with little selectivity among the subunit combinations tested. In contrast to diazepam, which exhibits markedly lower affinity for α<sub>1</sub>β<sub>2</sub>γ<sub>3 </sub>and α<sub>2</sub>β<sub>2</sub>γ<sub>3 </sub>receptors than for α<sub>1</sub>β<sub>2</sub>γ<sub>2 </sub>s and α<sub>2</sub>β<sub>2</sub>γ<sub>2 </sub>s receptors, replacement of γ<sub>2S </sub>with γ<sub>3 </sub>had little effect on the affinity of either ocinaplon or DOV 315,090 for any subunit combination (Table ##TAB##0##1##). Also, whereas diazepam has similar affinity for α<sub>1</sub>-containing and α<sub>2</sub>-containing receptors, both ocinaplon and DOV 315,090 have 3–4 fold lower affinity for α<sub>2</sub>-containing receptors. Specific [<sup>3</sup>H]Ro15–1788 or [<sup>3</sup>H]flunitrazepam binding to membrane preparations from cells transfected with α<sub>3</sub>, β<sub>2 </sub>and γ<sub>3 </sub>subunits was not detected, suggesting that these subunits failed to assemble in the HEK293 cells.</p>", "<title>Modulation of GABA<sub>A</sub>-R function by diazepam, ocinaplon and DOV 315,090</title>", "<p>Consistent with previous studies [##REF##16610803##22##,##REF##15870187##23##], the potency and efficacy of ocinaplon were lower than diazepam at the four receptor subtypes analyzed. The highest efficacy was observed at receptors containing α<sub>3 </sub>subunits (Table ##TAB##1##2##). DOV 315,090 also exhibited the highest maximal potentiation at α<sub>3</sub>-containing receptors; however, its E<sub>max </sub>values were similar to those of diazepam at receptors containing α<sub>1 </sub>or α<sub>3 </sub>subunits (Table ##TAB##1##2##).</p>", "<p>DOV 315,090 and ocinaplon exhibited similar efficacies (150% vs. 139% potentiation, respectively) and EC<sub>50</sub>s (12.5 μM vs. 9.12 μM, respectively, n = 4) at α<sub>2</sub>β<sub>2</sub>γ<sub>2S </sub>receptors (Figure ##FIG##3##4##, Table ##TAB##1##2##). In contrast, whereas ocinaplon and DOV 315,090 were approximately equipotent at α<sub>3</sub>β<sub>2</sub>γ<sub>2S </sub>receptors (EC<sub>50 </sub>= 8.01 μM and 10.21 μM, respectively), the efficacy of DOV 315,090 was almost 1.87 fold greater than that of ocinaplon (340% vs 181% potentiation) (Figure ##FIG##3##4##, Table ##TAB##1##2##). Finally, DOV 315,090 was less efficacious and potent than ocinaplon at α<sub>5</sub>β<sub>2</sub>γ<sub>2S </sub>receptors (Figure ##FIG##3##4##, Table ##TAB##1##2##). The rank order of potency (EC<sub>50</sub>) of the pyrazolopyrimidines at enhancing GABA-gated chloride currents in receptors containing different α subunits was: α<sub>2</sub>≈α<sub>3</sub>≈α<sub>5 </sub>&lt; α<sub>1 </sub>for DOV 315,090, compared to α<sub>2</sub>≈α<sub>3 </sub>&lt; α<sub>5</sub>≈α<sub>1 </sub>for ocinaplon. Furthermore, DOV 315,090 and ocinaplon had different efficacy (E<sub>max</sub>) profiles; the rank order of absolute efficacy was α<sub>5 </sub>&lt; α<sub>2 </sub>&lt; α<sub>1 </sub>&lt; α<sub>3 </sub>for DOV 315,090, as compared with α<sub>5 </sub>&lt; α<sub>1 </sub>&lt; α<sub>2 </sub>&lt; α<sub>3 </sub>for ocinaplon.</p>" ]
[ "<title>Discussion</title>", "<p>In the CNS, classical 1,4-BZDs such as diazepam, as well as other ligands of the BZD binding site, act on GABA<sub>A</sub>-Rs that are composed of α, β, and γ subunits. The majority of GABA<sub>A </sub>receptors contain α<sub>1–6</sub>, β<sub>2/3 </sub>and γ<sub>2 </sub>subunits, whereas the β<sub>1 </sub>and γ<sub>1/3 </sub>subunits have very restricted patterns of expression [##REF##7568326##2##]. It has been shown that BZD pharmacology is primarily dependent upon the α subunit subtype present (α<sub>1–3 </sub>or α<sub>5</sub>), whereas receptors containing α<sub>4 </sub>or α<sub>6 </sub>subunits are insensitive to \"classical\" 1,4-BZDs [##REF##3037384##7##,##REF##16971504##24##,##REF##2166916##25##]. Studies of animals in which genes coding for specific α subunits have been deleted or mutated to eliminate BZD sensitivity (e.g. the α<sub>1</sub>H101R mutation, which disrupts the BZD binding site) led to the hypothesis that the sedative effects of the BZDs are mediated by α<sub>1</sub>-subunit containing receptors (designated GABA<sub>A1</sub>-R), whereas anxiolytic effects are mediated by α<sub>2</sub>-subunit containing receptors (GABA<sub>A2</sub>-R) [##REF##3037384##7##,##REF##14744255##17##,##REF##8719416##26##,##REF##10548105##27##]. GABA<sub>A</sub>-Rs containing α<sub>5 </sub>subunits are thought to be responsible for the impairment of learning and memory that is induced by BZDs [##REF##10816315##28##]. These finding raised the attractive prospect that BZD-like drugs that specifically target GABA<sub>A</sub>-Rs that contain a specific α-subunit will be able to produce the intended pharmacological effect (e.g sedation or anxiolysis) with reduced incidence of side effects. Because BZD-like drugs function as allosteric modulators and do not occupy the GABA binding site, specificity may be potentially achieved on the basis of either differences in potency or on differences in modulatory efficacy at specific receptor subtypes.</p>", "<p>Compounds such as zolpidem and zaleplon, which exhibit higher affinity for α1-containing receptors relative to other subtypes, have been promoted as sedative agents, driven in part by the hypothesis that selectivity for GABA<sub>A1</sub>-Rs would translate into an improved side-effect profile, particularly with respect to tolerance, withdrawal, and abuse liability. Although these compounds are effective sedative agents, consistent with the identification of GABA<sub>A1</sub>-Rs as mediating sedation, the selectivity of these compounds for GABA<sub>A1</sub>-Rs vs. GABA<sub>A</sub>-Rs containing other α-subunits is generally an order of magnitude or less, and it is unclear to what extent the hypothesized benefits are achieved in clinical practice [##REF##14744255##17##].</p>", "<p>However, the situation is less clear for compounds possessing anxiolytic properties. Recently published articles describe the pharmacological properties of two novel anxioselective compounds – ocinaplon [##REF##16610803##22##] and DOV 51892 [##REF##15870187##23##]. These compounds do not exhibit a marked selectivity among GABA<sub>A</sub>-Rs containing different diazepam-sensitive subunits (e.g. α<sub>1–3 </sub>and α<sub>5</sub>), yet are reported to be anxioselective, lacking sedative and myorelaxant side effects at anxiolytic doses. In particular, DOV 51892 exhibits higher efficacy than diazepam at GABA<sub>A1</sub>-Rs.</p>", "<p>The classic BZD diazepam has been shown to act with high efficacy and similar potency across a broad spectrum of GABA<sub>A</sub>-Rs [##REF##8578436##1##,##REF##1661244##10##,##REF##16610803##22##] (Table ##TAB##1##2##). This lack of selectivity with respect to either potency or efficacy among the major GABA<sub>A</sub>-R types have been hypothesized to account for the side effects associated with the use of diazepam when used as an anxiolytic, which include sedation, myorelaxation, narcosis, and amnesia. However, as has been confirmed by <italic>in vivo </italic>behavioral studies, such side effects are not observed with ocinaplon (e.g. in motor activity test, inclined screen and rod walking) or for DOV 51892 (e.g. rotarod and grip strength tests), even at doses well in excess of those that enhanced punished responding in the thirsty rat test [##REF##16610803##22##,##REF##15870187##23##]. Further, ocinaplon is an effective anxiolytic in humans at doses that do not produce BZD-like side effects [##REF##16610803##22##]. The present study was designed to test whether the anxioselective profile of ocinaplon is due to metabolism into subtype-selective metabolites. Our pharmacokinetic data demonstrate that in rats, the major metabolite of ocinaplon is a 4'-N' oxide, DOV 315,090. Whereas DOV 315,090 is active as a GABA<sub>A</sub>-R modulator, and its <italic>in vitro </italic>binding affinities for recombinant α<sub>1</sub>β<sub>2</sub>γ<sub>2S</sub>, α<sub>2</sub>β<sub>2</sub>γ<sub>2S</sub>, and α<sub>3</sub>β<sub>2</sub>γ<sub>2S </sub>receptors differ only marginally from ocinaplon, its affinity for α<sub>5</sub>β<sub>2</sub>γ<sub>2S </sub>receptors is only slightly lower than that of ocinaplon (~2-fold).</p>", "<p>Comparison of the pharmacological profile of ocinaplon and DOV 315,090 using two electrode voltage clamp electrophysiology (Table ##TAB##1##2##) shows that the greatest difference in efficacy occurred at α<sub>3</sub>β<sub>2</sub>γ<sub>2S </sub>receptors. Although a clear maximum was not attained due to solubility limits, the extrapolated maximum potentiation by DOV 315,090 was 1.87-fold greater, followed by a 1.45-fold difference at α<sub>1</sub>β<sub>2</sub>γ<sub>2S </sub>receptors compared to ocinaplon. In contrast, maximum potentiation by DOV 315,090 was lower than that of ocinaplon at the α<sub>5</sub>β<sub>2</sub>γ<sub>2S </sub>receptor subtype. The efficacies of DOV 315,090 and ocinaplon at α<sub>2</sub>β<sub>2</sub>γ<sub>2S </sub>receptors were similar.</p>", "<p>These results do not support the hypothesis that the anxioselective profile of ocinaplon is attributable to enhanced selectivity of its metabolite DOV 315,090 for α<sub>2</sub>-containing receptors. Thus, compared to ocinaplon, DOV 315,090 does not exhibit enhanced affinity or potency for α<sub>2</sub>-containing receptors over α<sub>1</sub>-containing receptors, whereas the difference in efficacy favors α<sub>3</sub>-, α<sub>5</sub>-, or α<sub>1</sub>-containing receptors over α<sub>2</sub>-containing receptors. The present experiments examined GABA<sub>A</sub>-Rs in two different heterologous expression systems (<italic>Xenopus </italic>oocytes and HEK 293 cells), which may be lacking modulatory proteins or regulatory mechanisms that are only present in neurons. While we cannot exclude the possibility that such interactions somehow confer differences in modulator binding or efficacy, such a hypothesis would require that such interactions modify the structure of the benzodiazepine binding site, which is located in the extracellular domain of the GABA<sub>A</sub>-R, in such a way as to selectively alter its interactions with different ligands.</p>", "<p>Recent studies suggest that GABA<sub>A3</sub>-Rs receptors are also important in mediating anxiolysis [##REF##15252823##18##,##REF##16279764##20##,##REF##16326923##31##, ####REF##16706856##32##, ##REF##16386900##33##, ##REF##16291941##34####16291941##34##]. DOV 315,090 has relatively high efficacy at α<sub>3</sub>β<sub>2</sub>γ<sub>2S</sub>, so it is likely that modulation of GABA<sub>A3</sub>-Rs by DOV 315,090 contributes to the anxioselective profile of ocinaplon; however, adipiplon (NG2-73), an α<sub>3</sub>-selective positive modulator, has been reported to have sedative/hypnotic activity [##REF##15644443##35##], suggesting that α<sub>3 </sub>selectivity is not sufficient to confer anxioselectivity.</p>", "<p>In summary, transgenic mice in which the BZD recognition site of the α<sub>2 </sub>subunit is disabled exhibit reduced diazepam sensitivity in behavioral tests considered to be predictive of anxiolytic activity, and a similar modification to the α<sub>1 </sub>subunit reduces sensitivity in tests held to be predictive of sedation [##UREF##3##15##,##REF##8719416##26##]. These observations have led to optimism that it will be possible to achieve the long-desired goal of developing a nonsedating anxiolytic [##UREF##4##36##]. And indeed, there has been substantial progress in identifying such compounds [##REF##15655523##19##, ####REF##16279764##20##, ##REF##16392789##21##, ##REF##16610803##22####16610803##22##,##REF##16326923##31##,##REF##12801796##37##, ####REF##11454940##38##, ##REF##16480260##39##, ##REF##16183706##40####16183706##40##], yet ironically, they do not in general conform to the expected paradigm of favoring α<sub>2</sub>-containing over α<sub>1</sub>-containing receptors. This suggests that anxiolysis in humans may prove to be more complex than is suggested by a simple reading of the results from transgenic mice in behavioral models thought to be indicative of anxiety. It remains to be determined whether differences in the design of the behavioral assays [##REF##17092968##41##,##REF##15698896##42##], interspecies differences [##REF##15501303##43##,##REF##11498724##44##], or a combination of these factors account for these discrepancies. Translating such promising results into clinically useful compounds is likely to require an improved understanding of the ways in which BZD-like ligands act at different GABA<sub>A</sub>-R subtypes and the consequences of these effects upon neural system-mediated behavioral outputs.</p>" ]
[ "<title>Conclusion</title>", "<p>1. DOV315090 is a major metabolite of the anxioselective GABA<sub>A</sub>-R modulator ocinaplon.</p>", "<p>2. DOV 315,090 possesses modulatory activity at α<sub>1</sub>-, α<sub>2</sub>-, α<sub>3</sub>-, and α<sub>5</sub>-containing GABA<sub>A</sub>-Rs with a selectivity profile similar to that of ocinaplon.</p>", "<p>3. The anxioselective properties of ocinaplon, demonstrated in both preclinical and clinical studies, are not a consequence of enhanced subtype selectivity by DOV315090.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Compounds targeting the benzodiazepine binding site of the GABA<sub>A</sub>-R are widely prescribed for the treatment of anxiety disorders, epilepsy, and insomnia as well as for pre-anesthetic sedation and muscle relaxation. It has been hypothesized that these various pharmacological effects are mediated by different GABA<sub>A</sub>-R subtypes. If this hypothesis is correct, then it may be possible to develop compounds targeting particular GABA<sub>A</sub>-R subtypes as, for example, selective anxiolytics with a diminished side effect profile. The pyrazolo[1,5-a]-pyrimidine ocinaplon is anxioselective in both preclinical studies and in patients with generalized anxiety disorder, but does not exhibit the selectivity between α<sub>1</sub>/α<sub>2</sub>-containing receptors for an anxioselective that is predicted by studies using transgenic mice.</p>", "<title>Results</title>", "<p>We hypothesized that the pharmacological properties of ocinaplon <italic>in vivo </italic>might be influenced by an active biotransformation product with greater selectivity for the α<sub>2 </sub>subunit relative to α<sub>1</sub>. One hour after administration of ocinaplon, the plasma concentration of its primary biotransformation product, DOV 315,090, is 38% of the parent compound. The pharmacological properties of DOV 315,090 were assessed using radioligand binding studies and two-electrode voltage clamp electrophysiology. We report that DOV 315,090 possesses modulatory activity at GABA<sub>A</sub>-Rs, but that its selectivity profile is similar to that of ocinaplon.</p>", "<title>Conclusion</title>", "<p>These findings imply that DOV 315,090 could contribute to the action of ocinaplon in vivo, but that the anxioselective properties of ocinaplon cannot be readily explained by a subtype selective effect/action of DOV 315,090. Further inquiry is required to identify the extent to which different subtypes are involved in the anxiolytic and other pharmacological effects of GABA<sub>A</sub>-R modulators.</p>" ]
[ "<title>Abbreviations</title>", "<p>cDNA: complementary deoxyribonucleic acid; cRNA: complementary ribonucleic acid; DOV 51892: (7-(2-chloropyridin-4-yl)pyrazolo- [1,5-<italic>a</italic>]-pyrimidin-3-yl](pyridin-2-yl)methanone); ocinaplon, (2-pyridinyl [7-(4-pyridinyl)pyrazolo[1,5-a]pyrimidin-3-yl]methanone); DOV 315,090: (7-(1-Oxidopyridin-1-ium-4-yl)pyrazolo [1,5-a]pyrimidin-3-yl)(pyridin-2-yl)methanone, GABA, γ-aminobutyric acid; I<sub>GABA</sub>: GABA-gated current.</p>", "<title>Authors' contributions</title>", "<p>DB carried out electrophysiological recordings. MCG carried out radioligand binding experiments. EK performed initial electrophysiological experiments. SD developed the data-acquisition hardware and software used in this study. TTG participated in the design of the study, performed the statistical analysis and participated in manuscript preparation. DHF participated in the design of the study and participated in manuscript preparation. PS directed development of ocinaplon at DOV Pharmaceuticals and participated in manuscript preparation. ASB identified major ocinaplon metabolite and participated in manuscript preparation. All authors read and approved the final manuscript.</p>" ]
[ "<title>Acknowledgements</title>", "<p>This work was supported by NIMH R01MH049469 (DB, MCG, EK, TTG, DHF).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Structures of diazepam, ocinaplon and DOV 315,090.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Pharmacokinetics of ocinaplon and DOV 315,090.</bold> Blood levels of ocinaplon (●,○) and DOV 315090 (▲,△) were determined at various times after i.v. (●,▲) or oral (○,△) administration of 5 mg/kg ocinaplon to rats. Plotted results do not include one animal that exhibited a low blood level (0.47 μg/ml) of ocinaplon at the initial 10 min time point after oral administration and proportionally lower levels of both compounds throughout the duration of the experiment. This animal may have regurgitated a portion of the dose (of the suspension).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Displacement curves of [<sup>3</sup>H]Ro 15–1788 binding by diazepam (DZ), ocinaplon and DOV 315,090 in homogenates of HEK293 cells transfected with different subunit combinations.</bold> Smooth curves are calculated from the mean parameter values in Table 1.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Potentiation of GABA-gated currents by diazepam, ocinaplon and DOV 315,090.</bold> Rat GABA<sub>A</sub>-Rs consisting of α<sub>1</sub>β<sub>2</sub>γ<sub>2S</sub>, α<sub>2</sub>β<sub>2</sub>γ<sub>2S</sub>, α<sub>3</sub>β<sub>2</sub>γ<sub>2S </sub>and α<sub>5</sub>β<sub>2</sub>γ<sub>2S </sub>subunits were expressed in <italic>Xenopus </italic>oocytes. Potentiation was determined using an EC<sub>10 </sub>concentration of GABA (~10 μM for α<sub>1</sub>β<sub>2</sub>γ<sub>2S</sub>, α<sub>2</sub>β<sub>2</sub>γ<sub>2S </sub>and α<sub>3</sub>β<sub>2</sub>γ<sub>2S</sub>; ~5 μM for the α<sub>5</sub>β<sub>2</sub>γ<sub>2S</sub>). Curves were calculated by normalizing values of relative currents obtained following administration of diazepam (○), ocinaplon (●) or DOV 315,090 (□) in the presence of GABA (from at least four oocytes harvested from at least two batches) to the value obtained following application of GABA. The dose-response curves of diazepam were fitted up to 3 μM. Higher concentrations (in parentheses) were excluded from the fit due to a decline in potentiation at higher concentrations. Smooth curves are calculated based on mean parameter values given in Table 2. Asterisks indicate fits for which the extrapolated E<sub>max </sub>is more than 25% greater than the maximum potentiation observed at highest drug concentration.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Binding affinity of diazepam, ocinaplon and DOV 315,090 for GABA<sub>A</sub>-Rs with different subunit composition.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\" colspan=\"2\">Receptor Type</td><td align=\"left\"><bold>α<sub>1</sub>β<sub>2</sub>γ<sub>2S</sub></bold></td><td align=\"left\"><bold>α<sub>1</sub>β<sub>2</sub>γ<sub>3</sub></bold></td><td align=\"left\"><bold>α<sub>2</sub>β<sub>2</sub>γ<sub>2S</sub></bold></td><td align=\"left\"><bold>α<sub>2</sub>β<sub>2</sub>γ<sub>3</sub></bold></td><td align=\"left\"><bold>α<sub>3</sub>β<sub>2</sub>γ<sub>2S</sub></bold></td><td align=\"left\"><bold>α<sub>5</sub>β<sub>2</sub>γ<sub>2S</sub></bold></td><td align=\"left\"><bold>α<sub>5</sub>β<sub>2</sub>γ<sub>3</sub></bold></td></tr></thead><tbody><tr><td align=\"left\">diazepam (DZ)</td><td align=\"left\">IC<sub>50 </sub>(μM)</td><td align=\"left\">0.03</td><td align=\"left\">0.22</td><td align=\"left\">0.04</td><td align=\"left\">0.21</td><td align=\"left\">0.05</td><td align=\"left\">0.03</td><td align=\"left\">0.09</td></tr><tr><td/><td align=\"left\">pIC<sub>50</sub></td><td align=\"left\">7.54 ± 0.09</td><td align=\"left\">6.67 ± 0.08</td><td align=\"left\">7.50 ± 0.10</td><td align=\"left\">6.80 ± 0.26</td><td align=\"left\">7.32 ± 0.08</td><td align=\"left\">7.57 ± 0.13</td><td align=\"left\">7.09 ± 0.13</td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"left\">ocinaplon (OC)</td><td align=\"left\">IC<sub>50 </sub>(μM)</td><td align=\"left\">6.3</td><td align=\"left\">2.3</td><td align=\"left\">24</td><td align=\"left\">20</td><td align=\"left\">7.7</td><td align=\"left\">9.6</td><td align=\"left\">10</td></tr><tr><td/><td align=\"left\">pIC<sub>50</sub></td><td align=\"left\">5.20 ± 0.14</td><td align=\"left\">5.65 ± 0.01</td><td align=\"left\">4.62 ± 0.14</td><td align=\"left\">4.74 ± 0.15</td><td align=\"left\">5.12 ± 0.06</td><td align=\"left\">5.02 ± 0.03</td><td align=\"left\">5.01 ± 0.18</td></tr><tr><td/><td align=\"left\">IC<sub>50</sub>/DZ IC<sub>50</sub></td><td align=\"left\">218</td><td align=\"left\">10.5</td><td align=\"left\">759</td><td align=\"left\">115</td><td align=\"left\">158</td><td align=\"left\">355</td><td align=\"left\">120</td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"left\">DOV 315,090</td><td align=\"left\">IC<sub>50 </sub>(μM)</td><td align=\"left\">7.0</td><td align=\"left\">5.5</td><td align=\"left\">24</td><td align=\"left\">20</td><td align=\"left\">9.3</td><td align=\"left\">22</td><td align=\"left\">27</td></tr><tr><td/><td align=\"left\">pIC<sub>50</sub></td><td align=\"left\">5.19 ± 0.12</td><td align=\"left\">5.27 ± 0.07</td><td align=\"left\">4.63 ± 0.05</td><td align=\"left\">4.72 ± 0.09</td><td align=\"left\">5.08 ± 0.14</td><td align=\"left\">4.67 ± 0.08</td><td align=\"left\">4.58 ± 0.09</td></tr><tr><td/><td align=\"left\">IC<sub>50</sub>/DZ IC<sub>50</sub></td><td align=\"left\">220</td><td align=\"left\">25</td><td align=\"left\">760</td><td align=\"left\">120</td><td align=\"left\">170</td><td align=\"left\">790</td><td align=\"left\">323</td></tr><tr><td/><td align=\"left\">IC<sub>50</sub>/OC IC<sub>50</sub></td><td align=\"left\">1.02</td><td align=\"left\">2.40</td><td align=\"left\">0.89</td><td align=\"left\">0.98</td><td align=\"left\">1.09</td><td align=\"left\">2.24</td><td align=\"left\">2.59</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Properties of diazepam, ocinaplon and DOV315090 determined by two-electrode voltage clamp electrophysiology using Xenopus oocytes injected with cRNA.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Receptor Type</td><td/><td align=\"left\"><bold>α<sub>1</sub>β<sub>2</sub>γ<sub>2S</sub></bold></td><td align=\"left\"><bold>α<sub>2</sub>β<sub>2</sub>γ<sub>2S</sub></bold></td><td align=\"left\"><bold>α<sub>3</sub>β<sub>2</sub>γ<sub>2S</sub></bold></td><td align=\"left\"><bold>α<sub>5</sub>β<sub>2</sub>γ<sub>2S</sub></bold></td></tr></thead><tbody><tr><td align=\"left\">diazepam (DZ)</td><td align=\"left\">EC<sub>50 </sub>(μM)</td><td align=\"left\">0.04 (8)</td><td align=\"left\">0.03 (10)</td><td align=\"left\">0.092 (5)</td><td align=\"left\">0.025 (5)</td></tr><tr><td/><td align=\"left\">pEC<sub>50</sub></td><td align=\"left\">7.46 ± 0.07</td><td align=\"left\">7.60 ± 0.044</td><td align=\"left\">7.04 ± 0.05</td><td align=\"left\">7.51 ± 0.11</td></tr><tr><td/><td align=\"left\">E<sub>max</sub>, %</td><td align=\"left\">144 ± 8.0</td><td align=\"left\">157 ± 14</td><td align=\"left\">232 ± 31</td><td align=\"left\">224 ± 24</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\">ocinaplon (OC)</td><td align=\"left\">EC<sub>50 </sub>(μM)</td><td align=\"left\">2.93 (4)</td><td align=\"left\">9.12 (5)</td><td align=\"left\">8.01 (4)</td><td align=\"left\">3.5 (4)</td></tr><tr><td/><td align=\"left\">pEC<sub>50</sub></td><td align=\"left\">5.57 ± 0.11</td><td align=\"left\">5.04 ± 0.03</td><td align=\"left\">5.16 ± 0.14</td><td align=\"left\">5.48 ± 0.07</td></tr><tr><td/><td align=\"left\">EC<sub>50</sub>/DZ EC<sub>50</sub></td><td align=\"left\">77</td><td align=\"left\">350</td><td align=\"left\">87</td><td align=\"left\">139</td></tr><tr><td/><td align=\"left\">E<sub>max</sub>, %</td><td align=\"left\">132 ± 8</td><td align=\"left\">150 ± 6</td><td align=\"left\">181 ± 18</td><td align=\"left\">84 ± 4</td></tr><tr><td/><td align=\"left\">E<sub>max</sub>/DZ E<sub>max</sub></td><td align=\"left\">0.91</td><td align=\"left\">0.95</td><td align=\"left\">0.78</td><td align=\"left\">0.37</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\">DOV315090 (MET)</td><td align=\"left\">EC<sub>50 </sub>(μM)</td><td align=\"left\">4.87 (4)</td><td align=\"left\">12.5 (4)</td><td align=\"left\">10.21 (4)</td><td align=\"left\">10.14 (4)</td></tr><tr><td/><td align=\"left\">pEC<sub>50</sub></td><td align=\"left\">6.32 ± 0.05</td><td align=\"left\">4.92 ± 0.09</td><td align=\"left\">5.00 ± 0.05</td><td align=\"left\">5.03 ± 0.10</td></tr><tr><td/><td align=\"left\">EC<sub>50</sub>/DZ EC<sub>50</sub></td><td align=\"left\">128</td><td align=\"left\">482</td><td align=\"left\">111</td><td align=\"left\">405</td></tr><tr><td/><td align=\"left\">EC<sub>50</sub>/OC EC<sub>50</sub></td><td align=\"left\">1.66</td><td align=\"left\">1.37</td><td align=\"left\">1.27</td><td align=\"left\">2.92</td></tr><tr><td/><td align=\"left\">E<sub>max</sub>, %</td><td align=\"left\">192 ± 4</td><td align=\"left\">139 ± 23 *</td><td align=\"left\">340 ± 35 *</td><td align=\"left\">68 ± 8</td></tr><tr><td/><td align=\"left\">E<sub>max</sub>/DZ E<sub>max</sub></td><td align=\"left\">1.33</td><td align=\"left\">0.88</td><td align=\"left\">1.46</td><td align=\"left\">0.30</td></tr><tr><td/><td align=\"left\">E<sub>max</sub>/OC E<sub>max</sub></td><td align=\"left\">1.45</td><td align=\"left\">0.92</td><td align=\"left\">1.87</td><td align=\"left\">0.81</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><p>IC<sub>50 </sub>values were calculated from [<sup>3</sup>H]Ro15–1788 displacement curves using non-linear regression analysis for each independent experiment. pIC<sub>50 </sub>values are averages of the negative logarithms of IC<sub>50</sub>s. Results from each experiment (n = 3) were fitted independently and fitted parameters were averaged to calculate means and SEM. EC<sub>50 </sub>values were averaged as their negative logarithms (pIC<sub>50</sub>).</p></table-wrap-foot>", "<table-wrap-foot><p>Drugs were prepared from DMSO stock solution prior to experiment, EC<sub>10</sub>s of GABA were used, errors are SEM of fitted parameter values from the number of oocytes given in parentheses. Results from each oocyte were fitted independently and fitted parameters were averaged to calculate means and SEM. EC<sub>50 </sub>values were averaged as their negative logarithms (pEC<sub>50</sub>) * For these two cases, the extrapolated E<sub>max </sub>exceeded the observed maximum observed potentiation by over 25%, but parameter SEM was not substantially increased, indicating that range of concentrations was adequate to project E<sub>max</sub>. Higher drug concentrations could not be used due to solubility constraints.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1471-2210-8-11-1\"/>", "<graphic xlink:href=\"1471-2210-8-11-2\"/>", "<graphic xlink:href=\"1471-2210-8-11-3\"/>", "<graphic xlink:href=\"1471-2210-8-11-4\"/>" ]
[]
[{"surname": ["Sieghart", "Sperk"], "given-names": ["W", "G"], "article-title": ["Subunit composition, distribution and function of GABA"], "sub": ["A "], "source": ["Cur Top Med Chem"], "year": ["2002"], "volume": ["2"], "fpage": ["795"], "lpage": ["816"], "pub-id": ["10.2174/1568026023393507"]}, {"surname": ["Berezhnoy", "Gravielle", "Farb", "Sibley DR, Hanin I, Kuhar M, Skolnick P"], "given-names": ["D", "MC", "DH"], "article-title": ["Pharmacology of the GABA"], "sub": ["A "], "source": ["Handbook of Contemporary Neuropharmacology"], "year": ["2007"], "volume": ["1"], "edition": ["25"], "publisher-name": ["New York: Wiley and Sons"], "fpage": ["465"], "lpage": ["569"]}, {"surname": ["Boileau", "Kucken", "Evers", "Czajkowski"], "given-names": ["AJ", "AM", "AR", "C"], "article-title": ["Molecular dissection of benzodiazepine binding and allosteric coupling using chimeric \u03b3-aminobutyric acid A receptor subunits"], "source": ["Mol Pharm"], "year": ["1998"], "volume": ["53"], "fpage": ["295"], "lpage": ["303"]}, {"surname": ["Sieghart", "Kasper S, Boer J, Sitsen JM"], "given-names": ["W"], "article-title": ["Benzodiazepines, Benzodiazepine receptor, and Endogenous Ligands"], "source": ["Handbook of anxiety and depression"], "year": ["2003"], "publisher-name": ["New York, Basel: Marcel Dekker"], "fpage": ["415"], "lpage": ["442"]}, {"surname": ["Kehne", "McCloskey", "Peterson", "Near", "Bradshaw", "Natoli", "Crandall", "Matchett", "Xu", "Yu", "Maynard", "Xie", "Smith", "White", "Rajachandran", "Krause"], "given-names": ["JH", "TC", "S", "NearK", "E", "J", "M", "M", "Y", "W", "G", "L", "MD", "HS", "L", "JE"], "suffix": ["1", "IV"], "article-title": ["Further pharmacological exploration of \u03b13-subunit preferring GABAA receptor partial allosteric activators: Evidence for anxiolysis and reduced sedative tolerance of NDT 9530021"], "source": ["Neuroscience Meeting Planner"], "year": ["2007"], "comment": ["Program No. 632.6. Online."]}]
{ "acronym": [], "definition": [] }
44
CC BY
no
2022-01-12 14:47:25
BMC Pharmacol. 2008 Jun 13; 8:11
oa_package/e5/c5/PMC2529273.tar.gz
PMC2529274
18691432
[ "<title>Background</title>", "<p>Dementia of vascular origin i.e. Vascular dementia (VaD) has gained much attention in the recent times. After Alzheimer disease (AD), VaD is the second most common cause of dementia. In the vascular system, nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) plays an important role in maintenance of vascular tone [##REF##15198963##1##]. Hyperhomocysteinemia (Hhcy), or elevation of plasma total homocysteine, is an important risk factor for cardiovascular disease, stroke and vascular dementia [##REF##16155257##2##, ####REF##12387654##3##, ##REF##15935398##4####15935398##4##]. Hhcy has been shown to induce endothelial dysfunction by decreasing the bioavailability of NO, and increasing vascular oxidative stress [##REF##17416288##5##]. The decreased NO level has been demonstrated to contribute to the pathogenesis of dementia [##REF##17556102##6##].</p>", "<p>Increased levels of homocysteine have been documented to produce changes in structure and function of cerebral blood vessels along with oxidative stress, which play a key role in cerebral vascular dysfunction [##REF##16043641##7##]. Oxidative stress and vascular dysfunction are recognized as important contributing factors in the pathogenesis of AD and other dementia of vascular origin [##REF##17556102##6##]. In AD and other neurodegenerative diseases, structural deformities in the cerebral capillaries lead to impairment of cerebral perfusion with subsequent neuronal dysfunction and death [##REF##14697491##8##]. The well established risk factors of endothelial dysfunction and subsequent vascular dementia such as hypertension, history of stroke, diabetes mellitus and hypercholesterolemia are all associated with high risk of AD. The noted vascular dysfunction (vascular deformities) in AD and common risk factor of AD and VaD suggest a great overlap between AD and vascular dementia [##REF##8346443##9##]. Moreover, Hhcy has been documented to increase cholesterol synthesis [##UREF##0##10##]. Studies have revealed that in addition to elevated β-amyloid peptides and ApoE levels, high cholesterol level is another important risk factor for AD [##REF##16297842##11##].</p>", "<p>Only limited therapeutic interventions are available to reduce the incidence of VaD. Cholinesterase inhibitors, calcium channel blockers and glutamate antagonists are few classes of pharmacological agents which are being clinically explored to reduce symptomatically the impact of cognitive dysfunction associated with vascular dementia [##UREF##1##12##]. However, an agent that should improve both endothelial dysfunction and associated dementia still need to be explored. Very recently, the focus has been directed towards statins (HMG-CoA reductase inhibitors), which are most widely prescribed drugs for dyslipidemias [##REF##17603272##13##]. Statins in additions to their cholesterol lowering action are known to possess many cholesterol independent actions including favorable effect on vascular endothelium [##REF##17113151##14##]. Moreover, there is an emerging data indicating that statins exert neuroprotective and antioxidant actions [##REF##17113151##14##]. Statins have been shown to reduce the risk of ischemic stroke and related memory impairment by a variety of mechanisms [##REF##12615295##15##]. Epidemiological studies have suggested that individuals above 50 years of age, who were receiving statins, had a substantially lowered risk of developing dementia, independent of the presence or absence of untreated hyperlipidemia, or exposure to non-statin lipid-lowering drugs [##REF##12459888##16##]. However, there are conflicting observations regarding the effect of statins on cognitive functions. Although, there are a few studies showing cognitive decline [##REF##12885101##17##], some studies showing no effect on memory [##REF##10806282##18##,##REF##16864953##19##], yet few studies suggest improvement of cognitive functions with statin therapy. Therefore, implication of statins in endothelial dysfunction and related dementia deserves further investigation.</p>" ]
[ "<title>Methods</title>", "<title>Animals</title>", "<p>Age matched (six months old) male Wistar Albino rats, weighing 150–200 g were employed in the present study. Animals were procured from Institute of Veterinary Science, Izat-Nagar Bareilly (U.P), India. Rats were provided standard laboratory feed (Kisan Feeds Ltd, Chandigarh, India) and tap water ad libitum and were exposed to 12 h light and 12 h dark cycle. The animals were acclimatized to the laboratory condition before experiments. The experimental protocol was duly approved by Institutional Animal Ethics Committee (IAEC) and care of the animals was taken as per guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), ministry of Environment and Forests, Government of India, (Reg. No. 107/1999/CPCSEA).</p>", "<title>Drugs and Chemicals</title>", "<p>Atorvastatin was a gift from Ind swift Ltd., Mohali, Punjab, India. Pitavastatin was a gift from Zydus Research Center, Ahmedabad, Gujarat, India. All other reagents purchased from Merck limited, Mumbai, India, SD fine-chemicals limited, Mumbai, India, Loba Chem, Mumbai, India and Sigma-Aldrich, USA. Atorvastatin, Pitavastatin and L-Methionine were suspended in 0.5% w/v of carboxy methyl cellulose (CMC).</p>", "<title>L-Methionine Induced endothelial dysfunction and Vascular Dementia</title>", "<p>Rats, were administered L-Methionine (1.7 g/kg/day, p.o.) for 4 weeks to produce hyperhomocysteinemia-induced endothelial dysfunction [##REF##16841179##30##]. Assessment of vascular endothelial function was carried out by measuring acetylcholine induced endothelium dependent relaxation and sodium nitroprusside induced endothelium independent relaxation using isolated aortic ring preparation according to the method of Pieper [##REF##9217884##59##] together with estimation of serum nitrite concentration.</p>", "<p>Body weight of rats was monitored weekly. After 4 weeks, rats were subjected to Morris water maze test for the evaluation of their memory status. The L-Methionine treatment was continued during acquisition trials on Morris Water Maze.</p>", "<title>Morris Water Maze Test</title>", "<p>Morris Water Maze (MWM) test was employed to assess learning and memory of rats [##UREF##3##22##,##UREF##4##23##]. The MWM procedure was based on a principle, where the animals were placed in a large pool of water, as animals dislike swimming, their tendency was to escape from the water being accomplished by finding an escape platform. MWM consisted of large circular pool (150 cm in diameter, 45 cm in height), filled to a depth of 30 cm with water at 28 ± 1°C. The water was made opaque with non-toxic white colored dye. The tank was divided hypothetically, into four equal quadrants with help of two threads, fixed at right angle to each other on the rim of the pool. A submerged platform (10 cm<sup>2</sup>), painted in white was placed inside the target quadrants of this pool, 1 cm below surface of water. The position of platform was kept unaltered throughout the training session. Each animal was subjected to four consecutive trials on each day with gap of 5 min. The rat was gently placed in the water of the pool between quadrants, facing the wall of pool with drop location changing for each trial, and allowed 120 sec to locate submerged platform. Then, it was allowed to stay on the platform for another 20 sec. If it failed to find the platform within 120 sec, it was guided gently onto platform and allowed to remain there for 20 sec. Escape latency time (ELT) to locate the hidden platform in water maze was noted as an index of acquisition or learning. Animal was subjected to four acquisition trials daily for four consecutive days. On fifth day, the platform was removed and each rat was allowed to explore the pool for 120 sec. Mean time spent in all four quadrants was noted. The mean time spent by the animal in target quadrant searching for the hidden platform was noted as an index of retrieval.</p>", "<title>Acquisition Trial</title>", "<p>Each rat was subjected to four trials on each day. A rest period of 5 min was allowed in between each trial. Four trials per day were repeated for four consecutive days. Starting position on each day to conduct four acquisition trials was changed as described below and Q4 was maintained as target quadrant in all acquisition trials. Mean escape latency time (ELT) calculated for each day during acquisition trials and day 4 ELT was used as an index of acquisition (table ##TAB##2##3##).</p>", "<title>Retrieval Trial</title>", "<p>On fifth day the platform was removed. Rat was placed in water maze and allowed to explore the maze for 120 sec. Each rat was subjected to four such trials and each trial was started from different quadrant. Mean time spent in all three quadrants i.e. Q1, Q2 and Q3 were recorded and the time spent in the target quadrant i.e. Q4 in search of missing platform provided an index of retrieval. The experimenter always stood at the same position. Care was taken not to disturb the relative location of water maze with respect to other objects in the laboratory serving, as prominent visual clues. All the trials were completed between 09.00 to 17.00 hrs in semi sound proof laboratory.</p>", "<title>Biochemical Parameters</title>", "<title>Collection of sample</title>", "<p>The animals were sacrificed by cervical dislocation; thoracic aorta and brain tissue were carefully removed. Thoracic aorta was used for endothelium dependent and independent relaxation, whereas brain tissue was subjected to various biochemical estimations.</p>", "<p>The removed brains were homogenized in phosphate buffer (pH 7.4, 10% w/v) using Teflon homogenizer. The clear supernatant, obtained after centrifugation at 3000 rpm for 15 min, was used to estimate acetyl cholinesterase (AChE) activity, thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH) and protein content.</p>", "<p>Blood samples for biochemical estimation were collected just before sacrificing the rats. The blood was kept at room temperature for 30 min and then centrifuged at 4000 rpm for 15 min to separate serum. Serum was used to estimate serum homocysteine, serum nitrite concentration and total serum cholesterol.</p>", "<title>Estimation of serum homocysteine</title>", "<p>Determination of homocysteine was carried out using HPLC (Varian Inc., CA, USA) attached with fluorescent HPLC detector according to the method of <italic>Dimitrova et al </italic>[##REF##11861029##60##]. 100 μl of serum sample was added to 1.5 ml eppendorff containing 10 μl of water and 5 μl of n-amyl alcohol and was gently vortexed. 35 μl of sodium borohydrate reagent (35 μl of 1.43 M sodium borohydride in 0.1 M sodium hydroxide), 35 μl of 1 M of hydrochloric acid and 50 μl of 10 mM monobromobimane in 4 M sodium EDTA (pH 7) were added to eppendorff tube containing serum sample, capped, mixed and incubated at 42°C for 12 min. Then, it was cooled, vortexed and maintained at RT for 10 min. The sample was centrifuged at 12,200 g for 10 min to remove protein, acidic clear supernatant was separated and supernatant was adjusted to pH 4 using 25 μl of 2 M Tris-HCL. The sample was then centrifuged at 12,200 g for 1 min and 100 μl of supernatant was aliquoted for HPLC analysis.</p>", "<p>A fixed volume autosampler was used to inject 20 μl of sample into 4.6 × 250 mm RP8 ultrasphere column equipped with brownlee RP 18 new guard column. The solvent mixture in ratio of 94.75: 5: 0.25 of water: methanol: acetic acid was maintained at pH 3.4 using 5 M NaOH in pump A and 100% methanol was maintained in pump B. The injection rate of sample was maintained at 2 ml per min. HPLC detector was set with excitation wavelength at 390 nm and emission wavelength at 418 nm. The sensitivity range and rise time of detector were set at 0.1 sec and 2 sec respectively. The calibration curve for homocysteine as homocysteine-S-bimane was plotted using 100 μl quality control serum fortified with 10 μl homocysteine (1–1000 μM) solution. The data of calibration curve were regressed and the curve was used to calculate serum concentration of homocysteine.</p>", "<p>Rats with serum homocysteine levels of &gt; 10 μM were considered to be hyperhomocysteinemic.</p>", "<title>Estimation of serum nitrite concentration</title>", "<p>Serum nitrite concentration was estimated using method of Sastry <italic>et al. </italic>[##REF##12069417##24##]. 400 μl of carbonate buffer (pH 9.0) was added to 100 μl of serum or standards sample followed by addition of small amount (~0.15 g) of copper-cadmium alloy. The tubes were incubated at room temperature for 1 h to reduce nitrate to nitrite. The reaction was stopped by adding 100 μl of 0.35 M sodium hydroxide. Following this, 400 μl of zinc sulfate solution (120 mM) was added to deproteinate the serum samples. The samples were allowed to stand for 10 min and then centrifuged at 4000 g for 10 min. Greiss reagent (250 μl of 1.0% sulfanilamide prepared in 3 N HCl and 250 μl of 0.1% N-naphthylethylenediamine prepared in water) was added to aliquots (500 μl) of clear supernatant and serum nitrite was measured spectrophotometrically (DU 640B Spectrophotometer, Beckman Coulter Inc., CA, USA) at 545 nm. The standard curve of sodium nitrite (5 to 50 μM) was plotted to calculate concentration of serum nitrite.</p>", "<title>Estimation of total cholesterol</title>", "<p>Total serum cholesterol was estimated spectrophotometrically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 540 nm by CHOP/POD-phosphotungstate enzymatic method [##REF##4818200##61##] using commercially available conventional diagnostic kit (Monozyme India ltd., Secundrabad).</p>", "<title>Estimation of brain acetyl cholinesterase (AChE) activity</title>", "<p>The whole brain AChE activity was measured by the method of Ellman <italic>et al </italic>[##REF##13726518##62##] with slight modifications [##REF##5422218##63##]. This was measured on the basis of the formation of yellow colour due to the reaction of thiocholine with dithiobisnitrobenzoate ions. The rate of formation of thiocholine from acetylcholine iodide in the presence of brain cholinesterase was measured using a spectrophotometer. 0.5 ml of supernatant liquid of the brain homogenate was pipetted out into 25 ml volumetric flask and dilution was made with a freshly prepared DTNB {5,5'-dithiobis (2-nitro benzoic acid)} solution (10 mg DTNB in 100 ml of sorenson phosphate buffer, pH 8.0). From the volumetric flask, two 4 ml portions were pipetted out into two test tubes. Into one of the test tube, 2 drops of eserine solution was added. 1 ml of substrate solution (75 mg of acetylcholine iodide per 50 ml of distilled water) was pipetted out into both of the test tubes and incubated for 10 min. at 30°C. The solution containing eserine solution was used for zeroing the colorimeter and change in absorbance per min. of the sample was read spectrophotometrically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 420 nm. AChE activity was calculated using the following formula:</p>", "<p></p>", "<p>Where R = rate of enzyme activity in 'n' mole of acetylcholine iodide hydrolyzed/minute/mg protein</p>", "<p>δO.D. = Change in absorbance/minute</p>", "<p>E = Extinction coefficient = 13600/M/cm</p>", "<title>Estimation of brain thiobarbituric acid reactive species (TBARS) level</title>", "<p>The whole brain TBARS level was measured by the method of Ohokawa <italic>et al </italic>[##REF##36810##64##] with slight modifications. 0.2 ml brain homogenate was pipetted out in a test tube, followed by addition of 0.2 ml sodium dodecyl sulphate (SDS), 1.5 ml of 30% acetic acid (pH-3.5), 1.5 ml of 0.8% thiobarbituric acid (TBA) and make up the volume up to 4.0 ml with distilled water (DW). The test tubes were incubated at 95°C for 60 min., and then cool it. 1.0 ml of DW and 5.0 ml of n-butanol: pyridine (15: 1 v/v) mixture was added to the test tubes and centrifuged at the 4,000 × g for 10 min. The absorbance of developed colour in organic layer was Measured spectrophotometrically at 532 nm (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA). The absorbance from a standard curve generated using 1,1,3,3, tetra-methoxy propane as standard (range = 1 nmol – 10 nmol) was extrapolated.</p>", "<title>Estimation of brain reduced glutathione (GSH) level</title>", "<p>The whole brain GSH level was measured by the method of Beutler <italic>et al </italic>[##UREF##8##65##] with slight modifications. Tissue homogenate was taken and the proteins were precipitated with 10% w/v chilled trichloroacetic acid. Samples were kept in ice bath and were centrifuged after 30 min. at 1000 × g for 10 min. at 4°C. GSH levels were measured in the supernatant. 0.5 ml supernatant was mixed with 2.0 ml of 0.3 M disodium hydrogen phosphate solution and 0.25 ml of freshly prepared DTNB {5,5'-dithiobis (2-nitro benzoic acid)} solution (40 mg/100 ml in 1% w/v sodium citrate) was added just before measuring the absorbance spectrophotometrically at 412 nm (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA). Different concentration of GSH standard was also processed similarly to prepare a standard curve (5–50 μg) simultaneously. Results have been expressed as n mole of GSH/mg of protein.</p>", "<title>Estimation of brain total protein</title>", "<p>For the estimation of total protein in brain, method of Lowry <italic>et al </italic>[##REF##14907713##66##] with slight modifications was used. 150 μL of supernatant was taken in a test tube, volume was made up to 1 ml with distilled water than 5 ml of Lowry's reagent (freshly prepared mixture of 1% w/v copper sulphate, 2% w/v sodium potassium tartrate and 2% w/v sodium carbonate in 0.1 N NaOH in the ratio of 1:1:98 respectively), was added and mixed thoroughly. Mixture was allowed to stand for 15 minutes at room temperature and then 0.5 ml of 1:1 v/v diluted Folin-Ciacalteu reagent was added. Contents were vortexed and incubated at 37°C for 30 minutes. Then absorbance was determined spectrophotometrically at 750 nm (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) against suitably prepared blank. A standard curved using 25–200 mg of BSA was plotted. The amount of total protein was expressed in mg.</p>", "<title>Experimental Design</title>", "<p>Six groups, each group comprised 10 albino Wistar rats, were employed in the present study.</p>", "<title>Group I (Vehicle treated control)</title>", "<p>Rats were administered 0.5% w/v CMC (10 ml/kg/day, p.o.) for 4 weeks and then subjected to MWM test. The vehicle was also administered 45 min before acquisition trial conducted from day 1 to day 4 and retrieval trial conducted on day 5.</p>", "<title>Group II (L-Methionine treated)</title>", "<p>In order to induce hyperhomocysteinemia, the rats were administered L-methionine (1.7 g/kg/day, p.o.) for 4 weeks and then subjected to MWM test. The treatment of L-Methionine was continued (administered 45 min before) during acquisition trial conducted from day 1 to day 4. The animals were administered vehicle (0.5%w/v CMC, 10 ml/kg, p.o.) 45 min before retrieval trial conducted on day 5.</p>", "<title>Group III (Atorvastatin <italic>per se</italic>)</title>", "<p>Rats were administered Atorvastatin (10 mg/kg/day, p.o.) for 2 weeks and then subjected to MWM test. The treatment was continued (administered 45 min before) during acquisition trial conducted from day 1 to day 4. The animals were administered vehicle (0.5%w/v CMC, 10 ml/kg, p.o) 45 min before retrieval trial conducted on day 5.</p>", "<title>Group IV (Pitavastatin <italic>per se</italic>)</title>", "<p>Rats were administered Pitavastatin (10 mg/kg/day, p.o.) for 2 weeks and rest of protocol was same as mentioned in group III.</p>", "<title>Group V (L-Methionine + Atorvastatin treated)</title>", "<p>The hyperhomocysteinemic rats were treated with Atorvastatin (10 mg/kg/day, p.o.) for 2 weeks (3<sup>rd </sup>and 4<sup>th </sup>week of L-Methionine administration) and then subjected to MWM test. The co-administration of Atorvastatin and L-Methionine was continued (administered 45 min before) during acquisition trial conduct from day 1 to day 4. The animals were administered vehicle (0.5%w/v CMC, 10 ml/kg, p.o) 45 min before retrieval trial conducted on day 5.</p>", "<title>Group VI (L-Methionine + Pitavastatin treated)</title>", "<p>The hyperhomocysteinemic rats were treated with Pitavastatin (10 mg/kg/day, p.o.) and subjected to MWM test as described in group VI.</p>", "<title>Statistical analysis</title>", "<p>The results were expressed as mean ± standard error of means (S.E.M.) The data for isolated aortic ring preparation was statistically analyzed using repeated measure ANOVA followed by Newman-Keul's test. Rest of the data obtained from various groups was statistically analyzed using one-way ANOVA followed by Tukey's Multiple Range test. The <italic>p </italic>&lt; 0.05 was considered to be statistically significant.</p>" ]
[ "<title>Results</title>", "<title>Effect of Vehicle/Atorvastatin/Pitavastatin/L-Methionine on escape latency time (ELT) and time spent in target quadrant (TSTQ), using Morris water maze (MWM)</title>", "<p>Vehicle treated (0.5%w/v CMC, 10 ml/kg/p.o.) rats showed a downward trend in their ELT. There was a significant (<italic>p </italic>&lt; 0.01) fall in day 4 ELT, when compared to day 1 ELT of these rats (Table ##TAB##0##1##), reflecting normal learning ability. Further on day 5 a significant (<italic>p </italic>&lt; 0.01) rise in TSTQ was observed, when compared to time spent in other quadrants (Figure ##FIG##0##1##), reflecting normal retrieval as well.</p>", "<p>Administration of Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant per se effect on ELT and TSTQ as compared to vehicle treated rats. (Table ##TAB##0##1## and Figure ##FIG##0##1##) L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day) administration produced a significant increase (<italic>p </italic>&lt; 0.05) in day 4 ELT, when compared to day 4 ELT of vehicle control (Table ##TAB##0##1##) indicating impairment of acquisition. Further L-Methionine administration also produced a significant (<italic>p </italic>&lt; 0.01) decrease in TSTQ, when compared TSTQ of vehicle control animals (Figure ##FIG##0##1##), indicating impairment of memory as well.</p>", "<title>Effect of Atorvastatin/Pitavastatin on L-Methionine induced impairment of learning and memory using Morris water maze</title>", "<p>Administration of Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day), Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day), significantly (<italic>p </italic>&lt; 0.05) prevented L-Methionine induced rise in day 4 ELT, indicating reversal of L-methionine induced impairment of acquisition (Table ##TAB##0##1##). Further treatment of these drugs also attenuated L-Methionine induced decrease in day 5 TSTQ in a significant manner (<italic>p </italic>&lt; 0.05), indicating reversal of L-methionine induced impairment of memory (Figure ##FIG##0##1##).</p>", "<title>Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in endothelium dependent relaxation</title>", "<p>Acetylcholine (ACh) and sodium nitroprusside (SNP) in a dose dependent manner produced endothelium dependent and independent relaxation in phenylephrine (3 × 10<sup>-6 </sup>M) precontracted isolated rat aortic ring preparation. L-methionine (1.7 g/kg/<italic>p.o</italic>., 4 weeks and 4 day) administration significantly (<italic>p </italic>&lt; 0.05) attenuated acetylcholine induced endothelium dependent relaxation (Figure ##FIG##1##2##), however it did not affect SNP induced endothelium independent relaxation (Figure ##FIG##2##3##). Treatment of Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day), Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day), significantly (<italic>p </italic>&lt; 0.05) abolished the effect of L-Methionine on endothelial dependent relaxation. Further Atorvastatin and Pitavastatin did not show any per se effect on endothelium dependent relaxation.</p>", "<title>Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in serum homocysteine level</title>", "<p>Administration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day), produced a significant (<italic>p </italic>&lt; 0.01) increase in serum homocysteine, when compared to vehicle treated rats. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) produced a significant (<italic>p </italic>&lt; 0.05) reduction of L-methionine induced rise in serum homocysteine level (Table ##TAB##1##2##). Further, Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant <italic>per se </italic>effect on serum homocysteine level (Table ##TAB##1##2##).</p>", "<title>Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in serum nitrite level</title>", "<p>Administration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day), produced a significant (<italic>p </italic>&lt; 0.01) decrease in serum nitrite, when compared to vehicle treated rats. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) prevented L-methionine induced decrease in serum nitrite level in a significant (<italic>p </italic>&lt; 0.05) manner (Figure ##FIG##3##4##). Further, Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant <italic>per se </italic>effect on serum nitrite level (Figure ##FIG##3##4##).</p>", "<title>Effect of Atorvastatin/Pitavastatin/L-Methionine induced change in total serum cholesterol levels</title>", "<p>Administration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day) produced a significant (<italic>p </italic>&lt; 0.05), increase in total serum cholesterol levels of animals, when compared to vehicle control. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 days) attenuated L-Methionine induced rise in total serum cholesterol levels in a significant (<italic>p </italic>&lt; 0.05) manner (Figure ##FIG##4##5##). Furthermore Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant <italic>per se </italic>effect on total serum cholesterol levels (Figure ##FIG##2##3##), when compared to vehicle control group (Figure ##FIG##4##5##).</p>", "<title>Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in brain acetyl cholinesterase (AChE) activity</title>", "<p>Administration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day) produced a significant (<italic>p </italic>&lt; 0.05), increase in brain AChE activity, when compared to vehicle treated rats. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) significantly (<italic>p </italic>&lt; 0.05), prevented L-Methionine induced rise in brain AChE activity. Further Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant <italic>per se </italic>effect on brain AChE activity (Figure ##FIG##5##6##).</p>", "<title>Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in oxidative stress levels of brain</title>", "<p>Administration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day), produced a significant increase (<italic>p </italic>&lt; 0.05), in brain thiobarbituric acid reactive species (TBARS) level (Figure ##FIG##6##7##) and a decrease (<italic>p </italic>&lt; 0.01), in the level of reduced form of glutathione (GSH) (Figure ##FIG##7##8##), when compared to vehicle treated rats; hence reflecting induction of oxidative stress. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) significantly (<italic>p </italic>&lt; 0.05) prevented L-Methionine induced oxidative stress (Figure ##FIG##6##7##, ##FIG##7##8##). Further Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant <italic>per se </italic>effect on oxidative stress level (Figure ##FIG##6##7##, ##FIG##7##8##).</p>" ]
[ "<title>Discussion</title>", "<p>Young male rats were employed in the present study, as it is reported that aging and consequent variation of estrogen in blood modulates the activity of endothelial nitric oxide synthase (eNOS), which further affects the function of vascular endothelium and memory [##UREF##2##20##,##REF##15790731##21##]. Morris Water Maze test employed in present study is one of the most widely accepted models to evaluate learning and memory of the animals [##UREF##3##22##,##UREF##4##23##]. Nitric oxide (NO) synthesized in the endothelium and its levels get attenuated during endothelial dysfunction. Endogenously formed NO is highly unstable and gets converted to nitrate and nitrite [##REF##12069417##24##]. Therefore, serum nitrite concentration has been employed as specific a marker of endothelial dysfunction [##REF##17416288##5##].</p>", "<p>A significant decrease in escape latency time (ELT day 4) of control animals during ongoing acquisition trials denoted normal acquisition of memory and an increase in time spent in target quadrant (TSTQ), in search of missing platform during retrieval trial indicated, retrieval of memory. These results are consistent to our earlier findings and reports from other laboratory [##REF##17603272##13##,##UREF##5##25##,##REF##8946410##26##].</p>", "<p>L-Methionine treatment for 4 weeks, in the present study significantly raised serum homocysteine level, decreased serum nitrite levels and markedly attenuated acetylcholine induced endothelium dependent relaxation, therefore, reflecting endothelial dysfunction. Further L-methionine administration also produced a significant impairment of acquisition and retrieval of memory as reflected by decreased Morris water-maze performance. Moreover, an enhancement of brain acetyl cholinesterase (AChE) activity, increase in brain oxidative stress (as reflected by rise in brain TBARS and fall in GSH levels) and increase in serum total cholesterol levels were also observed. Recently, it has been reported that chronic experimental hyperhomocysteinemia produce cognitive dysfunction [##REF##15265632##27##,##REF##15882843##28##], and increase in brain AChE activity [##REF##17701348##29##]. L-Methionine induced hyperhomocysteinemia is a well established model of experimental endothelial dysfunction [##REF##16841179##30##,##REF##16442349##31##]. Hyperhomocysteinemia has been reported to induce endothelial dysfunction by decreasing the bioavailability of NO and by increasing vascular oxidative stress [##REF##17416288##5##]. Our observation also supports above contention, as a significant fall in serum nitrite levels along with rise in oxidative stress levels (increase TBARS and decrease GSH) were noted in L-methionine treated rats.</p>", "<p>The increased level of homocysteine has been reported to produce change in structure and function of cerebral blood vessels along with oxidative stress, which play a key role in cerebral vascular dysfunction [##REF##16043641##7##]. Several lines of evidences have strongly advocated a direct relationship between vascular endothelial dysfunction and dementia better known as vascular dementia [##REF##11730973##32##,##UREF##6##33##]. Cerebral vascular endothelial dysfunction has also been shown to enhance progression of dementia of Alzheimer disease (AD) [##UREF##6##33##]. Enhanced levels of brain AChE activity and oxidative stress have also been noted in patients suffering form dementia of AD and other dementias [##REF##11263760##34##].</p>", "<p>Further hyperhomocysteinemia has also been shown to be neurotoxic, and the neurotoxicity may be due to over activation of N-methyl-D-aspartate receptors or by enhanced vulnerability of hippocampal neuron to excitotoxic insults and amyloid β-peptide toxicity [##REF##9159176##35##,##REF##16697371##36##]. Moreover, methionine rich diet in rats has been demonstrated to enhance cholesterol concentration in the plasma and liver [##UREF##0##10##]. Several studies have also revealed high serum cholesterol level as another important risk factor of AD [##REF##10867775##37##]. Therefore L-Methionine induced memory dysfunction in the present study may be attributed to its multiple effects i.e. decrease in serum nitrite level (endothelial dysfunction), rise in oxidative stress level, enhancement of brain AChE activity, serum total cholesterol as well as direct neurotoxicity.</p>", "<p>In the present study, treatment with Atorvastatin and Pitavastatin significantly improved L-Methionine induced endothelial dysfunction manifested in the terms of endothelium dependent relaxation; increased serum nitrite levels, decreased serum homocysteine, and decreased oxidative stress (decrease TBARS and increase GSH). Statins, in addition to their cholesterol lowering action has been reported to exert number of cholesterol independent actions i.e. pleotropic actions. Statins have been demonstrated to enhance the expression of eNOS in human endothelial cells [##REF##9537338##38##]. They have been known to activate Akt/protein kinase B, which subsequently activates eNOS [##REF##10973320##39##]. Studies with Atorvastatin and Simvastatin have shown to inhibit the expression of prepro-endothelin (ET)-1 mRNA and reduce plasma ET-1 levels, endothelin being a potent vasoconstricting agent [##REF##9637705##40##]. Furthermore, statins appear to inhibit the synthesis of isoprenoids, compounds that are required for the posttranslational modification of important signaling molecules such as Rho, Rac, and Ras [##REF##1967820##41##]. Inhibition of Rho activation has been shown to increase endothelial NO production [##REF##9537338##38##] and reduces ET-1 expression [##REF##9637705##40##]. Recently, Atorvastatin has been documented to improve the function of endothelium by lowering the expression of p22phox and production of reactive oxygen species [##REF##16841179##30##,##REF##11408394##42##]. Further, in another recent report it has been indicated that hyperhomocysteinemia induce impairment of NO production through the modulation of Cav-1 expression associated with a loss of eNOS in caveolae [##REF##16616146##43##]. Statins have been shown to prevent the expression of caveolin, a negative regulator of eNOS [##REF##12652163##44##]. Therefore Atorvastatin and Pitavastatin induced improvement of endothelial dysfunction in our study may be attributed to their stimulatory effect on endothelial nitric oxide production and their antioxidative action.</p>", "<p>Further, administration of Atorvastatin/Pitavastatin in the present investigation also, reversed memory deficit induced by L-Methionine (hyperhomocysteinemia). Several recent clinical reports have suggested that the net brain cholesterol concentration is regulated by serum cholesterol level and there is a cross talk between the CNS and peripheral cholesterol pools [##REF##10787445##45##,##REF##11030791##46##]. Cholesterol turnover appears to play a crucial role in the deposition and clearance of amyloid peptide in brain and ApoE is a cholesterol transporting protein that is associated with amyloid deposits [##REF##8820179##47##,##REF##9111537##48##]. Further, serum cholesterol, atherosclerosis, apolipoprotein-E and AD all appear to be interconnected [##REF##9549720##49##,##REF##10439121##50##]. Studies involving cultured rat cortical neurons have revealed neuroprotective action of Atorvastatin against glutamate induced excitotoxicity [##REF##15748157##51##].</p>", "<p>In our recent studies, we have reported that Atorvastatin, Simvastatin and Pitavastatin reversed memory deficits of rats and mice associated with dementia of AD type and memory improving effects of these statins mediated through their cholesterol dependent and in dependent actions [##REF##17603272##13##,##UREF##5##25##]. Furthermore, statins have also been shown to exert beneficial effects in cerebral ischemia and stroke providing neuroprotection via enhancement of NO production [##REF##15375300##52##].</p>", "<p>Many studies, in the recent years have implicated a vital role for NO in neurophysiological process of learning and memory [##REF##7527843##53##]. Inhibition of NO system impaired memory in rats [##REF##11245824##54##,##REF##12024951##55##]. Whereas, stimulation of NO production improved cognitive functions in Alzheimer patients [##REF##10867219##56##]. NO, donors like molsidomine reversed scopolamine induced amnesia in rats [##REF##11527544##57##]. NO probably, acts as retrograde messenger in the formation of long term potentiation (LTP) at the molecular level of learning and memory processes [##UREF##7##58##]. Therefore, Atorvastatin and Pitavastatin in present investigation appear to reverse L-Methionine induced memory deficits via multiple actions viz; decrease in total cholesterol, brain oxidative stress (decrease TBARS and increase GSH) levels, AChE activity and increase in serum nitrite levels. Nevertheless, further studies incorporating female rats as well as other species of animals such as mice, using different memory model (other than Morris water-maze), duly supported by histopathology of brain tissue are required in order to support and extend potential of these statins in endothelial dysfunction related memory deficits.</p>" ]
[ "<title>Conclusion</title>", "<p>It may be concluded that chronic L-Methionine administration (hyperhomocysteinemia) has produced endothelial dysfunction along with impairment of learning and memory (vascular dementia). Atorvastatin and Pitavastatin exerted beneficial effects on endothelial dysfunction and related memory deficits by virtue of their cholesterol dependent as well as cholesterol independent actions. Perhaps this is the first report highlighting potential of Statins in L-Methionine induced endothelial dysfunction associated memory deficits.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Statins, HMG-CoA reductase inhibitors, are widely prescribed drugs for dyslipidemias. Recent studies have indicated number of cholesterol independent actions of statins including their beneficial effects on vascular endothelial dysfunction and memory deficits associated with dementia of Alzheimer's type. However the potential of statins in dementia of vascular origin still remains to be explored. Therefore, the present study has been designed to investigate the effect of Atorvastatin &amp; Pitavastatin on vascular endothelial dysfunction associated memory deficits in rats. In this study L-Methionine induced vascular dementia was assessed by Morris water-maze (MWM) test. Biochemical analysis was also performed to unfold possible mechanism of statins mediated modulation of vascular dementia.</p>", "<title>Results</title>", "<p>L-Methionine produced endothelial dysfunction as reflected by significant decrease in serum nitrite concentration. L-Methionine treated rats performed poorly on MWM indicating impairment of memory as well. These rats also showed a significant rise in brain oxidative stress, acetylcholinesterase (AChE) activity and serum total cholesterol levels. Both Atorvastatin as well as Pitavastatin attenuated L-Methionine induced endothelial dysfunction associated memory deficits. Statins also reversed L-Methionine induced rise in brain oxidative stress, AChE activity and serum cholesterol.</p>", "<title>Conclusion</title>", "<p>The beneficial effects of statins may be attributed to their multiple effects and the study highlights the potential of these drugs in vascular dementia.</p>" ]
[ "<title>Authors' contributions</title>", "<p>RUK carried out surgical operations, behavioral tests, and biochemical tests the data analysis in animal studies. BKS assisted in carrying out surgery, behavioral tests, and biochemical tests. ASJ carried out data analysis and participated in critical intellectual discussion and designing of the experiments. NS conceived the idea, coordinated the study, carried our data interpretation and drafted the manuscript. All authors read and approved the final manuscript.</p>" ]
[ "<title>Acknowledgements</title>", "<p>The authors would like to thank Dr. A.K. Tiwary for their constant support and keen interest in this project. We are also thankful to Ind swift Ltd., Mohali, Punjab, India, Zydus Research Center, Ahmedabad (Gujrat), India, for supplying the free sample of Atorvastatin and Pitavastatin respectively.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in day 5 Time Spent in Target quadrant (TSTQ), using Morris Water Maze</bold>. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = <italic>p </italic>&lt; 0.05 Time Spent in Q1, Q2, Q3, Quadrant Vs Q4 quadrant in Control. b = <italic>p </italic>&lt; 0.05 Vs Time Spent in Target Quadrant Q4 of Control. c = <italic>p </italic>&lt; 0.05 Vs Time Spent in Target Quadrant Q4 of L-Methionine treated.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Effect of Atorvastatin/Pitavastatin on L-Methionine change in endothelium dependent relaxation using isolated rat aortic ring preparation</bold>. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. Responses are expressed as % of maximum contraction induced by phenylephrine (3 × 10<sup>-6 </sup>M). a = <italic>p </italic>&lt; 0.05 Vs Control. b = <italic>p </italic>&lt; 0.05 Vs L-Methionine treated group.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Effect of Atorvastatin/Pitavastatin/L-Methionine induced on sodium nitroprusside induced endothelium independent relaxation using isolated rat aortic ring preparation</bold>. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. Responses are expressed as % of maximum contraction induced by phenylephrine (3 × 10<sup>-6 </sup>M).</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in serum nitrite level</bold>. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = <italic>p </italic>&lt; 0.01 Vs serum nitrite of Control. b = <italic>p </italic>&lt; 0.05 Vs serum nitrite of L-Methionine treated group.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in serum total cholesterol level</bold>. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = <italic>p </italic>&lt; 0.05 Vs serum total cholesterol of Control. b = <italic>p </italic>&lt; 0.05 Vs serum total cholesterol of L-Methionine treated.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes brain acetylcholinesterase (AChE) activity</bold>. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = <italic>p </italic>&lt; 0.05 brain AChE activity of Control. b = <italic>p </italic>&lt; 0.05 Vs brain AChE activity of L-Methionine treated.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p><bold>Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in brain thiobarbituric acid reactive species (TBARS) level</bold>. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = <italic>p </italic>&lt; 0.01 brain TBARS level of Control. b = <italic>p </italic>&lt; 0.05 Vs brain TBARS level of L-Methionine treated group.</p></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Figure 8</label><caption><p><bold>Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in brain reduced glutathione (GSH) level</bold>. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent Mean ± SEM. a = <italic>p </italic>&lt; 0.01 brain GSH level of Control. b = <italic>p </italic>&lt; 0.05 Vs brain GSH level of L-Methionine treated group.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in day 4 escape latency time (ELT), using Morris Water Maze.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Groups</bold></td><td align=\"center\"><bold>Treatment</bold></td><td align=\"center\"><bold>Dose (kg/day, <italic>p.o.</italic>)</bold></td><td align=\"center\"><bold>ELT (day 1) in sec</bold></td><td align=\"center\"><bold>ELT (day 4) in sec</bold></td></tr></thead><tbody><tr><td align=\"center\">I</td><td align=\"center\">Control</td><td align=\"center\">10 ml(0.5%w/w CMC)</td><td align=\"center\">81.5 ± 4.5</td><td align=\"center\">20.2 ± 2.2<sup>a</sup></td></tr><tr><td align=\"center\">II</td><td align=\"center\">L-Methionine</td><td align=\"center\">1.7 g</td><td align=\"center\">93.8 ± 4.2</td><td align=\"center\">49.9 ± 2.4<sup>b</sup></td></tr><tr><td align=\"center\">III</td><td align=\"center\">Atorvastatin <italic>per se</italic></td><td align=\"center\">10 mg</td><td align=\"center\">85.5 ± 4.1</td><td align=\"center\">22.4 ± 3.4</td></tr><tr><td align=\"center\">IV</td><td align=\"center\">Pitavastatin <italic>per se</italic></td><td align=\"center\">10 mg</td><td align=\"center\">82.3 ± 4.3</td><td align=\"center\">21.3 ± 3.8</td></tr><tr><td align=\"center\">V</td><td align=\"center\">L-Methionine + Atorvastatin</td><td align=\"center\">10 mg</td><td align=\"center\">81.9 ± 3.9</td><td align=\"center\">27.0 ± 2.4<sup>c</sup></td></tr><tr><td align=\"center\">VI</td><td align=\"center\">L-Methionine + Pitavastatin</td><td align=\"center\">10 mg</td><td align=\"center\">82.5 ± 4.4</td><td align=\"center\">27.9 ± 3.6<sup>c</sup></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in Serum homocysteine.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Groups</bold></td><td align=\"center\"><bold>Treatment</bold></td><td align=\"center\"><bold>Dose (kg/day, <italic>p.o.</italic>)</bold></td><td align=\"center\"><bold>Serum homocysteine (μM)</bold></td></tr></thead><tbody><tr><td align=\"center\">I</td><td align=\"center\">Control</td><td align=\"center\">10 ml(0.5%w/w CMC)</td><td align=\"center\">4.23 ± 0.15</td></tr><tr><td align=\"center\">II</td><td align=\"center\">L-Methionine</td><td align=\"center\">1.7 g</td><td align=\"center\">20.8 ± 0.84<sup>a</sup></td></tr><tr><td align=\"center\">III</td><td align=\"center\">Atorvastatin <italic>per se</italic></td><td align=\"center\">10 mg</td><td align=\"center\">3.95 ± 0.21</td></tr><tr><td align=\"center\">IV</td><td align=\"center\">Pitavastatin <italic>per se</italic></td><td align=\"center\">10 mg</td><td align=\"center\">3.92 ± 0.23</td></tr><tr><td align=\"center\">V</td><td align=\"center\">L-Methionine + Atorvastatin</td><td align=\"center\">10 mg</td><td align=\"center\">12.9 ± 0.38<sup>b</sup></td></tr><tr><td align=\"center\">VI</td><td align=\"center\">L-Methionine + Pitavastatin</td><td align=\"center\">10 mg</td><td align=\"center\">13.5 ± 0.34<sup>b</sup></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Mean escape latency time (ELT) calculated for each day during acquisition trials and day 4 ELT was used as an index of acquisition</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\">Day1</td><td/><td align=\"left\">Q1</td><td/><td align=\"left\">Q2</td><td/><td align=\"left\">Q3</td><td/><td align=\"left\">Q4</td></tr><tr><td align=\"left\">Day2</td><td/><td align=\"left\">Q2</td><td/><td align=\"left\">Q3</td><td/><td align=\"left\">Q4</td><td/><td align=\"left\">Q1</td></tr><tr><td align=\"left\">Day3</td><td/><td align=\"left\">Q3</td><td/><td align=\"left\">Q4</td><td/><td align=\"left\">Q1</td><td/><td align=\"left\">Q2</td></tr><tr><td align=\"left\">Day4</td><td/><td align=\"left\">Q4</td><td/><td align=\"left\">Q1</td><td/><td align=\"left\">Q2</td><td/><td align=\"left\">Q3</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>Each group (n = 10), represent mean ± SEM.</p><p>a = <italic>p </italic>&lt; 0.01 Day 1 Vs Day 4 ELT in vehicle control.</p><p>b = <italic>p </italic>&lt; 0.05 Vs Day 4 ELT in vehicle control.</p><p>c = <italic>p </italic>&lt; 0.05 Vs Day 4 ELT in L-Methionine treated group.</p></table-wrap-foot>", "<table-wrap-foot><p>Each group (n = 10), represent mean ± SEM.</p><p>a = <italic>p </italic>&lt; 0.01 Vs serum homocysteine level of Control.</p><p>b = <italic>p </italic>&lt; 0.05 Vs serum homocysteine level of L-Methionine treated group.</p></table-wrap-foot>" ]
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[{"surname": ["Hirche", "Schr\u00f6der", "Knoth", "Stang", "Eder"], "given-names": ["F", "A", "B", "GI", "K"], "article-title": ["Effect of dietary methionine on plasma and liver cholesterol concentrations in rats and expression of hepatic genes involved in cholesterol metabolism"], "source": ["Br J Nut"], "year": ["2006"], "volume": ["95"], "fpage": ["879"], "lpage": ["88"], "pub-id": ["10.1079/BJN20061729"]}, {"surname": ["Korczyn", "Kalvach", "Bornstein"], "given-names": ["AD", "P", "ND"], "article-title": ["Vascular dementia 2003"], "source": ["J Neurol Sci"], "year": ["2005"], "volume": ["229\u2013230"], "fpage": ["1"], "lpage": ["2"]}, {"surname": ["Vanhoutte"], "given-names": ["PM"], "article-title": ["Aging and endothelial dysfunction"], "source": ["European Heart J"], "year": ["2004"], "volume": ["4"], "fpage": ["A8"], "lpage": ["A17"], "pub-id": ["10.1016/S1520-765X(02)90068-4"]}, {"surname": ["Morris"], "given-names": ["RGM"], "article-title": ["Development of a water maze producer for studying spatial learning in the rats"], "source": ["J Neurosci Meth"], "year": ["1984"], "volume": ["11"], "fpage": ["47"], "lpage": ["60"], "pub-id": ["10.1016/0165-0270(84)90007-4"]}, {"surname": ["Parle", "Singh"], "given-names": ["M", "N"], "article-title": ["Animal models for testing memory"], "source": ["Asia Pacific J Pharmacol"], "year": ["2004"], "volume": ["16"], "fpage": ["101"], "lpage": ["22"]}, {"surname": ["Sharma", "Singh", "Singh"], "given-names": ["B", "N", "M"], "article-title": ["Modulation of celecoxib and streptozotocin-induced experimental dementia of Alzheimer's disease type by pitavastatin and donepezil"], "source": ["J Psychopharmacol"], "year": ["2008"]}, {"surname": ["Zhu", "Smith", "Honda", "Aliev", "Moreira", "Nunomura", "Casadesus", "Harris", "Siedlak", "Perry"], "given-names": ["X", "MA", "K", "G", "PI", "A", "G", "PLR", "SL", "G"], "article-title": ["Vascular oxidative stress in Alzheimer disease"], "source": ["J Neurological Sciences"], "year": ["2007"], "volume": ["257"], "fpage": ["240"], "lpage": ["6"], "pub-id": ["10.1016/j.jns.2007.01.039"]}, {"surname": ["O'Dell", "Hawkins", "Kandel", "Arancio"], "given-names": ["TJ", "RD", "ER", "O"], "article-title": ["Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger"], "source": ["Proc Natl Acad"], "year": ["1991"], "volume": ["88"], "fpage": ["11285"], "lpage": ["9"], "pub-id": ["10.1073/pnas.88.24.11285"]}, {"surname": ["Beutler", "Duron", "Kelly"], "given-names": ["RG", "O", "B"], "article-title": ["Reduced glutathion estimation"], "source": ["J Lb Clinical Med"], "year": ["1963"], "volume": ["61"], "fpage": ["82"]}]
{ "acronym": [], "definition": [] }
66
CC BY
no
2022-01-12 14:47:25
BMC Pharmacol. 2008 Aug 9; 8:14
oa_package/39/84/PMC2529274.tar.gz
PMC2529275
18691409
[ "<title>Background</title>", "<p>Low and high levels of haemoglobin may indicate increased risk of morbidity beyond the immediate cause of haemoglobin abnormality. Higher haemoglobin levels and polycythemia are associated with circulatory disturbances and risk of cardiovascular events, mortality and stroke [##REF##16297840##1##,##REF##14711910##2##]. Low levels of haemoglobin and the presence of anaemia have been found to be risk factors for poor mobility, increased frailty and decreased executive function in women [##REF##15507056##3##, ####REF##15983175##4##, ##UREF##0##5####0##5##], a relationship with poorer outcome after sub arachnoid haemorrhage [##REF##17038943##6##] decreased motor performance [##UREF##1##7##] and increased risk of death [##REF##17070178##8##]. Furthermore, 5797 participants in the cardiovascular health study (participants aged 65 or over) showed a 'reverse J' shaped curve relationship between mortality and haemoglobin levels [##REF##16246985##9##]. Both lower (&lt; 13.7 gm/dl for women and &lt; 12.6 gm/dl for men) and higher (&gt; 14.1 gm/dl for women and &gt; 15.6 gm/dl for men) haemoglobin levels conferred a significantly increased risk of death. In the higher category this was a statistically significant 17 percent increase [##REF##16246985##9##].</p>", "<p>In the Women's Health and Aging Study [WHAS] the reference value used was &gt; 12 to &lt; 15 and the reverse J shape was seen although only anaemia (not polycythaemia) was significantly associated with mortality [##REF##15507056##3##, ####REF##15983175##4##, ##UREF##0##5####0##5##]. Anaemia prevalence rates are reported at around 25 percent [##REF##16472893##10##, ####REF##15813911##11##, ##UREF##2##12####2##12##] (30% in men 17% in women) of individuals over the age of 85 when defined according to the WHO criteria [##UREF##3##13##] and the literature generally agrees that a fall in haemoglobin levels in the eighth decade of life occurs and that this may be part of normal ageing [##REF##12605656##14##,##UREF##4##15##].</p>", "<p>The elderly, particularly the very elderly, aged eighty and over, are the fastest growing part of the population worldwide [##UREF##5##16##], and are at high risk of dementia and related cognitive decline. If haemoglobin levels are related to either incident or prevalent dementia or cognitive decline, there may be a possibility of intervention to prevent or ameliorate onset of dementia; important both for potential sufferers, carers, and, in economic terms [##REF##15229308##17##]. A previous review focusing on the elderly did not provide meta-analytic data, did not examine higher levels of haemoglobin and did not focus on dementia or cognitive decline [##REF##15050882##18##]. Since that review, the literature has grown and the very elderly have become even more of an important group in society, we have therefore performed a systematic review, focused on dementia and cognitive decline.</p>", "<p>Objective: to review the evidence for a relationship between haemoglobin levels and cognitive decline/dementia in the elderly via a systematic review; and to evaluate the strength of the findings taking into account the methodological differences in constituent studies.</p>", "<title>Hypotheses</title>", "<title>Haemoglobin as a risk factor</title>", "<p>1: That anaemia (labelled as such in the literature) or as defined by the WHO as low levels of haemoglobin (&lt; 12 gm/dl women and &lt; 13 gm/dl men) will be predictive of lower cognitive function and or dementia.</p>", "<p>2: That high haemoglobin (&gt; 14.1 gm/dl women and &gt; 15.6 gm/dl men) will be predictive of lower cognitive function and or dementia.</p>" ]
[ "<title>Methods</title>", "<p>In order to evaluate the evidence that was available in the scientific literature, a review was carried out using systematic review methodology in order to examine the literature in the most thorough and unbiased manner. Search terms 'anaemia' or 'anemia' or 'haemoglobin' or 'hemoglobin' and 'dementia' or vascular dementia' or 'multi infarct dementia' or Alzheimer's disease' or 'cognitive impairment' or 'cognitive decline' were used as keywords and the databases Medline, Embase and Psychinfo were searched for English language publications relating to human populations and occurring between 1996 and March 2006. The last 10 years were chosen as research methodology has evolved and computing power changed rapidly prior to this time. When available, standard search categories were also used as matched the above terms. All searches were limited to subjects aged 65 and over. See figure ##FIG##0##1## for details. Two researchers appraised all abstracts independently; any discrepancies in decisions were discussed to achieve a unanimous choice of articles.</p>", "<p>Studies were ranked on a methodological basis with randomised studies first, followed by case control and epidemiological population studies, and quality assessed using key factors including appropriate design, recruitment, analysis and provision of suitable information relating to key aspects of the study. Case studies, letters, consensus opinion from conferences and expert opinions or editorials were not included. In order to aid investigation of causality only longitudinal studies were included. Studies reporting results that could be used in a meta-analysis were analysed accordingly and tests for heterogeneity carried out.</p>" ]
[ "<title>Results</title>", "<p>From an initial 21 papers identified as relevant based on abstract alone only three remained so after full evaluation. Study quality varied and there were no randomised controlled trials or intervention studies of any sort. Methodological details and meta-analytical results are discussed below. Table ##TAB##0##1## details the studies included.</p>", "<title>Results of included studies and meta-analysis</title>", "<p><bold>For the hypothesis that anaemia or low haemoglobin was predictive for dementia or cognitive decline; two studies using the WHO criteria for anaemia found significant results</bold>.</p>", "<p>The population study the Kungsholmen project found a Hazard Ratio [HR] of 2.0 (95% CI 1.0–3.8) for incident dementia with anaemia [##REF##15893409##19##]. The retrospective case control study from Beard et al [##REF##9141646##20##] found a similar result for Alzheimer's disease which was significant only in women (Odds Ratio [OR] 1.96 (95%CI 1.11–3.47)) although men showed a similar but non-significant effect OR 1.81 (95% CI 0.75–4.39). An additional analysis from the Kungsholmen population found an even stronger relationship with haemoglobin values below 11.7 g/dl for men and 11.6 g/dl for women respectively (The fifth percentile from their population). HR 2.2 (95% CI 1.0–4.9). [##REF##15893409##19##]. No other evidence relating to low haemoglobin concentrations other than those defined above was available.</p>", "<p>It was only possible to combine two longitudinal papers in a meta-analysis to examine the relationship between anaemia and dementia (figure ##FIG##1##2##). The analysis of heterogeneity was non significant (p = 0.98). The pooled ratio was significant at 1.94 (95% CI 1.32–2.87). It was not feasible to include the cohort study from Beard et al in the analysis, as it reported only standardised incidence rates (a ratio of the incidence rate in the study population to that expected in a 'standard' population) and the source of the standard population data was unclear [##REF##9141646##20##]. No analyses regarding haemoglobin concentration or cognitive decline were possible.</p>", "<p>Higher haemoglobin gave rise to an odds ratio of 0.86 for incident cognitive decline in the WHAS study but this was non significant (95% CI 0.60–1.21) [##UREF##6##21##].</p>", "<title>Methodological considerations</title>", "<title>Study design</title>", "<p>Two of the longitudinal studies were population based cohort designs [##REF##15893409##19##,##UREF##6##21##] with prevalent cases of dementia plus those with baseline MMSE below 20 and below 24 excluded respectively. The remaining longitudinal study reported on both a retrospective case control, (where haemoglobin values taken up to two years before incident dementia were used), and a cohort, (where all participants had newly recognized anaemia and were subsequently followed up) [##REF##9141646##20##].</p>", "<title>Study population</title>", "<p>Longitudinal study populations were homogeneous and from northern Europe [##REF##15893409##19##] and the United States [##REF##9141646##20##,##UREF##6##21##] with age ranges or mean ages between 65 and 95 and therefore old enough to manifest dementia cases. Studies were relatively large with patient numbers ranging from 255 case control pairs [##REF##9141646##20##] to 1435 followed for a mean of 3.4 years (Standard Deviation [SD]0.5) [##REF##15893409##19##]. Follow up was less clear with one paper including a cohort follow up and a case control sub study of incident cases with retrospective haemoglobin measurements from the year of and the year preceding the onset of dementia [##REF##9141646##20##]. Cohort follow up was wide ranging from one day to 7.9 years. This follow up included patients who had poorer cognition and who were not included in the analysis, there were 2417 person-years of follow up for the group that were analysed but the range was not clear [##REF##9141646##20##]. Follow up in the study including only women was three years [##UREF##6##21##].</p>", "<title>Outcome</title>", "<p>There was some heterogeneity in the cognitive assessment outcome of the identified studies and standardised criteria were not always applied. Of the three studies, two assessed dementia and one cognitive decline. Only one, the Kungsholmen project, used the standardised Diagnostic and Statistical Manual of mental disorders [DSM] criteria, (the [DSM-III-R]) supplemented with information from clinical examination, death certificates and medical records [##REF##15893409##19##]. Beard et al [##REF##9141646##20##] reported on Alzheimer's disease but used an adapted form of diagnostic criteria, similar to the DSM. The WHAS [##UREF##6##21##] defined cognitive decline as a fall in MMSE to less than 24.</p>", "<p>When measurement of haemoglobin or anaemia was considered two [##REF##15893409##19##,##REF##9141646##20##] used the WHO criteria for anaemia of less that 13 g/dl for men and less than 12 g/dl for women [##UREF##4##15##]. Atkinson et al reported using haemoglobin measures but no specific cut off values for anaemia. There was some discussion in the literature with regard to the utility of the WHO cut-off values and the Kungsholmen project also analysed their data using cut off values from the 25<sup>th </sup>and 5 percentiles (13.5 g/dl men, 12.9 g/dl women and 11.7 g/dl men, 11.6 g/dl women respectively).</p>", "<title>Variables adjusted for, and data from subjects not included or lost to follow up</title>", "<p>Comprehensive adjustments for confounding variables were made only in the Kungsholmen population (Age, sex, education, history of hypertension, diabetes, cerebrovascular disease, heart failure, chronic coronary disease, COPD, hypothyroidism, chronic renal failure, high white blood cells, high blood sedimentation rate, low albumin, low BMI.)[##REF##15893409##19##] and WHAS (Age, race, smoking, education, no diseases, pulmonary disease, haemoglobin, baseline walking speed, baseline MMSE, baseline IADL, baseline ADL) [##UREF##6##21##] studies.</p>", "<p>Of the longitudinal studies only one took into account the effect of patients who died or left the study [##REF##15893409##19##]. Death certificates were assessed to identify dementia.</p>" ]
[ "<title>Discussion</title>", "<p>The literature available to date in this area is limited and few data were available for analysis. Despite this, the available data are considered and presented here in order to give a robust review of findings to date. It is clear from this review that further research needs to be carried out in this area to strengthen the literature case and aid the ability to draw firm conclusions.</p>", "<p>After the search and review process, three longitudinal studies were identified and considered in this review. The most robust one of which was the large population study, the Kungsholmen project [##REF##15893409##19##]. The WHAS [##UREF##6##21##] was also of high quality and both found an increased risk of dementia or cognitive decline at later follow up with anaemia at baseline. The third paper reported on two studies and was weaker in that the length of follow up was varied for the cohort and unclear for the case control [##REF##9141646##20##] the latter of which was significant only and supported the relationship between anaemia and dementia. It was only possible to combine the case control study from Beard et al and the Kungsholmen data in a meta-analytic way and the result was unsurprisingly significant given its constituent parts.</p>", "<p>In reference to our hypotheses, the literature suggests that low haemoglobin or anaemia increases the risk of incident dementia or cognitive decline, although the available studies are few.</p>", "<p>There were no data available that allowed examination of the hypothesis relating to high concentrations of haemoglobin.</p>", "<p>It can be imagined that low haemoglobin or diagnosed anaemia could impact on future cognitive impairment, either directly by reducing blood oxygen levels in the brain over a sustained period of time or possibly by lowering a threshold or reserve capacity such that an otherwise silent cerebrovascular accident such as a small stroke or transient ischemic attack has a greater impact on subsequent cognition. Finally, all such relationships may also be related to other co-morbidities which could prompt both anaemia and cognitive impairment.</p>", "<p>Although the data here are reflective of the literature there are many limitations to this investigation. The studies considered here were heterogeneous in that they looked at different populations, with one study looking at women only. Only one of the studies looked at different concentrations of haemoglobin in detail and took into account the possibility that the elderly may have different normal concentrations. Studies adjusted for confounders to different levels of detail. Definitions of dementia and cognitive decline varied between the studies. Despite many limitations, the studies did frequently use the standard WHO definition of anaemia.</p>" ]
[ "<title>Conclusion</title>", "<p>All of the above leads us to conclude that further studies to examine the effect of anaemia and haemoglobin on dementia and cognitive decline are needed. Such studies would need to be of large size and longitudinal design with robust cognitive assessment in the very elderly, in order to allow the evaluation of possible risks and benefits in treatment or prevention of dementia or amelioration of cognitive symptoms both physical and financial.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Anaemia may increase risk of dementia or cognitive decline. There is also evidence that high haemoglobin levels increase risk of stroke, and consequently possible cognitive impairment. The elderly are more at risk of developing dementia and are also more likely to suffer from anaemia, although there is relatively little longitudinal literature addressing this association.</p>", "<title>Methods</title>", "<p>To evaluate the evidence for any relationship between incident cognitive decline or dementia in the elderly and anaemia or haemoglobin level, we conducted a systematic review and meta-analyses of peer reviewed publications. Medline, Embase and PsychInfo were searched for English language publications between 1996 and 2006. Criteria for inclusion were longitudinal studies of subjects aged ≥65, with primary outcomes of incident dementia or cognitive decline. Other designs were excluded.</p>", "<title>Results</title>", "<p>Three papers were identified and only two were able to be combined into a meta-analysis. The pooled hazard ratio for these two studies was 1.94 (95 percent confidence intervals of 1.32–2.87) showing a significantly increased risk of incident dementia with anaemia. It was not possible to investigate the effect of higher levels of haemoglobin.</p>", "<title>Conclusion</title>", "<p>Anaemia is one factor to bear in mind when evaluating risk of incident dementia. However, there are few data available and the studies were methodologically varied so a cautionary note needs to be sounded and our primary recommendation is that further robust research be carried out.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests. Details of funding; none received and researchers therefore independent.</p>", "<title>Authors' contributions</title>", "<p>All data gathering, analysis and writing was carried out by RP &amp; LB. CB, NB and JW provided advice and critical appraisal of the analyses to be performed, methods and writing style. RPoulter reviewed and commented on the paper.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2318/8/18/prepub\"/></p>" ]
[]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Showing results of systematic review.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Forest plot for longitudinal studies; anaemia and risk of incident dementia.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Characteristics of the three longitudinal studies</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Author</td><td align=\"center\">Population Baseline numbers Descriptives Length of follow up [fu]</td><td align=\"center\">Type of study, main outcome</td><td align=\"center\">Measurement of cognition/dementia</td><td align=\"center\">Measurement of haemoglobin [hb]/anaemia</td><td align=\"center\">Adjusted for:</td><td align=\"center\">Relationship with haemoglobin [hb]/anaemia</td><td align=\"center\">Statistics if available. OR = Odds Ratio, RR = Relative Risk, HR = Hazard Ratio, (numbers in brackets are 95% confidence intervals) In order to assess the most conservative finding, results used are those from the adjusted analyses unless otherwise stated.</td></tr></thead><tbody><tr><td align=\"center\">Atti et al [##REF##15893409##19##]</td><td align=\"center\">Kungsholmen project, Sweden<break/>1435 people without dementia at baseline<break/>Aged 75–95 years<break/>Mean fu 3.4(0.5 SD) years</td><td align=\"center\">Cohort Dementia</td><td align=\"center\">Clinical exam, review by specialist using DSM-III-R – dementia information on those who died in follow up from death certificates and medical records</td><td align=\"center\">WHO criteria 130 g/L men, 120 g/L women Plus: Lowest 25<sup>th </sup>percentile 135 d/L men &amp; 129 g/L women and lowest 5<sup>th </sup>percentile 117 g/L &amp; 116 g/L women</td><td align=\"center\">In those with MMSE &gt;= 26: Age, sex, education, history of hypertension, diabetes, cerebrovascular disease, heart failure, chronic coronary disease, COPD, hypothyroidism, chronic renal failure, high white blood cells, high blood sedimentation rate, low albumin, low BMI.</td><td align=\"center\">Anaemia increases risk of dementia in those with MMSE &gt;= 26<break/>Low Hb-below 130 g/L men &amp; 120 g/L women increases risk of dementia</td><td align=\"center\">In addition in subjects with MMSE &gt;= 26....<break/>With Hb &lt; 25<sup>th </sup>percentile HR 1.2 (0.7–2.0)<break/>With Hb WHO criteria HR 2.0 (1.0–3.8)<break/>With Hb &lt; 5<sup>th </sup>percentile HR 2.2 (1.0–4.9)</td></tr><tr><td align=\"center\">Atkinson et al [##UREF##6##21##]</td><td align=\"center\">Women's health &amp; ageing study 1, USA<break/>558 women with MMSE &gt; 24 and walking speed &gt; 0.4 m/s.<break/>Mean age of whole study = 78 yrs(SD8.1)<break/>Fu 3 years</td><td align=\"center\">Cohort Cognitive decline (fall in MMSE to &lt; 24)</td><td align=\"center\">MMSE</td><td align=\"center\">Hb(g/dl)</td><td align=\"center\">Age, race, smoking, education, no diseases, pulmonary disease, hb, baseline walking speed, baseline MMSE, baseline IADL, baseline ADL</td><td align=\"center\">Hb not significant association with cognitive decline</td><td align=\"center\">Cognitive decline<break/>OR 0.86 (0.60–1.21)<break/>Combined physical and cognitive decline<break/>OR 0.68 (0.47–0.98)</td></tr><tr><td align=\"center\">C.Mary Beard et al [##REF##9141646##20##]</td><td align=\"center\">Minnesota 1. 302 incident cases of Alzheimer's Disease [AD] with matched controls. (255 pairs analysed) Fu: Hb from year preceding or year of dementia onset.</td><td align=\"center\">1. Retrospective case-control</td><td align=\"center\">1. AD identified from lists of diagnostic terms thought to include dementia – diagnostic criteria similar to DSM. controls matched by age and gender.</td><td align=\"center\">WHO criteria 130 g/L men, 120 g/L women.<break/>1. Lowest measurement recorded in 2 yr window (onset + preceding yr) for cases and corresponding for controls.</td><td align=\"center\">1. age, gender</td><td align=\"center\">1. Anaemia increases risk of AD (not significant in men)</td><td align=\"center\">1.OR 188 (1.17–3.03)<break/>Men OR 1.81 (0.75–4.39)<break/>Women OR 1.96 (1.11–3.47)</td></tr><tr><td/><td align=\"center\">2. 618 people Aged 65 years+ Fu: 5.1 years (1 day-7.9 years)</td><td align=\"center\">2. Cohort Alzheimer's disease [AD]</td><td align=\"center\">2. Medical records reviewed by nurse abstractor and confirmed by author</td><td align=\"center\">2. A value below WHO criteria between 1985 &amp; 1989</td><td/><td align=\"center\">2.No association between anaemia and AD</td><td align=\"center\">2.SIR 0.98 (0.67–1.37)<break/>Men SIR 1.49 (0.79–2.56)<break/>Women SIR 0.79 (0.49–1.23)</td></tr></tbody></table></table-wrap>" ]
[]
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[]
[]
[]
[]
[]
[ "<graphic xlink:href=\"1471-2318-8-18-1\"/>", "<graphic xlink:href=\"1471-2318-8-18-2\"/>" ]
[]
[{"surname": ["Chaves", "Carlson", "Ferrucci", "Guralnik", "Semba", "Fried"], "given-names": ["P", "M", "L", "J", "R", "L"], "article-title": ["Association between mild anemia and executive function impairment in community-dwelling older women: The women's Health and Aging Study II"], "source": ["JAGS"], "year": ["2006"], "volume": ["54"], "fpage": ["1429"], "lpage": ["1435"], "pub-id": ["10.1111/j.1532-5415.2006.00863.x"]}, {"surname": ["Inzitari", "Di Carlo", "Baldereschi", "Pracucci", "Maggi", "Gandolfo", "Bonaiuto", "Farchi", "Scafato", "Carbonin", "Inzitari"], "given-names": ["M", "A", "M", "G", "S", "C", "S", "G", "E", "P", "D"], "collab": ["for the ILSA working group"], "article-title": ["Risk and predictors of motor-performance decline in a normally functioning population-based sample of elderly subjects: the Italian longitudinal study on aging"], "source": ["JAGS"], "year": ["2006"], "volume": ["54"], "fpage": ["318"], "lpage": ["324"], "pub-id": ["10.1111/j.1532-5415.2005.00584.x"]}, {}, {"collab": ["World Health Organization"], "article-title": ["Indicators and Strategies for Iron Deficiency and Anemia Programmes"], "source": ["Report of the WHO/UNICEF/UNU Consultation, Geneva Switzerland 6\u201310 December 1993"], "year": ["1994"]}, {"surname": ["Woodman", "Ferrucci", "Guralnik"], "given-names": ["R", "L", "J"], "article-title": ["Anemia in older adults"], "source": ["Current Opin Hematol"], "year": ["2005"], "volume": ["12"], "fpage": ["123"], "lpage": ["128"]}, {"article-title": ["Report on World Population Ageing"], "source": ["1950\u20132050 United Nations Population Division DESA"], "fpage": ["xxvii"], "lpage": ["xxxi"]}, {"surname": ["Atkinson", "Cesari", "Kritchevsky", "Penninx", "Fried", "Guralnik", "Williamson"], "given-names": ["H", "M", "S", "B", "L", "J", "J"], "article-title": ["Predictors of combined cognitive and physical decline"], "source": ["JAGS Society"], "year": ["2005"], "volume": ["53"], "fpage": ["1197"], "lpage": ["1202"], "pub-id": ["10.1111/j.1532-5415.2005.53362.x"]}]
{ "acronym": [], "definition": [] }
21
CC BY
no
2022-01-12 14:47:25
BMC Geriatr. 2008 Aug 8; 8:18
oa_package/d2/56/PMC2529275.tar.gz
PMC2529276
18662382
[ "<title>Background</title>", "<p>The value of evidence-based medicine (EBM) – using updated, relevant and trustworthy evidence to inform medical decisions is widely acknowledged [##REF##17204750##1##]. Recently the British Medical Journal nominated EBM as one of the 15 most important milestones in medicine since 1840 [##REF##17204760##2##]. Easy access to high quality research has the potential to improve patient care, but there are obstacles that face health professionals attempting to use evidence in their practice. In an Australian survey, physicians identified insufficient time (74%), limited search skills (41%) and limited access to evidence (43%) as impediments to making better use of research data [##REF##10914748##3##].</p>", "<p>Systematic reviews directly address several of these barriers, as their summarized form reduces the amount of time and search skills needed to access and appraise many individual studies [##REF##9310574##4##]. A systematic review is a summary of individual studies addressing a clearly formulated question that uses systematic and explicit methods to identify, select, and critically appraise the relevant research, and to collect and analyse data from the included studies. The Cochrane Collaboration is an international organisation of volunteers dedicated to producing systematic reviews of rigorous methodological quality. These reviews are published in one of the databases on <italic>The Cochrane Library </italic>[##UREF##0##5##], a web site that has the potential to further simplify the task of finding trustworthy evidence. Additionally the Library hosts other databases for systematic reviews, health technology assessments and randomized controlled trials, making it a central online collection of varying types of evidence from a variety of sources.</p>", "<p>Part of the mission of The Cochrane Collaboration is \"to promote the accessibility of systematic reviews to anyone wanting to make a decision about health care\". The organization also aims to produce reviews that are easy to read and understand by someone with a basic sense of the topic [##UREF##1##6##]. But does the Library web site support the Collaboration's goals of clarity and ease of use, as well as the overreaching mission of making evidence accessible for decision making? We wanted to explore this question through observing the experiences of health professionals using <italic>The Cochrane Library</italic>. We were interested not only in site-specific problems but also in issues that might be relevant to other web sites publishing collections of evidence-based content.</p>", "<title>User experience</title>", "<p>Usability testing is a method that is widely used in the field of web design to uncover errors and areas of improvement by observing users solving given tasks on the site [##UREF##2##7##,##UREF##3##8##]. There is increased recognition of the limitations of examining only task-related problems when attempting to understand why users' interactions with web sites might succeed or fail. Attention to the user's whole experience has begun to gain ground in the field of human-computer interaction [##UREF##4##9##]. Morville's \"honeycomb\" model (see Figure ##FIG##0##1##) distinguishes between seven separate facets of user experience, including findability, accessibility, usability, usefulness, credibility, desirability and value [##UREF##5##10##].</p>", "<p>A brief explanation of these terms:</p>", "<p><bold>Findability: </bold>can users locate what they are looking for?</p>", "<p><bold>Accessibility: </bold>are there physical barriers to actually gaining access, also for people with handicaps?</p>", "<p><bold>Usability: </bold>how easy and satisfying is this product to use?</p>", "<p><bold>Usefulness: </bold>does this product have practical value for this user?</p>", "<p><bold>Credibility: </bold>is it trustworthy?</p>", "<p><bold>Desirability: </bold>is it something the user wants? Has a positive emotional response to?</p>", "<p><bold>Value: </bold>does this product advance the mission of the organization behind it?</p>", "<p>Our study aimed to explore the user experience of health professionals trying to find evidence in <italic>The Cochrane Library</italic>, building on methods from usability testing. In this article we use the honeycomb model to organize findings from our study to illustrate more general potential pitfalls and challenges particular to evidence-based online resources. At the end we suggest some guidelines for designers, writers and developers working to improve the user experience of these types of sites.</p>" ]
[ "<title>Methods</title>", "<p>We carried out two series of user tests in 2005 (Test 1) and 2006 (Test 2), with participants from Norway and UK. The publisher of the site, Wiley-Blackwell, made changes to the site after Test 1, partly based on the results we uncovered. Most of these changes regarded branding at the top of the site, making <italic>The Cochrane Library </italic>the prominent identity and toning down the logo and universal navigation of the publisher. Therefore we altered the interview guide of Test 2 in small ways so that the questions would match the changes that had been made. See Additional file ##SUPPL##0##1## for the complete interview guide we used in Test 2.</p>", "<p>We limited our selection to health professionals who used the Internet and had some knowledge of systematic reviews, to ensure that the results of the interface testing would not be confounded by unfamiliarity with the media or the site's content. We sent email invitations to lists of previous attendees of evidence-based practice workshops, employees in the Directorate of Health and Social Affairs in Oslo and individuals in evidence-based health care networks in Oxford. Volunteers who responded were screened by phone or email to assess whether they fitted the requirements, and also to find relevant topics of interest so that we could individually tailor test questions. We also asked them about their online searching habits, and what sources of online information they usually used in connection with work. We did not reveal the name of the site we were testing during recruitment. Test persons were promised a gift certificate worth the equivalent of $80 USD or a USB memory stick if they showed up for the test.</p>", "<p>Tests were performed individually and took approximately one hour. The test participant sat at a computer in a closed office together with the test leader who followed a semi-structured test guide. We recorded all movement on the computer desktop through use of Morae usability test software [##UREF##6##11##] and video-filmed the participant, who was prompted to think out loud during the whole session. We projected the filming of the desktop and the participant as well as the sound track, to another room where two observers transcribed, discussed, and took notes.</p>", "<p>The data was anonymous to the degree that participants' names were not connected to video, audio or text results. We received written permission to store the recordings for five years before deleting it, guaranteeing that video/audio tapes would not be used for any purpose outside of the study and not be published/stored in places of public access. The protocol was approved by the Norwegian Social Science Data Services and found in line with national laws for privacy rights.</p>", "<p>We began the test with preliminary questions about the participant's profession, use of Internet, and knowledge of <italic>The Cochrane Library</italic>. We then asked the participant to find specific material published on the Library starting from an empty browser window. Once on the site, we asked about their initial reactions to the front page, and they were invited to browse freely, looking for content of interest to themselves. Then we asked them to perform a series of tasks, some of which involved looking for specific content about topics tailored to their field or professional interests. For instance, a midwife was asked to find:</p>", "<p>- all information on the whole library that dealt with prevention of spontaneous abortion</p>", "<p>- a specific review about the effect of caesarean section for non-medical reasons</p>", "<p>- all new Cochrane Reviews relevant to the topic \"music used to relieve pain\".</p>", "<p>Other general tasks included finding help, finding the home page, and finding information about Cochrane. We also had specific tasks leading to searching and to reading a review. At the end, we asked if they had any general comments to the site and suggestions to how it could be improved.</p>", "<p>Our analysis was done in two phases. The aim of the first analysis was to provide the stakeholders and site developers with an overview and a prioritizing of the problems we had identified. At least two of us carried out content analysis of the transcripts, independently coding each test. These codes were then compared, discussed and merged. The topics were then rated according to the severity of the problem for the user. We rated severity in three categories: high (show-stopper, leads to critical errors or hinders task completion), medium (creates much frustration or slows user down), or low (minor or cosmetic problems).</p>", "<p>The second analysis was done to lift more generalizable issues underlying this article out of the site-specific data. We re-sorted the findings into the seven user-experience categories from the honeycomb model by re-reading the transcript, checking the context where the problems came from, and evaluating which of the seven categories best fit each finding. Severity-of-problem ratings from the first analysis were kept in the second analysis.</p>", "<p>We did not evaluate accessibility (the degree to which the website complied with standards of universal accessibility, for instance as defined by the Web Accessibility Initiative [##UREF##7##12##]), since user testing methods are not an effective way of gathering data on various aspects of this issue.</p>", "<p>The findings presented here are a selection of issues that received a high degree of saturation in our tests, and that we judge to be critical (\"high severity\") to the user experience of evidence-based web sites in general. This judgement is based on basic principles for web usability [##UREF##2##7##,##UREF##8##13##, ####UREF##9##14##, ##UREF##10##15####10##15##] as well as the principles underlying evidence-based health care: to successfully search for, critically appraise and apply evidence in medical practice [##REF##8555924##16##].</p>", "<p>Most of the findings here are still of relevance to <italic>The Cochrane Library </italic>in its current format, though we have included some observations of problems that are now resolved, because they illustrate issues that are potentially important for others. Our aim is not to write a critical review of the library, but to highlight issues we found that can be important to user experience of evidence-based web sites for health professionals.</p>" ]
[ "<title>Results</title>", "<title>Participant profiles</title>", "<p>We tested a total of 32 persons (See Table ##TAB##0##1## for participant details). Test 1 included 13 persons from Norway, and Test 2 included five persons from Norway and 14 from the UK. Twenty-one of the 32 participants were non-native English speakers accustomed to reading in English.</p>", "<p>Participants were educated in nursing/midwifery (10); medicine (8); dentistry (4); physiotherapy (4); social sciences (3); psychology (2); and occupational therapy (1). They were currently working as health professionals in primary or secondary care (17); as government advisors working with health-related issues (7); as teachers at nursing/physiotherapy schools or universities (4); as research nurses (3); or as an editor for a patient information website (1).</p>", "<p>Most used the Internet daily or several times a week, and much of this use was work-related. All had searched the Internet for health-related information or evidence. Most participants reported that they normally looked for information in response to a specific problem. A few of them had strategies to keep up to date within a certain field on a more regular basis. When in need of information, the most common sources mentioned were colleagues, research databases, and the Internet. All but one participant had some previous knowledge of The Cochrane Collaboration and 25 of the 32 participants could provide at least a basic description of the term \"systematic review\". Twenty-six said that they had visited <italic>The Cochrane Library </italic>site previously.</p>", "<p>The findings that we included in this article are listed in Table ##TAB##1##2##.</p>", "<title>Findability</title>", "<title>Finding the website</title>", "<p>Finding the site was an obstacle for the majority of participants in Test 1. Despite the fact that 11 of 13 of these participants said they had visited <italic>The Cochrane Library </italic>before, the same number were not able to find the site without considerable confusion, and six of these 11 did not find the site at all until they were helped by the test facilitator. Although most participants in Test 2 had more success, finding the site remained a problem for some. One of these, a EBM-skilled UK participant, used 23 minutes to arrive at <italic>The Cochrane Library </italic>from a blank browser page.</p>", "<p>Much of this trouble stemmed from the participants' failed attempts to find Cochrane through Google search technology. These searches often failed because Google did not rank <italic>The Cochrane Library </italic>on the top of the first results page when queried for \"Cochrane\" or \"Cochrane Library\". In part this may be due to the fact that only the top few pages of <italic>The Cochrane Library </italic>were open to indexing in Google, affecting the ranking of the site. Several participants followed other links that appeared higher up on the results list, including links leading to the previous publisher of the site and to The Cochrane Collaboration site, expecting they would lead to the Library. After arriving at these other sites, participants continued to express confusion as to where they were because they found Cochrane-related content.</p>", "<title>Problems searching for content</title>", "<p>Finding specific content was also a major problem once participants arrived at <italic>The Cochrane Library</italic>. Participants attempted to solve most tasks by performing a search. Even when participants were asked to \"take a look around the site\", 75% started this task with a search. Few of our participants used the advanced search functionality. The simple search was the single most used feature in these tests, and many of these searches failed, leaving participants with a negative impression of the search functionality in the Library. Some participants compared <italic>The Cochrane Library </italic>to PubMed search, which they found easier to use.</p>", "<p>Misspelling was the most common search-related mistake made by non-English participants. They were used to getting help with this from other search engines that was not provided by <italic>The Cochrane Library </italic>search: <italic>\"If I get the spelling wrong, Google will help\"</italic>. Another problem this group experienced was recalling precise terms (for instance recalling \"overweight\" but not \"obesity\"). The publisher redesigned parts of the search interface after Test 1. However in Test 2 the non-native English participants still had considerable problems finding content, mainly due to problems with spelling and recall of correct terms.</p>", "<p>Search results were often misinterpreted. One of the most critical problems we observed was participants' confusion regarding what they were finding. Many participants did not notice that hits occurred in different databases in <italic>The Cochrane Library </italic>and thought all hits were completed Cochrane Reviews. We observed participants clicking on and reading review protocols and reports of individual clinical trials, mistaking them for systematic reviews.</p>", "<p>The search engine was also too sensitive. For instance \"huntingtons\" gave no hits, while \"huntington's\" did. \"Keywords\" option did not provide stabile results.</p>", "<p>Participants were also confused when their searches produced few or no search results. Some misinterpreted getting few hits as being the result of a bad search. The concept underlying the Cochrane Database of Systematic Reviews of one review per subject did not seem apparent. In addition, non-native English speakers interpreted a lack of hits as a result of their own bad English even though this might not have been the case.</p>", "<title>Problems browsing for content</title>", "<p>Test persons did not browse much, though this may have had to do with their problems understanding the organisation of the site. Few people were able to describe how the content was structured by viewing the front page and nobody could point to a menu with any certainty. Only one test person used the \"Topics\" entry at the top of the front page, though it was not apparent whether other participants did not see it or preferred not to use it.</p>", "<title>Usability</title>", "<title>Language and jargon</title>", "<p>Participants reacted to the use of jargon throughout the site. Some of the jargon was site-specific (such as the term \"<italic>record</italic>\" which led to full texts) and some was tied to research terminology (for instance \"<italic>protocol</italic>\"). The use of jargon gave the impression that the site was for academic use only and effectively discouraged participants from using several of the site's functions.</p>", "<title>Legibility and layout</title>", "<p>Most felt that there was too much text on the front page and that the type was too small. The participants that clicked on the \"Help\" and the \"More Information\" section also found them very dense.</p>", "<p>\"It's very messy. Do I have to read all of this?\"</p>", "<p>There was lots of frustration about the screen being taken up by other things than the review text such as the top banner space. Several participants made negative comments about having to scroll down to see full front page.</p>", "<p>\"The actual content is stuck in this little area down here.\"</p>", "<title>Reading pattern</title>", "<p>We were interested in how participants read reviews and asked them to show us how they normally would approach document if they had limited time (two to five minutes). Most referred to the conclusion section. Several said they would read the abstract, while some mentioned the objectives, results, and background sections. Most said that they normally would not be interested in reading a whole review.</p>", "<p>We asked participants specifically about the forest plot graphs in the Cochrane Reviews, as they present a lot of information in a summarized form that could be useful for a reader in a hurry. Some participants found them helpful; others found them confusing. They were very difficult to comprehend for those participants who had not seen them before, and were not intuitively located.</p>", "<title>Credibility</title>", "<p>When asked if they would trust the information on <italic>The Cochrane Library</italic>, all participants replied that they would, often because of a familiarity with the Cochrane name and more or less vague ideas about the quality of Cochrane products: \"<italic>because it's very respected</italic>\"; \"<italic>it's a reputable name</italic>\"; \"<italic>because I've heard good things about it</italic>.\"</p>", "<p>In Test 1, however, we observed potential challenges to this trust because of confusion about site/content ownership. This was primarily tied to the prominence of the Library's publisher Wiley-Blackwell on the website. Wiley's logo was placed higher up on the page than Cochrane's, and Wiley's Home, About Us, Contact Us, and Help buttons were assumed to be Cochrane Library buttons by most participants. Participants who used these buttons often did not realise that they were no longer in <italic>The Cochrane Library</italic>. When asked to describe the relationship between Wiley and <italic>The Cochrane Library</italic>, many described <italic>The Cochrane Library </italic>as a sub-group of Wiley:</p>", "<p>\"<italic>It gives me sort of pharmaceutical industry associations. I think that The Cochrane Library is a subgroup (of Wiley).\"</italic></p>", "<p>Several changes were made to the website in order to address these issues after Test 1, and participants in Test 2 did not display the same confusion.</p>", "<p>We also observed that <italic>The Cochrane Library</italic>'s perceived credibility could be over-interpreted. The only contents on <italic>The Cochrane Library </italic>that are \"Cochrane approved\" are the reviews listed in the Cochrane Database of Systematic Reviews. Despite this fact, some participants assumed that everything in the Library was \"<italic>Cochrane-approved\"</italic>, including the trials, reviews and reports in the individual databases: \"<italic>This will just have things that Cochrane have looked at</italic>\"; \"<italic>If I was looking for a piece of evidence and I found it on Cochrane I would think that it was high quality</italic>.\"</p>", "<title>Usefulness</title>", "<p>Some participants assumed that <italic>The Cochrane Library </italic>only dealt with medical topics and did not expect to find information on topics such as dentistry, nutrition, or acupuncture. The Library was also perceived by some as primarily an academic resource: <italic>\"I've tended to think that this is where researchers go to add to the body of knowledge or to see what there is, they'd use this (to build up) Clinical Evidence or Bandolier.... but if I was wanting to get back to the source of information, this is where I would want to go.\"</italic></p>", "<p>The website has attempted to signal that it is a resource for all types of healthcare decision-makers by adding buttons on the front page entitled \"For Clinicians\"; \"For Researchers\"; \"For Patients\"; and \"For Policy makers\". These lead to short descriptions of what <italic>The Cochrane Library </italic>can offer each of these groups. However, while some participants thought these were advertising because of their position in the right-hand column, several others assumed that they led to specially adapted versions of <italic>The Cochrane Library</italic>, and were disappointed when this turned out not be the case:</p>", "<p>\"<italic>I'm surprised that there's a link through to patients here. (...) I didn't realise that it was so well-developed along those lines.\"</italic></p>", "<p>\"Oh, so it's an (advert)... I was hoping it would give me a tailored search programme, a bit like NLH, which asks you \"are you a GP...\"</p>", "<p>Others disliked these distinctions between different target groups: <italic>\"I don't know why clinicians should differ from researchers. We all need to have \"high quality information at our fingertips.\"</italic></p>", "<p>Several participants were positive to the fact that patients' information needs were being addressed in the form of the Plain Language Summaries they found in the Cochrane Reviews. They saw these products as helpful both for communicating with patients and for understanding the research results themselves.</p>", "<p>\"I wouldn't want to go and read all the nitty gritty. The short bits, the one page was useful.\"</p>", "<title>Desirability</title>", "<p>Two thirds of the participants complained that the site looked messy and difficult to use, that there was too much information. All expressed frustration with failed attempts to find relevant content. Participants wanted a web site they could get into quickly, find what they were looking for, and get out again. \"<italic>Crowded</italic>,\" \"<italic>busy</italic>,\" \"<italic>cluttered</italic>,\" \"<italic>a lot going on</italic>,\" \"<italic>difficult to find any one particular thing</italic>\" were typical comments. Some participants felt \"<italic>overwhelmed</italic>,\" \"<italic>bombarded</italic>\" and <italic>\"stupid.\"</italic></p>", "<p>While most expressed interest in this type of evidence-based resource, many were cautious, or concerned that they lack the necessary skills: A nurse commented: \"<italic>This is maybe more for doctors</italic>.\" A physician who had trouble finding specific content chose to search for <italic>\"dementia\" </italic>during a test task, and explained why: <italic>\"That's kind of how I'm feeling right now.\"</italic></p>", "<title>Value</title>", "<p>At the beginning of the test all participants said they expected to be able to find content that was relevant for them on <italic>The Cochrane Library</italic>. Most felt that Cochrane Reviews represented the golden standard for systematic reviews. Many were put off by the amount of information and concerned about the time it would take them to find what they were looking for.</p>", "<p>\"Not easy to get around\"; \"Most of us don't have time to get around\"; \"So many pages are better designed, so you just get fed up and frustrated and go somewhere else.\"</p>" ]
[ "<title>Discussion</title>", "<p>Our study shows that health professionals' experiences of <italic>The Cochrane Library </italic>were considerably less than optimal. Test participants had much difficulty locating both the site and the evidence. Non-native English speakers were at an extra disadvantage when retrieving relevant documents. Many participants displayed feelings of ineptitude, alienation and frustration. Some made serious mistakes in correctly identifying different information types. Although nearly all expressed a high regard for the credibility of The Cochrane Collaboration, some later displayed a mistrust of the independence of the information. Others were overconfident, thinking everything on <italic>The Cochrane Library </italic>site had been quality-approved through an editorial evaluation, transferring the quality association they had of Cochrane Reviews to the entire content of the library.</p>", "<p>There are few published usability studies of health professionals using online health libraries or other similar collections of evidence-based medical literature. A commercial company carried out parallel testing of <italic>The Cochrane Library </italic>for Wiley-Blackwell in 2005 and 2006. Their unpublished reports showed findings that were by and large similar to ours, though included only participants living and working in the UK and therefore did not duplicate the problems we found regarding non-native English speakers. One usability study of an NHS website published in 2003 [##REF##12919276##17##] found that major problems were often caused by specialized library terminology. This supports our findings regarding unfamiliar language and jargon. The few other usability studies of health-related web sites we uncovered dealt with online information for patients or the public.</p>", "<p>Our results were used in discussions with The Cochrane Collaboration Steering Group and the publisher, Wiley-Blackwell, in order to develop and improve The Library web site. Other publishers of evidence-based content could use the more generic results to improve their own websites.</p>", "<title>Searching (and finding): critical to evidence-based practice</title>", "<p><italic>The Cochrane Library </italic>site is not alone in having problems with findability. Results from usability tests of 217 web sites performed by Jakob Nielsen's team showed that search functionality and findability are the two largest categories of usability problems leading to task failure [##UREF##2##7##]. However, it is particularly ironic that a website built specifically to support evidence-based health care by synthesizing, organising and making accessible an overwhelming amount of health research should itself be perceived as overwhelming and difficult to navigate.</p>", "<title>Discriminating design</title>", "<p>In this study the non-native English speakers, though displaying no visible trouble reading English text, were at an extra disadvantage when trying to search. Their problems were related primarily to difficulty recalling and spelling query terms that resulted in relevant hits. Creating a reliable base of evidence is a task no organisation or country can solve alone – cross-national efforts are needed. Easy access to a body of high quality evidence should not be limited to native English-speaking participants. There is a wealth of technology that could be used to improve the user experience of searching for non-native English speakers. Spelling aids or query translation from other languages would be particularly helpful to these kinds of users. Automatic query expansion with synonyms (used by PubMed) could provide a better experience both for all searchers but would be particularly helpful for those with a limited English vocabulary.</p>", "<title>Challenge – building a good mental model for evidence searching</title>", "<p>Our findings revealed other challenges for designing good search functionality. In the Cochrane Database of Systematic Reviews, a precise query will result in only one or a few hits, as the underlying concept is one review per topic. However our participants' mental models of how search should function were based both on Google and PubMed, where simple queries produce a great number of results. The concept of a narrow search resulting only in a few hits is clearly still novel to many users and ways in which this can be made clearer need to be explored.</p>", "<title>Challenge – building a good mental model of evidence-based information hierarchy</title>", "<p>Our findings showed that systematic reviews can be confused with protocols and reports of clinical trials, even among experienced users who have a clear idea of the difference between these document types. This kind of misinterpretation may happen especially when different document types are mixed together in search results lists. Different document types need to be distinguished from each other, both physically and visually – protocols should possibly be moved to a separate list. The importance of large clear labelling at the top of the individual documents enabling readers to easily distinguish between protocols, reviews and individual studies should also not be underestimated.</p>", "<title>Appraising the source instead of the document</title>", "<p>A related problem is the tendency for users to assume all Cochrane Library contents are Cochrane-approved. Most of our test persons seemed inclined to be satisfied with a quality assessment short-cut: making judgements about the trustworthiness of the <italic>publishing source </italic>rather than critically assessing individual documents of research as EBM teaching encourages. This inclination, when coupled with poor signposting on a site containing information of varying levels of editorial evaluation and research quality, leaves a gap wide-open for serious misunderstandings about the strength and quality of different pieces of evidence. Blind trust of a whole source is a complex labelling and branding problem and needs to be addressed by publishers on many levels.</p>", "<title>Fragile credibility</title>", "<p>Though Cochrane clearly enjoyed a high reputation among our participants, our study showed that even very small details can cause otherwise trusting users to suddenly question ownership and thereby credibility, such as an \"About us\" button leading to a page with a publishers' (unfamiliar) logo. While a large study from the Stanford Credibility project showed that consumers placed a lot of emphasis on the look of a site [##UREF##11##18##], a smaller parallel study showed that expert users tended to emphasize the reputation of the source when evaluating the trustworthiness of information found online [##UREF##12##19##]. Additionally it is important to follow the EBM principles of transparency and make it absolutely clear who is behind information that claims to be neutral and evidence-based.</p>", "<title>This site is not for someone like me...</title>", "<p>Many of our participants felt that <italic>The Cochrane Library </italic>site was for <italic>\"researchers\" </italic>or others with more knowledge than themselves, in part due to use of unfamiliar or academic jargon, but also connected to their failure to find relevant information. The feelings of ineptitude expressed by participants in this study is perhaps mirrored in the Australian study, where 41% of the participating physicians blamed their own limited search skills as impediments to making better use of research data. In fact, many of the problems our participants encountered were not due to their own lack of skills, but to design flaws that could be solved following usability heuristics [##UREF##13##20##] and research-based guidelines for web design [##UREF##2##7##,##UREF##8##13##,##UREF##14##21##] or implementing better search technology. It is also important to signal inclusiveness and relevance to other health care areas than just medicine. Clear signs of content produced for patient target groups could also serve to lower the perceived threshold for professionals.</p>", "<title>Is valuable content enough?</title>", "<p>Repeatedly we heard praise for the quality of content of this site. But frustration levels were very high, and several participants said they were ultimately too lazy to bother to use a site that made it so difficult for them. Information foraging theory describes user behaviour on the Internet as similar to wild animal's search for food: we want maximum benefit for a minimum of effort [##UREF##15##22##]. Jakob Nielsen points out that with the development of good search engines, it has become easier for information gatherers to move quickly between different hunting grounds, claiming that web sites should be designed less like big meals and more like tasty snacks, quick both to find and to eat [##UREF##16##23##]. A resource like Cochrane may be theoretically a great meal for a hungry animal, but too difficult to find and catch to be worth the effort, especially when less challenging prey is more easily available.</p>", "<title>Limitations of this study</title>", "<p>Our goal is to identify the emerging issues rather than to quantify them. In reporting results, we have therefore not emphasized frequencies of events. As our data set has not been designed to statistically represent a set of respondents, presenting numbers can be misleading [##REF##10625273##24##].</p>", "<p>The user tests were performed in a laboratory setting, and may not reflect actual behaviour or reactions from real-life situations. For instance, increased time pressure in clinical situations may result in even higher degrees of user frustration when an interface does not easily or intuitively produce quick results.</p>", "<p>UK-based tests were held in the office of The Cochrane Collaboration, and this may have influenced the answers of participants regarding use and attitudes towards <italic>The Cochrane Library </italic>and Cochrane Reviews, despite our assurances that we were not connected with the design of the web site. Answers regarding familiarity and use of research were self-reported and not empirically validated.</p>", "<p>The honeycomb model was not used to design the interview questions, only applied in retrospect to our data analysis. This may have affected the relevance of the data we collected to this model. On the other hand, this may have led to less \"leading\" questioning on our part.</p>", "<p><italic>The Cochrane Library</italic>, like most websites, is under continuous development/change, and several of the weaknesses we identified have since been improved.</p>" ]
[ "<title>Conclusion</title>", "<title>Recommendations based on findings</title>", "<p>Building web sites for evidence-based practice is not much different than building for good web usability in general. However, the consequences of not finding information or of finding the wrong information have potentially critical consequences. Health professionals' user experience of evidence-based online resources can be improved by applying the following principles:</p>", "<p>- Follow existing usability heuristics and web usability guidelines, designing especially for findability through search engines, as well as for speed of use particularly important to health professionals.</p>", "<p>- If resources are limited, focus on improving simple (non-advanced) search functionality, including technology that will help non-native English speakers.</p>", "<p>- Drop \"researcher\" language and jargon to encourage use by health professionals.</p>", "<p>- Don't assume users possess good mental models of evidence hierarchies. Make document types evident where possible – through information architecture, labelling, and search results design.</p>", "<p>- Clearly mark the difference between quality-approved content and not quality-approved content.</p>", "<p>- Ownership and authoring must be clear at all levels of the site for supporting and maintaining credibility.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Evidence-based decision making relies on easy access to trustworthy research results. <italic>The Cochrane Library </italic>is a key source of evidence about the effect of interventions and aims to \"promote the accessibility of systematic reviews to anyone wanting to make a decision about health care\". We explored how health professionals found, used and experienced The Library, looking at facets of user experience including findability, usability, usefulness, credibility, desirability and value.</p>", "<title>Methods</title>", "<p>We carried out 32 one-hour usability tests on participants from Norway and the UK. Participants both browsed freely and attempted to perform individually tailored tasks while \"thinking aloud\". Sessions were recorded and viewed in real time by researchers. Transcriptions and videos were reviewed by one researcher and one designer. Findings reported here reflect issues receiving a high degree of saturation and that we judge to be critical to the user experience of evidence-based web sites, based on principles for usability heuristics, web guidelines and evidence-based practice.</p>", "<title>Results</title>", "<p>Participants had much difficulty locating both the site and its contents. Non-native English speakers were at an extra disadvantage when retrieving relevant documents despite high levels of English-language skills. Many participants displayed feelings of ineptitude, alienation and frustration. Some made serious mistakes in correctly distinguishing between different information types, for instance reviews, review protocols, and individual studies. Although most expressed a high regard for the site's credibility, some later displayed a mistrust of the independence of the information. Others were overconfident, thinking everything on <italic>The Cochrane Library </italic>site shared the same level of quality approval.</p>", "<title>Conclusion</title>", "<p>Paradoxically, <italic>The Cochrane Library</italic>, established to support easy access to research evidence, has its own problems of accessibility. Health professionals' experiences of this and other evidence-based online resources can be improved by applying existing principles for web usability, prioritizing the development of simple search functionality, emitting \"researcher\" jargon, consistent marking of site ownership, and clear signposting of different document types and different content quality.</p>" ]
[ "<title>Competing interests</title>", "<p>CG is director of the Norwegian branch of the Nordic Cochrane Centre. JC is Co-ordinator &amp; Managing Editor of The Cochrane Anaesthesia Review Group. CG and SR are involved in projects to improve summaries included in Cochrane Reviews. Test 2 was carried out in collaboration with Wiley-Blackwell, and partly funded by them.</p>", "<title>Authors' contributions</title>", "<p>SR conceived of and designed the study, carried out user testing, data analysis and interpretation, and drafting of the manuscript. CG helped design the study, carried out user testing, data analysis and interpretation, and drafting of the manuscript. JC recruited UK participants, carried out user testing, transcribed and coded the British tests, and commented on the manuscript. All authors read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1472-6947/8/34/prepub\"/></p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>Irene Wiik Langengen recruited Norwegian participants and carried out user testing. Arild Bjørndal reviewed several versions of the manuscript.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>The honeycomb model of user experience, reproduced here with permission from Peter Morville, Sematic Studios LLC.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Participant details</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\"><bold>Gender</bold><break/></td><td align=\"left\"><bold>Age</bold><break/></td><td align=\"left\"><bold>Profession</bold><break/></td><td align=\"left\"><bold>Internet use: </bold><break/><bold>Frequency</bold></td><td align=\"left\"><bold>Native </bold><break/><bold>language</bold></td><td align=\"left\"><bold>Place of </bold><break/><bold>residence</bold></td></tr></thead><tbody><tr><td align=\"left\">1</td><td align=\"left\">F</td><td align=\"left\">44</td><td align=\"left\">Midwife</td><td align=\"left\">Daily</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">2</td><td align=\"left\">F</td><td align=\"left\">43</td><td align=\"left\">Sociologist, advisor in health-related govt. institution</td><td align=\"left\">Daily</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">3</td><td align=\"left\">F</td><td align=\"left\">53</td><td align=\"left\">Physical therapist/teacher</td><td align=\"left\">1–2 times a month</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">4</td><td align=\"left\">F</td><td align=\"left\">45</td><td align=\"left\">Midwife/researcher</td><td align=\"left\">Daily</td><td align=\"left\">Other (not English)</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">5</td><td align=\"left\">F</td><td align=\"left\">-</td><td align=\"left\">advisor in health-related govt. institution</td><td align=\"left\">Up to 5 times a week</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">6</td><td align=\"left\">F</td><td align=\"left\">-</td><td align=\"left\">Masters in nursing science, lectures at college level</td><td align=\"left\">Daily</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">7</td><td align=\"left\">F</td><td align=\"left\">39</td><td align=\"left\">Midwife/teacher</td><td align=\"left\">Daily</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">8</td><td align=\"left\">M</td><td align=\"left\">49</td><td align=\"left\">Medical Doctor/dept. director at health-related govt. institution</td><td align=\"left\">Daily</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">9</td><td align=\"left\">F</td><td align=\"left\">28</td><td align=\"left\">Psychologist at health station for youth</td><td/><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">10</td><td align=\"left\">M</td><td align=\"left\">40–50</td><td align=\"left\">Medical Doctor/senior advisor at health-related govt. institution</td><td align=\"left\">Daily</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">11</td><td align=\"left\">F</td><td align=\"left\">56</td><td align=\"left\">Sociologist/Masters in health admin./advisor at health-related govt. institution</td><td align=\"left\">Almost everyday</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">12</td><td align=\"left\">M</td><td align=\"left\">25–35</td><td align=\"left\">Physical therapist</td><td align=\"left\">Daily</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">13</td><td align=\"left\">F</td><td align=\"left\">28</td><td align=\"left\">Physical therapist at county health station</td><td align=\"left\">Up to 5 days a week</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">14</td><td align=\"left\">M</td><td align=\"left\">43</td><td align=\"left\">Psychologist at hospital</td><td align=\"left\">Daily</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">15</td><td align=\"left\">F</td><td align=\"left\">34</td><td align=\"left\">Medical Doctor at hospital</td><td align=\"left\">Up to 5 days a week</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">16</td><td align=\"left\">M</td><td align=\"left\">49</td><td align=\"left\">Medical Doctor at hospital</td><td align=\"left\">Daily</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">17</td><td align=\"left\">F</td><td align=\"left\">54</td><td align=\"left\">Midwife/teacher</td><td align=\"left\">3 times a week</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">18</td><td align=\"left\">F</td><td align=\"left\">23</td><td align=\"left\">Nurse (recently graduated)</td><td align=\"left\">3 times a week</td><td align=\"left\">Norwegian</td><td align=\"left\">Oslo</td></tr><tr><td align=\"left\">19</td><td align=\"left\">F</td><td align=\"left\">42</td><td align=\"left\">Research nurse</td><td align=\"left\">5–10 hours a week</td><td align=\"left\">Danish</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">20</td><td align=\"left\">F</td><td align=\"left\">-</td><td align=\"left\">Pediatric Nurse</td><td align=\"left\">10–20 hours a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">21</td><td align=\"left\">F</td><td align=\"left\">45</td><td align=\"left\">Consultant, public health. Clinical dentist, doing an Mba</td><td align=\"left\">10–20 hours a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">22</td><td align=\"left\">M</td><td align=\"left\">35</td><td align=\"left\">Medical Doctor</td><td align=\"left\">10–20 hours a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">23</td><td align=\"left\">F</td><td align=\"left\">31</td><td align=\"left\">Psychiatrist</td><td align=\"left\">10–20 hours a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">24</td><td align=\"left\">F</td><td align=\"left\">46</td><td align=\"left\">General practitioner</td><td align=\"left\">20–40 hours a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">25</td><td align=\"left\">F</td><td align=\"left\">41</td><td align=\"left\">Mental Health nurse</td><td align=\"left\">5–10 hours a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">26</td><td align=\"left\">M</td><td align=\"left\">66</td><td align=\"left\">Consultant Dentist Public Health</td><td align=\"left\">Less than 5 hours a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">27</td><td align=\"left\">F</td><td align=\"left\">32</td><td align=\"left\">Nursing, Post-doc in nursing-related field</td><td align=\"left\">10–20 hours a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">28</td><td align=\"left\">F</td><td align=\"left\">40</td><td align=\"left\">Clinical orthodontist</td><td align=\"left\">Up to 5 times a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">29</td><td align=\"left\">F</td><td align=\"left\">45</td><td align=\"left\">Occupational therapist</td><td align=\"left\">Less than 5 times a week</td><td align=\"left\">Other (not English)</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">30</td><td align=\"left\">F</td><td align=\"left\">50</td><td align=\"left\">Nursing, Midwife, starting Phd</td><td align=\"left\">Up to 5 times a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">31</td><td align=\"left\">M</td><td align=\"left\">-</td><td align=\"left\">Dentist</td><td align=\"left\">Daily</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr><tr><td align=\"left\">32</td><td align=\"left\">M</td><td align=\"left\">54</td><td align=\"left\">General practitioner</td><td align=\"left\">5–10 hours a week</td><td align=\"left\">English</td><td align=\"left\">Oxford</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Main findings, sorted into the facets of the honeycomb user experience model</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"2\"><hr/></td></tr><tr><td align=\"left\"><bold>Findability</bold></td><td align=\"left\">Difficulty finding the web site through Google or other external search</td></tr><tr><td/><td align=\"left\">Difficulty finding specific content on the site, using on-site search</td></tr><tr><td/><td align=\"left\">- non-English participants spelled search queries wrong</td></tr><tr><td/><td align=\"left\">- search engine too sensitive</td></tr><tr><td/><td align=\"left\">- keywords search didn't work properly</td></tr><tr><td/><td align=\"left\">- simple search produced unexpected results (i.e.: too few or too many of wrong type)</td></tr><tr><td/><td align=\"left\">- search results were misinterpreted, users confused document types</td></tr><tr><td/><td align=\"left\">- confusion when retrieving only a small number of search results</td></tr><tr><td/><td align=\"left\">Topics navigation not used or not seen</td></tr><tr><td/><td align=\"left\">Minimum of browsing even when encouraged to look around the site</td></tr><tr><td colspan=\"2\"><hr/></td></tr><tr><td align=\"left\"><bold>Usability</bold></td><td align=\"left\">Unfamiliar language/jargon caused confusion</td></tr><tr><td/><td align=\"left\">Text too small</td></tr><tr><td/><td align=\"left\">Too dense, too much text (front page, Help, More information pages)</td></tr><tr><td/><td align=\"left\">Important content too far down on page (review pages)</td></tr><tr><td/><td align=\"left\">Not interested in reading whole review</td></tr><tr><td/><td align=\"left\">Forrest plots unfamiliar and not intuitively located</td></tr><tr><td colspan=\"2\"><hr/></td></tr><tr><td align=\"left\"><bold>Credibility</bold></td><td align=\"left\">Users trusted content in The Cochrane Library</td></tr><tr><td/><td align=\"left\">Confusion about site ownership/neutrality due to dominance of publisher identity and universal navigation, weakens trust</td></tr><tr><td/><td align=\"left\">Misunderstanding about editorial quality evaluation – thinking all content on the whole site content has been reviewed by Cochrane</td></tr><tr><td colspan=\"2\"><hr/></td></tr><tr><td align=\"left\"><bold>Usefulness</bold></td><td align=\"left\">Assuming the library only dealt with medical topics (and not topics such as dentistry, nutrition, acupuncture)</td></tr><tr><td/><td align=\"left\">Misunderstanding targeted texts on front page, thinking content would be tailored for these groups</td></tr><tr><td/><td align=\"left\">Perceived as an academic resource</td></tr><tr><td/><td align=\"left\">Plain language summaries appreciated</td></tr><tr><td colspan=\"2\"><hr/></td></tr><tr><td align=\"left\"><bold>Desirability</bold></td><td align=\"left\">Site seemed off-putting, overwhelming</td></tr><tr><td/><td align=\"left\">Site can be alienating (research/academic identity and language)</td></tr><tr><td colspan=\"2\"><hr/></td></tr><tr><td align=\"left\"><bold>Value</bold></td><td align=\"left\">Felt Cochrane represented golden standard for systematic reviews</td></tr><tr><td/><td align=\"left\">Site is too difficult, would go elsewhere</td></tr><tr><td colspan=\"2\"><hr/></td></tr><tr><td align=\"left\"><bold>Accessibility</bold></td><td align=\"left\">Not evaluated</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Appendix. Interview guide used in Test 2.</p></caption></supplementary-material>" ]
[]
[ "<graphic xlink:href=\"1472-6947-8-34-1\"/>" ]
[ "<media xlink:href=\"1472-6947-8-34-S1.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>" ]
[{"surname": ["Collaboration"], "given-names": ["TC"], "article-title": ["The Cochrane Library"]}, {"collab": ["The Cochrane Collaboration"], "surname": ["Higgens JPT, Green S"], "source": ["Cochrane Handbook for Systematic Reviews of Interventions"]}, {"surname": ["Nielsen", "Loranger"], "given-names": ["J", "H"], "source": ["Prioritizing Web Usability"], "year": ["2006"], "edition": ["1"], "publisher-name": [" New Riders Press"]}, {"surname": ["Kuniavsky", "Kaufmann M"], "given-names": ["M"], "article-title": ["Observing the User Experience: A Practitioner's Guide to User Research"], "source": ["Interactive Technologies"], "year": ["2003"], "edition": ["1"], "publisher-name": [" Morgan Kaufmann"], "fpage": ["575"]}, {"surname": ["Hassenzahl", "Tractinsky"], "given-names": ["M", "N"], "article-title": ["User experience - a research agenda"], "source": ["Behaviour & Information Technology"], "year": ["2006"], "volume": ["25"], "fpage": ["91 "], "lpage": ["997"], "pub-id": ["10.1080/01449290500330331"]}, {"surname": ["Morville"], "given-names": ["P"], "article-title": ["User Experience Design"], "fpage": ["honeycomb model"]}, {"article-title": ["Morae"], "fpage": ["Usability Testing for Software and Web Sites"]}, {"article-title": ["Web Accessibility Initiative"]}, {"collab": ["Department of Health and Human Services"], "source": ["Research-Based Web Design & Usability Guidelines"], "year": ["2006"], "edition": ["Enlarged/Expanded"], "publisher-name": ["Washington, DC , U.S. Dept. of Health and Human Services"]}, {"surname": ["Nielsen", "Tahir"], "given-names": ["J", "M"], "source": ["Homepage Usability: 50 Websites Deconstructed"], "year": ["2001"], "publisher-name": [" New Riders Press"], "fpage": ["336"]}, {"surname": ["Morville"], "given-names": ["P"], "source": ["Ambient Findability"], "year": ["2005"], "publisher-name": ["Sebastopol CA , O'Reilly Media, Inc."]}, {"surname": ["Fogg", "Cathy", "David", "Leslie", "Julianne", "Ellen"], "given-names": ["BJ", "S", "RD", "M", "S", "RT"], "article-title": ["How do users evaluate the credibility of Web sites?"], "source": ["Consumer Reports WebWatch"], "year": ["2002"], "publisher-name": ["Stanford Persuasive Technology Lab, Cordura Hall 226, Stanford University, Stanford, Calif., 94305"]}, {"surname": ["Stanford", "Tauber", "Fogg", "Marable"], "given-names": ["J", "E", "BJ", "L"], "article-title": ["Experts vs. online consumers: a comparative credibility study of health and finance web sites"], "source": ["Consumer Reports WebWatch"], "year": ["2002"], "publisher-name": [" Consumers Union of U.S. Inc."]}, {"surname": ["Nielsen"], "given-names": ["J"], "article-title": ["Ten usability heuristics"]}, {"surname": ["Fogg"], "given-names": ["BJ"], "article-title": ["Stanford guidelines for web credibility. A research summary from the stanford persuasive technology lab."]}, {"surname": ["Pirolli", "Card"], "given-names": ["P", "S"], "article-title": ["Information foraging"], "source": ["Psychological Review"], "year": ["1999"], "volume": ["106"], "fpage": ["643"], "lpage": ["675"], "pub-id": ["10.1037/0033-295X.106.4.643"]}, {"surname": ["Nielsen"], "given-names": ["J"], "article-title": ["Information foraging: why google makes people leave your site faster"]}]
{ "acronym": [], "definition": [] }
24
CC BY
no
2022-01-12 14:47:25
BMC Med Inform Decis Mak. 2008 Jul 28; 8:34
oa_package/7a/cb/PMC2529276.tar.gz
PMC2529277
18673533
[ "<title>Background</title>", "<p>Choose and Book is the National Health Service's electronic booking system for first outpatient appointments in secondary care [##UREF##0##1##]. It is a key project within NHS Connecting for Health and central to the UK Government's patient choice agenda [##UREF##1##2##]. By phoning an appointments line, booking over the Internet, or booking at the GP surgery, patients have a choice of time, date and place for their appointment. Choose and Book is an international exemplar of both the introduction of a large-scale medical informatics system and of a government policy to deliver patient choice.</p>", "<p>Choose and Book enables <italic>Choice at referral </italic>[##UREF##2##3##], which was the first commitment to providing patients with more choice about when and where they receive treatment [##UREF##3##4##]. Since January 2006, barring certain exceptions, all patients requiring elective treatment should be offered the choice of at least four providers, once their GP has decided a referral is required [##UREF##0##1##]. Choose and Book also furnishes a choice of appointment date and time, a central element of the business case for electronic booking [##UREF##4##5##]. Since April 2008, the scheme has been further expanded with the Free Choice policy under which most patients should be able to choose from any secondary care provider (NHS or independent sector) across England.</p>", "<p>In Choose and Book, the referring clinician creates an Appointment Request by selecting a shortlist suited to the patient's clinical needs and preferences from a Directory of Services. Patients then book from a list of available slots in one of three ways:</p>", "<p>i. Patient calls the Booking Management Service quoting a unique booking reference number (UBRN) given to them by the GP;</p>", "<p>ii. Patient accesses Choose and Book through the Web-based Patient Portal within NHS Healthspace [##UREF##5##6##] and books for themselves, using their UBRN; or</p>", "<p>iii. As part of or following the consultation, the GP or one of the practice staff uses the Choose and Book system to make an appointment while the patient is in the surgery.</p>", "<p>Patient choice has been heralded as the driver for transforming the NHS and a means of \"meeting patient expectations\" ([##UREF##6##7##], p. 56). In combination with the new NHS funding mechanism of Payment by Results, where money follows the patient, patient choice is reasoned to provide hospitals with an incentive to improve the quality of their services to attract patients. It is cited as the solution to much that is presently wrong with the NHS from excessive waiting times to even car parking issues [##UREF##7##8##].</p>", "<p>Current theories around choice empowering patients and driving up standards is, for many, convincing. The notion that the concept of consumerism can be applied to healthcare seems plausible given patient's desire for choice [##UREF##8##9##, ####UREF##9##10##, ##UREF##10##11##, ##UREF##11##12####11##12##]. However there are alternative views at both the macro level and at the micro level of patient experience [##REF##16373723##13##, ####UREF##12##14##, ##UREF##13##15##, ##UREF##14##16####14##16##]. 'Choice overload' may lead to bewilderment and anxiety, particularly for patients without access to, or skill to understand, information to make decisions about choices on offer [##REF##16373723##13##,##UREF##12##14##]. There are questions whether the choices proposed are those that patients desire [##UREF##15##17##,##UREF##16##18##] and even over demand for choice itself. Patients' attitudes to choice are inconsistent and variable, depending on their individual circumstances, the types of choice and when they are offered [##UREF##17##19##]. For example, 89% of survey respondents agreed that access to a good local hospital was more appropriate than having more hospitals from which to choose [##UREF##18##20##].</p>", "<p>The case for a choice of appointment date and time has been less contentious and is expected to produce a reduction in non-attendance ([##UREF##4##5##], p. 93), as well as satisfying patient demands.</p>", "<p>Where choice has been introduced in pilot schemes, it has proved popular with patients. Pilots of the earlier <italic>Choice at six months </italic>scheme [##UREF##19##21##] demonstrated high take up rates [##UREF##20##22##,##UREF##21##23##] with a large majority of participants stating that they would recommend the scheme [##UREF##22##24##], although these may not be representative of <italic>Choice at referral</italic>. A pilot scheme for <italic>Choice at referral </italic>was successfully run [##UREF##23##25##]. Although there is some consensus among these different studies, they highlight that patients' attitudes to choice are variable and depend on the types of choice and when they are offered. The national inception of Choose and Book in 2006 presented an opportune time to assess patients' experiences.</p>", "<p>To 3 April 2006, Choose and Book had been used for 261,983 bookings, 12% of the total [##UREF##24##26##]. It is estimated that bookings are growing by at least 40% <italic>per </italic>month. The take up of the system is estimated to be a year behind schedule, due in part to the extension of the scope of the originally designed e-booking system to support <italic>Choice at referral</italic>. There has been considerable bad feeling associated with Choose and Book with criticism about risks to patient confidentiality, reliability and speed [##UREF##25##27##, ####REF##16441973##28##, ##UREF##26##29##, ##UREF##27##30##, ##UREF##28##31##, ##REF##17877873##32####17877873##32##].</p>" ]
[ "<title>Methods</title>", "<p>Using a structured questionnaire in two variants, we evaluated attitudes and experiences among patients referred to the Hillingdon Hospital (Hillingdon site only) through Choose and Book or through the conventional booking process, Partial Booking. The Hillingdon Hospital NHS Trust is based on two sites (Hillingdon and Mount Vernon) in outer London, serving a population of over 300,000. Hillingdon Hospital is the only acute hospital in the London borough of Hillingdon.</p>", "<p>Partial Booking is where a GP sends a written letter of referral to a specified hospital. The choice of the hospital is made by the GP with as much consultation with the patient as the GP chooses. The hospital then writes to the patient to acknowledge their referral and advises them of the anticipated waiting time. This letter requests that the patient phone the hospital booking centre to arrange their appointment.</p>", "<p>We did not find any existing validated measures that could be used here. Patient questionnaires were developed based on a review of the literature and, in particular, consideration of the quantitative research methodology used in Patient Choice pilot studies [##UREF##21##23##,##UREF##29##33##]. Initial drafts were developed and reviewed by the research team and operational staff (Outpatient Booking Co-ordinators) at the Hillingdon Hospital. Amendments were made and a second draft version of each questionnaire then piloted among a small number of initial respondents within the study, following which some minor adjustments were made. Full questionnnaires are given in the Appendix; additional data to those reported here were also collected.</p>", "<p>Using the Trust's Patient Administration System (PAS), a consecutive series of new patients referred through Choose and Book was identified over a planned three-month period. Three months was chosen as a compromise between providing a useful sample size and limited researcher resource. Patients were approached in clinics while waiting to be seen. This was done in preference to a postal survey so as to minimise response bias, where we were concerned patients with particularly negative experiences would be more likely to respond, and to better be able to include patients with poorer English literacy. While waiting at the hospital is also a salient time for patients to recall their booking experience. A matched sampling methodology was adopted recruiting patients who were present in the same outpatient waiting area at the same time and who had come through Partial Booking. However, for logistical reasons, it was not always possible to interview a matched patient. To increase the Partial Booking sample size, during the same period, interviewers attended clinics on additional days and interviewed all new patients in attendance.</p>", "<p>Patients were recruited by JG, ZM or two assistants. They were given a copy of a patient information sheet, which gave them the option not to proceed. Consenting subjects were asked to complete one of two questionnaires while waiting in clinic for their appointment, the questionnaire being dependent on their referral type. Participants were assisted with the questionnaires as necessary, with regular meetings between the data collectors to ensure that any such assistance was consistent and unbiasing. Prior arranged translation arrangements for the consultation were used to facilitate completing the questionnaire; these included a British Sign Language signer and interpretation through family members/escorts.</p>", "<p>The study was approved by the Hillingdon Hospital NHS Trust Research and Development department at the time as part of an audit. In seeking to publish, we sought advice from the NHS Local Research Ethics Committee, whose Chair confirmed that the study is not considered to be research according to the National Research Ethics Service's guidelines and thus had not needed Research Ethics Committee approval.</p>" ]
[ "<title>Results</title>", "<title>Description of the sample</title>", "<p>A total of 104 patients took part in the study between 4 May and 9 August 2006. Of these, 47 were Choose and Book patients. This represents 44% of the 107 total Choose and Book patients seen at the Hillingdon site between these dates. A further 57 patients were referred through the conventional Partial Booking referral process, 19 matched and 38 from additional clinics. Figure ##FIG##0##1## presents a flowchart of recruitment.</p>", "<p>Data were collected on participants' gender, age, ethnic group and specialty division; see Table ##TAB##0##1##. Age data was Normally distributed; other variables are categorical. The two groups were not statistically significantly different on gender, age or ethnic group. The sample was compared with data obtained on all first outpatient attenders at the Trust in the same time period: χ<sup>2</sup>-tests showed no statistically significant difference by gender, age category or ethnic group.</p>", "<p>There was a significant difference between Choose and Book and Partial Booking patients in terms of the specialty division they were under, with more of the Partial Booking patients under Ambulatory Care (Table ##TAB##0##1##). Participants were also asked how important it was to them to be offered a choice of hospital and the two groups showed no statistically significant difference (Table ##TAB##0##1##).</p>", "<p>At the beginning of the questionnaire, patients were asked whether they had been aware, before their GP appointment, of their entitlement to choose to which hospital to be referred. Overall, 63% (65/104; 95% CI 53 to 71%) said they had not; there was no statistically significant association between prior knowledge and referral method (Fisher's exact <italic>p </italic>= 0.3).</p>", "<title>Experience of choice</title>", "<p>Patients were asked if they felt they had been given any choice over their outpatient appointment. No guidance was given about what choice meant. A series of questions then probed the nature of choice respondents felt had been offered. Overall, 52% (53/102; 95% CI: 42 to 61%) of patients felt they had been given choice over their appointment: Choose and Book patients more often than Partial Booking patients. Choose and Book patients also reported being given a choice of hospital more often and being offered a greater number of hospitals than Partial Booking patients. However, there were no statistically significant differences between the two groups on being given a choice of appointment date or time (Table ##TAB##1##2##).</p>", "<p>Focusing just on the Choose and Book patients, 29% (13/45; 95% CI 17 to 43%) reported not being given any choice, 32% (15/47; 95% CI 20 to 46%) reported not being given a choice of hospital, 66% (31/47; 95% CI 52 to 78%) reported not being given a choice of appointment date, 66% (31/47; 95% CI 52 to 78%) reported not being given a choice of appointment time, and 86% (37/43; 95% CI 74 to 94%) reported being given a choice of fewer than four hospitals in total. In all, only one Choose &amp; Book patient (2%; 95% CI 0 to 10%) stated that they had been offered a choice of four hospitals with a choice of appointment date and time.</p>", "<p>Choose and Book patients were asked how their appointment had been booked. Just over half (53%, 25/47) were booked within the GP surgery, with 83% of these (20/24) booked by the GP versus the remainder by non-clinical staff (one response missing). Another third (36%, 17/47) were booked by the patient calling the NHS Direct Appointment Booking Line. Booking on-line accounted for 11% (5/47). How appointments were booked did not statistically significantly vary by gender, age or specialty division. However, there was a relationship with ethnic group (Fisher exact <italic>p </italic>= 0.007) with all the south Asian patients booking within the GP surgery.</p>", "<p>There was a statistically significant association between how Choose and Book patients booked their appointment and whether they reported being given a choice of appointment date and time. Those booking within the GP surgery were least likely to report having a choice and those online, most likely (Table ##TAB##2##3##).</p>", "<p>Those participants who indicated that they had been given a choice over their appointment were asked how satisfied they were with the experience (Table ##TAB##3##4##). Overall, 14% (6/44; 95% CI 6 to 26%) of patients were dissatisfied with the experience of booking their appointment. Among those who reported being given a choice, there was no statistically significant difference between Choose and Book and Partial Booking patients. For both groups, the median response was \"fairly satisfied\" (95% bootstrapped CI [##UREF##30##34##]: \"very satisfied\" to \"fairly satisfied\").</p>" ]
[ "<title>Discussion</title>", "<p>A sizable proportion of patients in this study referred through Choose and Book do not consider that any choice was available to them. That over a quarter (29%) of Choose and Book patients felt they had not been offered choice is in itself striking, but that even higher proportions did not perceive that they had been given a choice of hospital (32%), appointment date (66%) or appointment time (66%) means that, even when patients are offered choice, it does not match Government intentions. While the confidence intervals for these results are wide, even their lower limits are surprisingly high. The clearest demonstration of this misalignment is that, of the complete study sample, only one patient stated that they had been offered a choice of four hospitals with a choice of appointment dates and times, that which Choose and Book supposedly offers to everyone.</p>", "<p>As far as we know, this is the first study of patients experiencing the live implementation of Choose and Book. This is a small study; non-significant statistical test results in comparisons between the Choose and Book and Partial Booking patients should not be over-interpreted given the limited power. It must be recognised that, at the time of the study, not all outpatient clinics at Hillingdon Hospital were Choose and Book enabled and a limited number of GPs were using Choose and Book locally, so the Choose and Book patients surveyed are not necessarily representative of the population coming through the system over the next few years. Restricted interviewer resource prevented achieving a complete consecutive series of Choose and Book patients. Matching between Choose and Book patients and Partial Booking patients was also limited. However, the sample group was representative of the total first outpatient population of Hillingdon Hospital NHS Trust in terms of gender, age and ethnicity.</p>", "<p>The methodology was retrospective in nature and relies on patients' recall. For a true account of how choice is being enacted by healthcare providers and received by patients, observational studies are required of patient/GP consultations.</p>", "<p>It might be anticipated that people with current health issues would have greater awareness of NHS policy than the general public. We found 63% (95% CI 53 to 71%) of patients had no prior knowledge of patient choice compared with a poll showing 80% of British residents aged over 40 knew little or nothing about choice reforms [##UREF##29##33##].</p>", "<p>Our results suggest the Choose and Book booking process may be one factor contributing to the discrepancy in patients' experience of choice. The booking method influences the degree of choice patients perceived. Those booking appointments at the GP surgery are less likely to consider that they have been given a choice over the date or time of their appointment than patients booking their own appointments through the call centre or over the Internet. This may be due to the manner in which the options are either expressed by staff or understood by the patient. For example, where options are framed by the GP as a package of a hospital, a date and a time, any focus, by the patient or the GP, on one individual component may overshadow that further choice has been offered. The reality of there being low awareness of choice among the general public could also mean that patients tend to accept the first date/time offered and primary care staff, therefore, do not present further choices. In contrast, Internet booking involves discrete stages for each of the three choice elements.</p>", "<p>Comments made by participants during data collection suggest, however, that booking online is not always successful. A number of respondents spoke of initially trying to book their appointment over the Internet, but technical problems forced them to call the booking line or go back to their GP to book their appointment. This could partly explain why the proportion of patients booking online is small and suggests that, for Choose and Book to deliver the intended scope of choice, online booking needs to be improved.</p>", "<p>Patients' perception of the scope of choice offered may also be influenced by their own priorities. Comments made by a number of participants suggest that being offered an alternative hospital with, for example, an unsatisfactory waiting time did not consitute a real choice for them. The same was suggested of hospitals with long travel times. For other patients, the fact that their preferred hospital was not on the menu of providers compromised their perception of being offered choice. Ancedotal reports suggest that the timing of clinics is such that the choice of appointment time and day that patients want cannot be delivered, leading to disappointment.</p>", "<p>Neither patients' nor GPs' behaviour necessarily conforms to models of rational choice economics. For example, they tend to show loyalty to their familiar healthcare providers even when they may not offer the best quality care [##UREF##12##14##]. Our results here may be an expression of that cultural barrier to choice. Given the asymmetric doctor/patient relationship, patients may also choose not to choose: even competent adults may prefer to delegate their choice of treatment to someone, typically a health professional, whom they regard as better informed to take the decision on their behalf [##UREF##12##14##,##UREF##16##18##,##REF##17913793##35##]. The role of the GP is anticipated to remain fundamental to patient choice, which places potential constraints on how <italic>Choice at referral </italic>is implemented as the GP can decide how to frame options available to the extent that not all the options are perceived by the patient as being available.</p>" ]
[ "<title>Conclusion</title>", "<p>The findings from this preliminary study suggest that Choose and Book did not deliver choice as portrayed in UK government policy to this patient comunity. A key question for researchers now must be whether these findings generalise across the country. There is also a need for prospective methodologies looking at patient behaviour and experience of Choose and Book. If the findings do stand up to replication, there could be consequences for the programme: for example, if the majority of patients are not experiencing a choice over appointment time and date, will it produce the expected reduction in non-attendance? One could make recommendations that the availability of choice needs to be further promoted; and technical issues need to be addressed. However, as with other policies to increase patient choice, substantial investment may be required in restructuring healthcare services if Choose and Book is failing to deliver [##REF##17913793##35##].</p>", "<p>Choice is only meaningful if there are realistic options and an experience of choice. We suggest our results reveal both a symptom and a cause: the lack of experienced choice may be a symptom of a lack of meaningful choice in the system, while aspects of the system's design may cause patients to experience less choice than intended.</p>", "<p>Our results paint a different picture to the case studies on the Choose and Book website [##UREF##31##36##]. While our findings about Choose and Book need replicating, they more generally match prior studies showing the public is not experiencing the intent of UK government policy on choice [##UREF##15##17##,##UREF##32##37##]. Consumerist models of choice driving quality improvements fail if patients are not exercising that choice. Understanding the discordance between experience and policy intent is crucial to the success of the patient choice agenda. We suggest that consideration needs to be given as to whether choice of hospital should be the focus of patient choice and whether the nature of NHS services, or healthcare services in general, are such that a meaningful choice of place, date and time can ever be delivered.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Choose and Book is a central part of the UK Government patient choice agenda that seeks to provide patients with a choice over the time, date and place of their first outpatient appointment. This is done through the use of a computerised booking system. After a 2004 pilot study, Choose and Book was formally launched in January 2006. This is the first study of patient experience of Choose and Book since then.</p>", "<title>Methods</title>", "<p>A questionnaire survey of reported experience of choice over the time, data and place of appointment, carried out in a National Health Service hospital in London. 104 patients at their first outpatient appointment completed the questionnaire, consisting of a consecutive series of patients referred through Choose and Book and a sample referred through the conventional booking system.</p>", "<title>Results</title>", "<p>Among the Choose and Book patients, 66% (31/47; 95% CI 52 to 78%) reported not being given a choice of appointment date, 66% (31/47; 95% CI 52 to 78%) reported not being given a choice of appointment time, 86% (37/43; 95% CI 74 to 94%) reported being given a choice of fewer than four hospitals in total and 32% (15/47; 95% CI 20 to 46%) reported not being given any choice of hospital.</p>", "<title>Conclusion</title>", "<p>In this study, patients did not experience the degree of choice that Choose and Book was designed to deliver.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>JG conceived the original study, which was developed with HP. Data collection was by JG and ZM. The paper was drafted by HP and JG and all three authors were involved in critically revising the final version.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1472-6947/8/36/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>We thank Stephen Meechan, Richard Parr, Glenn Robert and Alex Douglass-Bonner for assistance and comments, and all the patients and staff who participated in the research. No funding was received for this study. This study had no sponsors.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Flowchart showing study recruitment.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Background variables</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Variable</td><td align=\"left\">Categories</td><td align=\"center\"><bold>Choose &amp; Book </bold>(<italic>n </italic>= 47)</td><td align=\"center\"><bold>Partial Booking </bold>(<italic>n </italic>= 57)</td><td align=\"center\">TOTAL</td><td align=\"left\">Test comparing groups (excluding missing data)</td></tr></thead><tbody><tr><td align=\"right\">Gender</td><td align=\"left\">Female</td><td align=\"center\">29</td><td align=\"center\">38</td><td align=\"center\">67</td><td align=\"left\">Fisher's exact <italic>p </italic>= 0.7</td></tr><tr><td/><td align=\"left\">Male</td><td align=\"center\">18</td><td align=\"center\">19</td><td align=\"center\">37</td><td/></tr><tr><td align=\"right\">Age</td><td align=\"left\">16–29</td><td align=\"center\">10</td><td align=\"center\">9</td><td align=\"center\">19</td><td align=\"left\">Mann-Whitney <italic>z </italic>= 1.5, <italic>p </italic>= 0.1</td></tr><tr><td/><td align=\"left\">30–44</td><td align=\"center\">6</td><td align=\"center\">18</td><td align=\"center\">24</td><td/></tr><tr><td/><td align=\"left\">45–59</td><td align=\"center\">11</td><td align=\"center\">17</td><td align=\"center\">28</td><td/></tr><tr><td/><td align=\"left\">60–79</td><td align=\"center\">16</td><td align=\"center\">11</td><td align=\"center\">27</td><td/></tr><tr><td/><td align=\"left\">80+</td><td align=\"center\">4</td><td align=\"center\">2</td><td align=\"center\">6</td><td/></tr><tr><td align=\"right\">Ethnic group</td><td align=\"left\">White British</td><td align=\"center\">30</td><td align=\"center\">41</td><td align=\"center\">71</td><td align=\"left\">Fisher's exact <italic>p </italic>= 0.6</td></tr><tr><td/><td align=\"left\">South Asian</td><td align=\"center\">9</td><td align=\"center\">8</td><td align=\"center\">17</td><td/></tr><tr><td/><td align=\"left\">Other</td><td align=\"center\">3</td><td align=\"center\">7</td><td align=\"center\">10</td><td/></tr><tr><td/><td align=\"left\"><italic>missing</italic></td><td align=\"center\">5</td><td align=\"center\">1</td><td align=\"center\">6</td><td/></tr><tr><td align=\"right\">Specialty division</td><td align=\"left\">Ambulatory Care</td><td align=\"center\">10</td><td align=\"center\">29</td><td align=\"center\">39</td><td align=\"left\">Fisher's exact <italic>p </italic>= 0.008</td></tr><tr><td/><td align=\"left\">Surgery</td><td align=\"center\">14</td><td align=\"center\">8</td><td align=\"center\">22</td><td/></tr><tr><td/><td align=\"left\">Women &amp; Children</td><td align=\"center\">8</td><td align=\"center\">10</td><td align=\"center\">18</td><td/></tr><tr><td/><td align=\"left\">Medicine</td><td align=\"center\">11</td><td align=\"center\">6</td><td align=\"center\">17</td><td/></tr><tr><td/><td align=\"left\"><italic>missing</italic></td><td align=\"center\">4</td><td align=\"center\">4</td><td align=\"center\">8</td><td/></tr><tr><td align=\"right\">\"How important to you is being given choice over where you go to receive hospital treatment?\"</td><td align=\"left\">Very important</td><td align=\"center\">21</td><td align=\"center\">27</td><td align=\"center\">48</td><td align=\"left\">Mann-Whitney <italic>z </italic>= 0.8, <italic>p </italic>= 0.4</td></tr><tr><td/><td align=\"left\">Important</td><td align=\"center\">12</td><td align=\"center\">17</td><td align=\"center\">29</td><td/></tr><tr><td/><td align=\"left\">Slightly important</td><td align=\"center\">3</td><td align=\"center\">4</td><td align=\"center\">7</td><td/></tr><tr><td/><td align=\"left\">Not important at all</td><td align=\"center\">0</td><td align=\"center\">4</td><td align=\"center\">4</td><td/></tr><tr><td/><td align=\"left\"><italic>missing</italic></td><td align=\"center\">11</td><td align=\"center\">5</td><td align=\"center\">16</td><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Perception of choice by referral method</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\" colspan=\"2\"><break/>Question</td><td align=\"left\"><break/>Categories</td><td align=\"center\"><bold>Choose &amp; Book </bold><break/>(<italic>n</italic> = 47)</td><td align=\"center\"><bold>Partial Booking </bold><break/>(<italic>n</italic> = 57)</td><td align=\"center\"><break/>TOTAL</td><td align=\"left\">Test comparing groups <break/>(excluding missing data)</td></tr></thead><tbody><tr><td align=\"right\" colspan=\"2\">\"Do you consider that you were given any choice over your appointment?\"</td><td align=\"left\">Yes</td><td align=\"center\">30</td><td align=\"center\">23</td><td align=\"center\">53</td><td align=\"left\">Fisher's exact <italic>p </italic>= 0.015 (<italic>no </italic>and <italic>unsure </italic>combined)</td></tr><tr><td/><td/><td align=\"left\">No</td><td align=\"center\">13</td><td align=\"center\">31</td><td align=\"center\">44</td><td/></tr><tr><td/><td/><td align=\"left\">Unsure</td><td align=\"center\">2</td><td align=\"center\">3</td><td align=\"center\">5</td><td/></tr><tr><td/><td/><td align=\"left\"><italic>missing</italic></td><td align=\"center\">2</td><td align=\"center\">0</td><td align=\"center\">2</td><td/></tr><tr><td colspan=\"7\"><hr/></td></tr><tr><td align=\"right\">\"Were you given a choice of the following?\"</td><td align=\"right\">Hospital</td><td align=\"left\">Yes</td><td align=\"center\">32</td><td align=\"center\">11</td><td align=\"center\">43</td><td align=\"left\">Fisher's exact <italic>p </italic>&lt; 0.001</td></tr><tr><td/><td/><td align=\"left\">No</td><td align=\"center\">15</td><td align=\"center\">46</td><td align=\"center\">61</td><td/></tr><tr><td/><td align=\"right\">Appointment Date</td><td align=\"left\">Yes</td><td align=\"center\">16</td><td align=\"center\">17</td><td align=\"center\">33</td><td align=\"left\">Fisher's exact <italic>p </italic>= 0.7</td></tr><tr><td/><td/><td align=\"left\">No</td><td align=\"center\">31</td><td align=\"center\">40</td><td align=\"center\">71</td><td/></tr><tr><td/><td align=\"right\">Time of Appointment</td><td align=\"left\">Yes</td><td align=\"center\">16</td><td align=\"center\">14</td><td align=\"center\">30</td><td align=\"left\">Fisher's exact <italic>p </italic>= 0.4</td></tr><tr><td/><td/><td align=\"left\">No</td><td align=\"center\">31</td><td align=\"center\">43</td><td align=\"center\">74</td><td/></tr><tr><td colspan=\"7\"><hr/></td></tr><tr><td align=\"right\" colspan=\"2\">Number of hospitals offered</td><td align=\"left\">1 – no choice</td><td align=\"center\">15</td><td align=\"center\">47</td><td align=\"center\">62</td><td align=\"left\">Mann-Whitney <italic>z </italic>= 5.3, <italic>p </italic>&lt; 0.001</td></tr><tr><td/><td/><td align=\"left\">2</td><td align=\"center\">16</td><td align=\"center\">7</td><td align=\"center\">23</td><td/></tr><tr><td/><td/><td align=\"left\">3</td><td align=\"center\">6</td><td align=\"center\">1</td><td align=\"center\">7</td><td/></tr><tr><td/><td/><td align=\"left\">4</td><td align=\"center\">6</td><td align=\"center\">0</td><td align=\"center\">6</td><td/></tr><tr><td/><td/><td align=\"left\">Can't remember</td><td align=\"center\">4</td><td align=\"center\">0</td><td align=\"center\">4</td><td/></tr><tr><td/><td/><td align=\"left\"><italic>missing</italic></td><td align=\"center\">0</td><td align=\"center\">2</td><td align=\"center\">2</td><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Perception of choice by Choose and Book booking method</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\" colspan=\"2\">Question</td><td align=\"left\">Categories</td><td align=\"center\"><bold>Within GP surgery</bold></td><td align=\"center\"><bold>Call centre</bold></td><td align=\"center\"><bold>Online</bold></td><td align=\"center\">TOTAL</td><td align=\"left\">Fisher exact test comparing groups</td></tr></thead><tbody><tr><td align=\"left\" colspan=\"2\">\"Do you consider that you were given any choice over your appointment?\"</td><td align=\"left\">Yes</td><td align=\"center\">13</td><td align=\"center\">14</td><td align=\"center\">3</td><td align=\"center\">30</td><td align=\"left\"><italic>p </italic>= 0.07 (<italic>no </italic>and <italic>unsure </italic>combined)</td></tr><tr><td/><td/><td align=\"left\">No</td><td align=\"center\">11</td><td align=\"center\">2</td><td align=\"center\">0</td><td align=\"center\">13</td><td/></tr><tr><td/><td/><td align=\"left\">Unsure</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">2</td><td/></tr><tr><td/><td/><td align=\"left\"><italic>missing</italic></td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">2</td><td align=\"center\">2</td><td/></tr><tr><td colspan=\"8\"><hr/></td></tr><tr><td align=\"left\">\"Were you given a choice of the following?\"</td><td align=\"left\">Hospital</td><td align=\"left\">Yes</td><td align=\"center\">17</td><td align=\"center\">12</td><td align=\"center\">3</td><td align=\"center\">32</td><td align=\"left\"><italic>p </italic>= 1.0</td></tr><tr><td/><td/><td align=\"left\">No</td><td align=\"center\">8</td><td align=\"center\">5</td><td align=\"center\">2</td><td align=\"center\">15</td><td/></tr><tr><td/><td align=\"left\">Appointment Date</td><td align=\"left\">Yes</td><td align=\"center\">5</td><td align=\"center\">6</td><td align=\"center\">5</td><td align=\"center\">16</td><td align=\"left\"><italic>p </italic>= 0.003</td></tr><tr><td/><td/><td align=\"left\">No</td><td align=\"center\">20</td><td align=\"center\">11</td><td align=\"center\">0</td><td align=\"center\">31</td><td/></tr><tr><td/><td align=\"left\">Time of Appointment</td><td align=\"left\">Yes</td><td align=\"center\">21</td><td align=\"center\">9</td><td align=\"center\">1</td><td align=\"center\">16</td><td align=\"left\"><italic>p </italic>= 0.008</td></tr><tr><td/><td/><td align=\"left\">No</td><td align=\"center\">4</td><td align=\"center\">8</td><td align=\"center\">4</td><td align=\"center\">31</td><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Satisfaction with choice</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><break/><break/>Question</td><td align=\"left\"><break/><break/>Categories</td><td align=\"center\"><bold>Choose &amp; Book </bold><break/>(<italic>n</italic> = 30)</td><td align=\"center\"><bold>Partial Booking </bold><break/>(<italic>n</italic> = 23)</td><td align=\"center\"><break/><break/>TOTAL</td><td align=\"left\">Mann-Whitney test comparing groups</td></tr></thead><tbody><tr><td align=\"left\">For those who reported being given a choice: \"Overall, how satisfied or dissatisfied were you with the experience of choosing your hospital?\"</td><td align=\"left\">Very satisfied</td><td align=\"center\">11</td><td align=\"center\">9</td><td align=\"center\">20</td><td align=\"left\"><italic>z </italic>= 0.2, <italic>p </italic>= 0.9</td></tr><tr><td/><td align=\"left\">Fairly satisfied</td><td align=\"center\">9</td><td align=\"center\">9</td><td align=\"center\">18</td><td/></tr><tr><td/><td align=\"left\">Fairly dissatisfied</td><td align=\"center\">3</td><td align=\"center\">1</td><td align=\"center\">4</td><td/></tr><tr><td/><td align=\"left\">Very dissatisfied</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">2</td><td/></tr><tr><td/><td align=\"left\"><italic>missing</italic></td><td align=\"center\">6</td><td align=\"center\">3</td><td align=\"center\">9</td><td/></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[]
[ "<graphic xlink:href=\"1472-6947-8-36-1\"/>" ]
[]
[{"collab": ["Department of Health"], "source": ["\"Choose & Book\" \u2013 Patient's Choice of Hospital and Booked Appointment Policy Framework for Choice and Booking at the Point of Referral London"], "year": ["2004"]}, {"collab": ["Department of Health"], "source": ["Patient Choice London"], "year": ["2006"]}, {"collab": ["Department of Health"], "source": ["Choice at Referral: Guidance Framework for 2006/2007 London"], "year": ["2006"]}, {"collab": ["Department of Health"], "source": ["The NHS Plan: a plan for investment, a plan for reform London"], "year": ["2000"]}, {"collab": ["NHS National Programme for Information Technology"], "source": ["Electronic Booking Project Full Business Case London"], "year": ["2003"]}, {"collab": ["Department of Health"], "source": ["Healthspace London"], "year": ["2006"]}, {"collab": ["The Labour Party"], "source": ["Britain Forward Not Back: The Labour Party Manifesto 2005 London"], "year": ["2005"]}, {"article-title": ["HC Deb 18 July 2006 c134"]}, {"surname": ["Appleby", "Alvarez", "Park et al"], "given-names": ["J", "A"], "article-title": ["Public Responses to NHS Reform"], "source": ["British Social Attitudes Survey 22nd Report, 'Two terms of Labour: the public's reaction'"], "year": ["2005"], "volume": ["chapter 5"], "publisher-name": ["London, Sage"]}, {"collab": ["Department of Health"], "source": ["Building on the Best: Choice, Responsiveness and Equity in the NHS London"], "year": ["2003"]}, {"collab": ["Department of Health"], "source": ["Developing choice, responsiveness and equity in health and social care \"Fair for all and personal to you\" a national consultation exercise \u2013 letter London"], "year": ["2003"]}, {"surname": ["Coulter", "Magee"], "given-names": ["A", "H"], "source": ["The European Patient of the Future"], "year": ["2003"], "publisher-name": ["Maidenhead, Open University Press"]}, {"surname": ["Bate", "Robert"], "given-names": ["P", "G"], "article-title": ["'Build it and they will come' \u2013 or will they? Choice, policy paradoxes and the case of the NHS treatment centres"], "source": ["Policy & Politics"], "year": ["2006"], "volume": ["34"], "fpage": ["651"], "lpage": ["672"], "pub-id": ["10.1332/030557306778553141"]}, {"collab": ["Mori"], "source": ["Impact on Public Expectation London"], "year": ["2004"]}, {"surname": ["Calman", "Rowe"], "given-names": ["M", "R"], "article-title": ["Trust and health care"], "source": ["Sociology Compass"], "year": ["2007"], "comment": ["OnlineEarly Articles. doi:10.1111/j.1751-9020.2007.00007.x"]}, {"collab": ["BMA"], "source": ["NHS does not provide choice says public \u2013 press release London"], "year": ["2006"]}, {"surname": ["Fotaki", "Boyd", "Smith", "McDonald", "Roland", "Sheaff"], "given-names": ["M", "A", "L", "R", "M", "R"], "source": ["Patient Choice and the Organisation and Delivery of Health Services: Scoping review Report for the National Co-ordinating Centre for NHS Service Delivery and Organisation R&D London"], "year": ["2005"]}, {"collab": ["Which?"], "source": ["Which Choice?: health Policy Report London"], "year": ["2005"]}, {"collab": ["Which?"], "source": ["Choices Omnibus Survey London"], "year": ["2005"]}, {"collab": ["Department of Health"], "source": ["Choice at Six Months: Good practice London"], "year": ["2005"]}, {"surname": ["Coulter", "Le Maistre", "Reeves"], "given-names": ["A", "A", "R"], "source": ["Patients' experience of CHD Choice"], "year": ["2004"], "publisher-name": ["Oxford, Picker Institute Europe"]}, {"surname": ["Burge", "Devlin", "Appleby", "Rohr", "Grant"], "given-names": ["P", "N", "J", "C", "J"], "source": ["London Patient Choice Project Evaluation A model of patients' choices of hospitals from stated and revealed preference choice data"], "year": ["2005"], "publisher-name": ["London, Rand Corporation/The King's Fund and City University"]}, {"surname": ["Coulter", "le Maistre", "Henderson"], "given-names": ["A", "N", "L"], "source": ["Patients' experience of choosing where to undergo surgical treatment Evaluation of London Patient Choice Scheme"], "year": ["2005"], "publisher-name": ["Oxford, Picker Institute Europe"]}, {"surname": ["Taylor", "Pringle", "Coupland"], "given-names": ["R", "M", "C"], "source": ["Implications of offering 'patient choice' for routine adult surgical referrals Project Final Report"], "year": ["2004"], "publisher-name": ["Nottingham, Dr Foster/University of Nottingham"]}, {"collab": ["National Audit Office"], "source": ["The National Programme for IT in the NHS London"], "year": ["2006"]}, {"collab": ["BBC News"], "source": ["GPs dissatisfied with IT system London"], "year": ["2006"]}, {"collab": ["E-health Insider"], "source": ["Every PCT to miss Choose and Book target London"], "year": ["2005"]}, {"collab": ["National Audit Office"], "source": ["Patient Choice at the point of GP referral London"], "year": ["2005"]}, {"surname": ["Brooks"], "given-names": ["R"], "article-title": ["System failure!"], "source": ["Private Eye"], "fpage": ["17"], "lpage": ["24"], "comment": ["2\u201315 March 2007"]}, {"surname": ["Page", "Byrom"], "given-names": ["B", "A"], "source": ["What will people choose when Choice goes live?"], "year": ["2005"], "publisher-name": ["London, Mori"]}, {"surname": ["Davison", "Hinkley"], "given-names": ["AC", "DV"], "source": ["Bootstrap Methods and Their Applications"], "year": ["1997"], "publisher-name": ["Cambridge, Cambridge University Press"]}, {"collab": ["Patient experiences of Choose and Book"], "article-title": ["Patient experiences of Choose and Book"], "source": ["Choose and Book website"], "year": ["2008"]}, {"surname": ["Worcester"], "given-names": ["R"], "source": ["Patient Choice"], "year": ["2003"], "publisher-name": ["London, Mori/London School of Economics"]}]
{ "acronym": [], "definition": [] }
37
CC BY
no
2022-01-12 14:47:25
BMC Med Inform Decis Mak. 2008 Aug 1; 8:36
oa_package/4e/a4/PMC2529277.tar.gz
PMC2529278
18691405
[ "<title>Background</title>", "<p>Tobacco smoking is the commonest addiction to psychoactive substances and, being an extremely harmful behavior, grossly compounds to general morbidity and mortality. Growing evidence from classic twin studies supports the significant influence of genetic factors in smoking initiation and persistence, as well as the ability to quit smoking [##REF##12492752##1##,##REF##12745498##2##]. Currently, studies focus on investigating candidate genes to gain a better understanding of a molecular basis for smoking behavior [##REF##15370155##3##]. However, the precise contribution of individual genes in such a complex behavior remains uncertain. It is most likely that many genes influence it, including those that are involved in neurotransmitter pathways and nicotine metabolism [##REF##7733857##4##].</p>", "<p>Studies indicate that nicotine increases serotonin release in the brain, while nicotine withdrawal has an opposite effect [##REF##8242344##5##,##REF##9845269##6##]. It was hypothesized that smoking habits may be associated with diminished serotonin (5-HT) neurotransmission determined by genetic polymorphism [##REF##9521442##7##]. The human serotonin transporter gene (<italic>5-HTT</italic>), being involved in serotonin reuptake, has appeared as a plausible candidate gene for susceptibility to smoking, the more so since its link with psychological traits relevant to smoking behavior was demonstrated [##REF##12808427##8##,##REF##8602004##9##], as well as with alcohol dependence, which increases the risk of tobacco smoking [##REF##11113619##10##,##REF##11449397##11##]. A 44-bp insertion/deletion polymorphism within the promoter region (<italic>5-HTTLPR</italic>) has been identified with two allelic variants, the long (<italic>L</italic>) and the short (<italic>S</italic>) form, which affect the transcriptional efficiency of the <italic>5-HTT </italic>gene [##REF##8632190##12##]. Lerman et al. [##REF##9521442##7##] first hypothesized that <italic>S </italic>allele might exert a protective effect against smoking and evaluated the association of smoking practices and smoking cessation with <italic>5-HTTLPR</italic>. However, they did not find any significant differences in the distribution of <italic>5-HTT </italic>genotypes between smokers and non-smokers in either Caucasian or African Americans. One of the possible explanations for the negative results of this initial case-control study could be the relatively small sample size (268 current smokers vs. 230 never smokers), which could decrease the statistical power to detect small gene effect. However, in contrast to these results, Ishikawa et al. [##REF##10498403##13##] found in a Japanese sample of a similar size that individuals with S/S genotype were less inclined to smoke and/or could more easily stop smoking than others. Results of subsequent studies, including larger sample studies of over one thousand subjects [##REF##16702982##14##], which attempted to replicate these findings [##REF##16702982##14##, ####REF##15863794##15##, ##REF##15806583##16##, ##REF##16770336##17####16770336##17##], have not been consistent, which might result from different ethnic backgrounds being associated with different degrees of linkage disequilibrium with other genes loci [##REF##12215232##18##]. Thus, the hypothesized association between the low-activity <italic>S </italic>allele and smoking remains controversial, although a recent positron emission tomography study provided new evidence of this possible link [##REF##15758168##19##]. Therefore, there is the need for further replication of association studies to define the role of the <italic>5-HTTLPR </italic>in vulnerability to smoking.</p>", "<p>Since there have been no investigations into the association of <italic>5-HTTLPR </italic>and smoking carried out so far in a Polish population, we conducted the present study. The purpose was to determine whether <italic>5HTTLPR </italic>genotype is associated with smoking behavior in Caucasians from Northern Poland, as well as to investigate other risk factors for tobacco smoking.</p>" ]
[ "<title>Materials and methods</title>", "<title>The study sample and measures</title>", "<p>The study sample was completed among patients and staff of the Academic Clinical Center in Gdansk and outpatients of the Department of Family Medicine, the Nicolaus Copernicus University of Torun and Collegium Medicum in Bydgoszcz (NCUT-CMB). They were asked to complete a questionnaire referring to socio-demographic data (age, gender, educational level) and categorical definitions of smoking status. Never smoker was someone, who either had never smoked at all or had never been daily smoker and had smoked less than 100 cigarettes (or the equivalent amount of tobacco) in his lifetime [##UREF##0##20##]. Ever smokers were defined as individuals who had smoked at least 100 cigarettes in their lifetime [##UREF##0##20##]. Current smokers were defined as individuals who, at the time of the survey, smoked cigarettes either daily or occasionally [##UREF##0##20##]. Former smokers were defined as those who had quit smoking at least 1 year before the study. To verify recent non-smoking status, the measurement of carbon oxide in exhaled air was performed in former smokers with the use of <italic>Micro CO </italic>smokelyser (Bedfont Instruments, Kent, UK). The level of education was recorded as primary, vocational, high and university. Three hundred and ten adult subjects, including 150 ever smokers (cases) and 160 gender matched never smoking controls, were recruited. All of them were Caucasians from the North of Poland.</p>", "<p>Several quantitative measures of smoking behavior were also completed with the use of the questionnaire. They included: age of starting regular smoking, number of cigarettes smoked, and number of years of smoking. Pack-years were calculated using the average number of cigarettes smoked daily and the number of years smoked. Current daily smokers were also asked to complete the Fagerstrom Test for Nicotine Dependence [##REF##1932883##21##] and give the longest abstinence period on quitting attempt. All reported periods of maximal abstinence were further calculated into days.</p>", "<p>In addition, information on recent or prior treatment due to any psychiatric disorders (diagnosis and medications) and on alcohol dependence was obtained from respondents by self-report.</p>", "<p>From all participants of the study, 8-mm of venous blood sample was collected into heparinized tubes. Samples were frozen and stored at -80°C until required for molecular genotypic analyses.</p>", "<p>The institutional research ethics committees at the Medical University of Gdansk and the Nicolaus Copernicus University of Torun approved all study procedures, and all subjects provided written, informed consent prior to participation in the study.</p>", "<title>Genotyping</title>", "<p>Genomic DNA was extracted from lymphocytes by an enzymatic method using a commercial kit <italic>Blood DNA Prep Plus </italic>(A&amp;A Biotechnology, Gdynia) and used as a template for the PCR. <italic>5-HTTLPR </italic>genotyping was performed with the use of oligonucleotide primers flanking the <italic>5-HTTLPR </italic>as described by Heils et al. [##REF##8632190##12##], and with a few modifications [##REF##15905633##22##]. The set of primers used was as follows:</p>", "<p>sense: 5'-GGCGTTGCCGCTCTGAATGC-3';</p>", "<p>antisense: 5'-GAGGGACTGAGCTGGACAACCAC-3'.</p>", "<title>Statistical analyses</title>", "<p>The chi-squared (χ<sup>2</sup>) test was used to asses the deviations of genotype distribution from the Hardy-Weinberg equilibrium and for group comparisons of frequencies of allele and genotype. Logistic regression analysis, using STATISTICA 7.1 software (StatSoft Inc., USA), was used to estimate correlations. Variables which appeared to be associated with any increased risk for smoking status in the univariate analysis were analyzed by multivariate analysis. The association between these variables and smoking was expressed as crude and adjusted odds ratios (Ors) with 95% confidence intervals (95% CIs). Student's test t was used to compare means for continuous variables. Because of the non-normal distribution of most parameters, nonparametric Mann-Whitney U test was applied for two-group comparisons. Data have been expressed as means ± standard deviation (SD). A significance level of 0.05 was set for a type 1 error in all analyses.</p>" ]
[ "<title>Results</title>", "<p>Three participants did not undergo genotyping successfully; they were, therefore, excluded from further investigation. As a result, the final sample for analysis consisted of 307 subjects, including 149 ever smokers (66 females; mean age 53.0 ± 11.2 years) and 158 never smokers (79 females; mean age 45.0 ± 16.2 years).</p>", "<p>Distribution of <italic>5-HTTLPR </italic>genotypes did not deviate significantly from the Hardy-Weinberg expectation, as determined by the chi-square test (χ<sup>2 </sup>= 0.80, df = 1; p = 0.37)</p>", "<p>With carriers defined as subjects who tested positive for the presence of the allelic variants, whether homozygous or heterozygous (i.e., <italic>S/S</italic>, <italic>S/L</italic>), we found that 58% of the sample (n = 178) carried <italic>S </italic>allele (n = 42 homozygous), while 42% (n = 129) did not (i.e., <italic>L/L</italic>). Frequencies of <italic>L </italic>and <italic>S </italic>allele, as well as short allele carriers and non-carriers, did not differ significantly in ever and never smokers (χ<sup>2 </sup>= 1.71, df = 1; p = 0.19, and χ<sup>2 </sup>= 1.03; df = 1; p = 0.31, respectively). Similarly, there were no significant difference in the distribution of <italic>5-HTTLPR L </italic>and <italic>S </italic>alleles and genotypes in current and never smokers, as well as in current and former smokers. Allele frequencies and genotypes for the <italic>5-HTT </italic>gene by smoking status can be found in Table ##TAB##0##1##.</p>", "<p>In the smokers group, no association was observed between any quantitative measures of smoking and the polymorphism (Table ##TAB##1##2##).</p>", "<p>Out of the total of 307 participants, 23 subjects (7.5%; 15 females) reported current or prior treatment because of psychiatric disorders, including depression or anxiety-related disorders (20 subjects) and schizophrenia (3 subjects). Cited medications were in agreement with self-reported psychiatric diagnoses and included inhibitors of selective serotonine transporter (15 subjects), tricyclic antidepressants (4 subjects), neuroleptics (3 subjects), and anxiolytics (1 subject). There were 6 (26%) never smokers and 17 (74%) current smokers, while there were no former smokers among them. The rate of ever smokers among subjects reporting mental health problems was significantly higher than in other subjects (74% vs. 46.5%; p = 0.011). Seventeen subjects (5.5%; 3 females) in the study group mentioned alcohol dependence in the self-report. There were 15 ever smokers (88%) and two never smokers among them (12%). The frequency of smoking in alcoholics was significantly higher in comparison to non-alcoholic subjects (88% vs. 46%; p = 0.0018). We found no differences in frequencies of <italic>S </italic>and <italic>L </italic>alleles between subjects with psychiatric disorders or alcohol dependence and remaining subjects (p = 0.64 and p = 0.91, respectively).</p>", "<p>In univariate logistic regression analyses, the following variables appeared to be associated with an increased risk for ever smoking status (i.e., smoking initiation): older age, a history of psychiatric disorders and alcohol dependence. Gender was not considered in these analyses because of the gender-matching of the study sample.</p>", "<p>Multivariate regression analysis demonstrated a significant association between the older age and alcohol dependence and smoking, while adjusted odds ratio for smoking for subjects with psychiatric disorders nearly reached statistical significance (Table ##TAB##2##3##).</p>", "<p>The results of the separate analyses on the association between <italic>5-HTTLPR </italic>and smoking, performed in more homogenous groups of ever and never smokers obtained by excluding subjects with psychiatric disorders and/or alcohol dependence, did not differ significantly from results of analyses performed in the whole study sample. Frequencies of <italic>L </italic>and <italic>S </italic>allele, as well as short allele carriers and non-carriers did not still differ significantly in ever and never smokers (χ<sup>2 </sup>= 1.87, df = 1, p = 0.17, and χ<sup>2 </sup>= 1.43, df = 1, p = 0.23, respectively). Similarly, we consistently found no association between any quantitative measures of smoking and the polymorphism in the smokers group (Table ##TAB##3##4##). Further, the results of multivariate analysis of the association between ever tobacco smoking and <italic>5-HTTLPR</italic>, age, and the level of education (adjusted ORs and 95% CI: 0.76, 0.43–1.34; 4.72, 2.63–8.46; 1.07, 0.57–2.01, respectively) were consistent with those when it was performed in the whole study sample.</p>" ]
[ "<title>Discussion</title>", "<p>In the surveyed population from the North of Poland, we found 36% of carriers of the <italic>5-HTTLPR </italic>short variant allele. This rate was approximate to 34% found by another Polish research group [##REF##15292674##23##], somewhat lower than those reported in two studies conducted in European Americans, where <italic>S </italic>allele was found in 40% and 43% of subjects, respectively, and somewhat higher than the rates found in African American participants of these studies, i.e., 30% and 31%, respectively [##REF##9521442##7##,##REF##9402979##24##]. In turn, the considerably lower rate of <italic>S </italic>allele was found in two studies conducted in Japanese populations – 16% and 19% [##REF##10498403##13##,##REF##9402979##24##]. These differences between <italic>5-HTTLPR </italic>allele frequencies in various populations reflect their racial and ethnic genetic differentiation [##REF##9402979##24##].</p>", "<p>We found no association between <italic>5-HTTLPR </italic>and smoking status, as well as any quantitative measure of smoking, such as the number of cigarettes smoked daily, the number of pack-years, FTND score, or the duration of the longest abstinence period on quitting. To date, only a few studies, in contrast to our findings, supported a link between <italic>5-HTTLPR </italic>and smoking, and demonstrated that individuals with the <italic>L </italic>allele were more inclined to smoke and/or had more difficulties with quitting smoking than others [##REF##10498403##13##,##REF##15863794##15##]. Other studies failed to replicate these positive results, including the more recent report of Trummer et al., who found additionally that neither smoking status nor Fagerstrom Tolerance Questionnaire score, pack-years, number of cigarettes smoked daily or previous attempts to quit smoking were related to <italic>5-HTT </italic>genotypes [##REF##9521442##7##,##REF##16702982##14##]. It should be noted, however, that several factors may have a substantial impact on the outcome of association studies and contribute to their inconsistency [##REF##15370155##3##,##REF##12215232##18##]. The most important is population heterogeneity, as regards ethnicity, gender or age, and possible stratification. Further, interacting effects, such as environment and personality, are considered to play an important role [##REF##7733853##25##]. Between-study heterogeneity results also from the various categorical definitions of never smokers, current smokers and former smokers adopted in particular studies, as well as from differences in the mean number of cigarettes smoked per day or mean values of other quantitative measures of smoking behavior in smokers used in studies. If we compare, for instance, our survey to the Israeli study [##REF##15863794##15##] which reported a highly significant association between <italic>5-HTTLPR </italic>genotype and smoking behavior, irrespective of dependence level, several essential differences regarding both populations might be found. Apart from population ancestry, the younger age of the participants of the Israeli survey seems to be one of them, with the average age of ever smokers being 29 years. In addition, smokers in this study group were mainly light smokers, not biologically dependent on nicotine (i.e., they had a FTND score lower than 6), while, in our study population, half of current smokers were highly addicted subjects. It is likely that these differences could influence the results of these two studies. Moreover, probably the effect of polymorphism is related to socio-cultural settings and ethnicity and may be less marked in populations of such countries like Poland, where smoking was a \"national habit\" for decades [##REF##16010709##26##]. Ever smokers in our material were recruited mostly from individuals who had started smoking at a particular socio-political period of Polish history, which influenced the style of life of Poles with an extremely high consumption of cigarettes [##REF##16010709##26##]. We found that subjects aged 50 years or older had an over three times higher risk of smoking than younger subjects. It is possible that possession of <italic>S </italic>allele, hypothesized to increase dopamine availability in midbrain, was not enough to protect from smoking in such a disadvantageous environment. Thus, non-replication of association studies may result from a small effect of a single gene and the relatively greater influence of a number of environmental factors on smoking. The link of the <italic>5-HTTLPR </italic>or other candidate gene polymorphism with smoking might emerge in a given population when smoking rates decline due to the predominance of non-genetic factors, including positive changes in the attitudes to smoking of the population as a whole.</p>", "<p>It appears essential in case-control genetic association studies to precise defining of smoking phenotypes. The method of classification of ever smokers may have an impact on the results of the study [##REF##12215242##27##]. It is suggested, that a significant amount of information on the resistance to regular smoking might be emerged with defining individuals who have smoked 1–99 cigarettes in their lifetime as ever smokers, not never smokers according to WHO [##REF##12215232##18##,##REF##12215242##27##]. It is also suggested, that genuine never smokers, who had never smoked even one cigarette (or even one puff) may be highly genetically informative [##REF##15370155##3##]. However, in our study conducted in the population of extremely high prevalence of smoking by decades, subjects having smoked up to 100 cigarettes in their lifetime were considered never smokers.</p>", "<p>Because of the divergent results of association studies, evidence for a substantial role of the <italic>5-HTT </italic>gene in smoking behavior is not strong to date. Additionally, meta-analysis of studies on association between the <italic>5-HTTLPR </italic>and smoking behavior [##REF##15370155##3##] did not suggest an effect of this polymorphism on initiating, adopting and persisting with smoking. However, when only the studies which reported data on the <italic>5-HTTLPR </italic>polymorphism that pertained to smoking cessation were analyzed, a significant effect was revealed. It is likely that the presence of a variant allele may be associated with an increased likelihood of successful smoking cessation. In our study, the comparison of the mean length of the self-reported longest abstinence on quitting did not reveal significant differences between <italic>S </italic>allele carriers and non-carriers, which indirectly indicated that ability to quit might not be related to the <italic>5-HTTLPR </italic>genotype. Other genes, non-genetic factors, personality and motivation to quit might contribute a greater effect in smoking cessation.</p>", "<p>Growing evidence indicates that smoking behavior and ability to quit are influenced by personality traits, including neuroticism, and psychiatric disorders or alcohol dependence [##UREF##1##28##,##REF##8465868##29##]. On the other hand, several studies demonstrated the association between the <italic>5-HTTLPR </italic>and alcohol dependence or neuroticism, as well as depression and anxiety, which correlate well with this personality trait [##REF##11113619##10##,##REF##11449397##11##,##REF##11121194##30##,##REF##9577843##31##]. Data suggested also that smoking behavior is influenced by an interaction between neuroticism and <italic>5-HTTLPR </italic>genotype [##REF##10822347##32##]. More recently, however, a number of studies demonstrated that although neuroticism and depression vulnerability were associated with smoking behavior, genotype did not affect this relationship [##REF##18188666##33##,##REF##15718071##34##].</p>", "<p>Undergoing treatment for alcohol addiction and/or the presence of psychiatric disorders was an exclusion criterion imposed on subjects' eligibility for some of the earlier association studies on <italic>5-HTTLPR </italic>to avoid a possible confounding effect on the distribution of genotypes in smokers and non-smokers [##REF##9521442##7##,##REF##10498403##13##]. In the present study, this exclusion criterion was not adopted, although for example the results of other Polish researchers indicated an importance of careful inclusion of probands in studies on association between <italic>5-HTTLPR </italic>and personality traits [##REF##15905633##22##,##REF##11340364##35##], which in turn may affect smoking status. The fact that we found no differences in the frequencies of <italic>S </italic>and <italic>L </italic>alleles between subjects with psychiatric disorders or alcohol dependence and remaining subjects might suggest the lack of associations between psychiatric disorders and alcohol dependence and <italic>5-HTT </italic>genotype. However, the group size of subjects reporting these problems seems too small to generalize the results of this study, especially as regards the link of <italic>5-HTTLPR </italic>with separate psychiatric diagnoses. In several studies which focused on the investigation of a possible link of 5-<italic>HTTLPR </italic>with affective disorders, a positive association was found [##REF##11121194##30##,##REF##9577843##31##], while other studies have not supported this [##REF##14735161##36##, ####REF##16186633##37##, ##REF##10898918##38####10898918##38##]. Thus, the relationship between this polymorphism and affective disorders remains uncertain. Our study let us only support earlier observations that individuals with psychiatric disorders and alcohol dependence are more likely to smoke cigarettes [##REF##8465868##29##].</p>", "<p>Several limitations of the study should be pointed out. Firstly, the study sample was relatively small, while sample size in thousands of subjects might be more sufficient to detect small genetic effects, which are likely for single loci and complex smoking behavior. However, several studies on the link of <italic>5-HTTLPR </italic>with smoking have been recently conducted in smaller samples, which included several hundreds of subjects, and positive association was reported [##REF##15863794##15##, ####REF##15806583##16##, ##REF##16770336##17####16770336##17##]. This incited us to investigate the potential association between <italic>5-HTTLPR </italic>and smoking in the sample of a similar size in Polish population. Secondly, we have not genotyped a SNP within the <italic>5-HTTLPR </italic>[##REF##15087484##39##], which could modify the effect of the <italic>L </italic>allele. Further, the smoking status has been verified with the measurement of exhaled CO concentration only in former smokers (i.e., those smokers who had quit smoking at least 1 year before the study) to avoid the classification error attributed to self-report [##REF##8363000##40##]. In turn, we did not expect this bias toward a socially desirable response in never smokers and current smokers, therefore biochemical verification of smoking status was not performed in these groups. Finally, participants' self-reports of their psychiatric disorders were not confirmed by a formal clinical interview or checking medical documentation, but only confronted with cited pharmacological treatment. However, the main purpose of the present study was to assess the relationship between <italic>5-HTTLPR </italic>and smoking, and psychiatric disorders in general, as well as alcohol dependence, served only as covariates in our analyses.</p>" ]
[ "<title>Conclusion</title>", "<p>In spite of potential limitations, the results of our study allow us to conclude that <italic>5-HTTLPR </italic>is not a major factor determining cigarette smoking in Poles. Probably, the risk of smoking results from a large number of genes, each contributing a small part of the overall risk, while numerous non-genetic factors might strongly influence these genetic undergrounds of susceptibility to smoking. A better understanding of the genetic determinants of smoking needs further investigation into the interactions of genes involved in synthesis, release, uptake and receptor function for a variety of neurotransmitters, as well as into establishing the interactions between the <italic>5-HTTLPR </italic>polymorphism and psychological traits.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>A better understanding of the genetic determinants of tobacco smoking might help in developing more effective cessation therapies, tailored to smokers' genotype. Insertion/deletion polymorphism in the promoter region of the serotonin transporter gene (<italic>5-HTTLPR</italic>) has been linked to vulnerability to smoking and ability to quit. We aimed to determine whether <italic>5-HTTLPR </italic>genotype is associated with smoking behavior in Caucasians from Northern Poland and to investigate other risk factors for tobacco smoking.</p>", "<title>Methods</title>", "<p><italic>5-HTTLPR </italic>genotypes were determined in 149 ever smokers (66 females; mean age 53.0 years) and 158 gender and ethnicity matched never smoking controls (79 females; mean age 45.0 years) to evaluate the association of this polymorphism with ever smoking status. Analysis of smokers was performed to evaluate the role of <italic>5-HTTLPR </italic>in the age of starting regular smoking, the number of cigarettes smoked daily, pack-years, FTND score, duration of smoking, and the mean length of the longest abstinence on quitting. Genotype was classified according to the presence or absence of the short (<italic>S</italic>) allele vs. the long (<italic>L</italic>) allele of <italic>5-HTTLPR </italic>(i.e., <italic>S/S </italic>+ <italic>S/L </italic>vs. <italic>L/L</italic>). Logistic regression analysis was also used to evaluate correlation between ever smoking and several selected variables.</p>", "<title>Results</title>", "<p>We found no significant differences in the rates of <italic>S </italic>allele carriers in ever smokers and never smokers, and no relationship was observed between any quantitative measures of smoking and the polymorphism. Multivariate analysis demonstrated significant association between the older age (OR = 4.03; 95% CI: 2.33–6.99) and alcohol dependence (OR = 10.23; 95% CI: 2.09–50.18) and smoking.</p>", "<title>Conclusion</title>", "<p><italic>5-HTTLPR </italic>seems to be not a major factor determining cigarette smoking in Poles. Probably, the risk of smoking results from a large number of genes, each contributing a small part of the overall risk, while numerous non-genetic factors might strongly influence these genetic undergrounds of susceptibility to smoking.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>AS conceived the study, participated in its design and coordination, performed the statistical analysis and drafted the manuscript. KB participated in the data collection phase, helped to interpret findings and contributed to the text. EJ participated in the design, coordination and supervision of the study and helped to draft the manuscript. ET participated in the data collection phase. All authors reviewed drafts of the manuscript and approved the final version before submitting it for publication.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2350/9/76/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>The authors would like to thank Dr. Adam Burkiewicz (A&amp;A Biotechnology, Gdynia, Poland) for performing molecular analyses and Prof. J. Jassem from the Oncology and Radiotherapy Department of the Medical University of Gdansk for his assistance in language correction.</p>" ]
[]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Distribution of <italic>5-HTTLPR L </italic>and <italic>S</italic>eles and genotypes according to smoking status in the surveyed population from the North of Poland</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Smoking status</td><td align=\"center\" colspan=\"2\">No. (%) of allele</td><td align=\"center\" colspan=\"3\">No. (%) of genotype</td></tr><tr><td/><td colspan=\"5\"><hr/></td></tr><tr><td/><td align=\"center\"><italic>L</italic></td><td align=\"center\"><italic>S</italic></td><td align=\"center\"><italic>L/L</italic></td><td align=\"center\"><italic>L/S</italic></td><td align=\"center\"><italic>S/S</italic></td></tr></thead><tbody><tr><td align=\"left\">Ever smokers</td><td align=\"center\">199 (66.8)</td><td align=\"center\">99 (33.2)</td><td align=\"center\">67 (45)</td><td align=\"center\">65 (43.6)</td><td align=\"center\">17 (11.4)</td></tr><tr><td align=\"left\">Current smokers</td><td align=\"center\">131 (66.2)</td><td align=\"center\">67 (33.8)</td><td align=\"center\">43 (43.4)</td><td align=\"center\">45 (45.5)</td><td align=\"center\">11 (11.1)</td></tr><tr><td align=\"left\">Former smokers</td><td align=\"center\">68 (68)</td><td align=\"center\">32 (32)</td><td align=\"center\">24 (48)</td><td align=\"center\">20 (40)</td><td align=\"center\">6 (12)</td></tr><tr><td align=\"left\">Never smokers</td><td align=\"center\">195 (61.7)</td><td align=\"center\">121 (38.3)</td><td align=\"center\">62 (39.2)</td><td align=\"center\">71 (44.9)</td><td align=\"center\">25 (15.8)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Values for smoking characteristics by 5-<italic>HTTLPR </italic>genotype in ever smokers</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Characteristic</td><td align=\"center\" colspan=\"2\">Mean (SD)</td><td align=\"center\"><italic>P </italic>value</td></tr><tr><td/><td colspan=\"2\"><hr/></td><td/></tr><tr><td/><td align=\"center\"><italic>S/S</italic>+<italic>S/L</italic></td><td align=\"center\"><italic>L/L</italic></td><td/></tr></thead><tbody><tr><td align=\"left\">Duration of smoking</td><td align=\"center\">27.4 ± 11.9</td><td align=\"center\">26.9 ± 12.7</td><td align=\"center\">0.76</td></tr><tr><td align=\"left\">No. of cigarettes smoked daily</td><td align=\"center\">19.6 ± 10.5</td><td align=\"center\">18.2 ± 8.9</td><td align=\"center\">0.58</td></tr><tr><td align=\"left\">No of pack/years</td><td align=\"center\">27.3 ± 20.0</td><td align=\"center\">22.4 ± 12.8</td><td align=\"center\">0.32</td></tr><tr><td align=\"left\">Age of proceeding to regular smoking</td><td align=\"center\">19.9 ± 4.5</td><td align=\"center\">20.5 ± 5.3</td><td align=\"center\">0.76</td></tr><tr><td align=\"left\">FTND score*</td><td align=\"center\">5.5 ± 2.5</td><td align=\"center\">5.3 ± 2.0</td><td align=\"center\">0.66</td></tr><tr><td align=\"left\">Duration of the longest abstinence in quitting attempts*</td><td align=\"center\">368.6 ± 1102.3</td><td align=\"center\">211.6 ± 388.2</td><td align=\"center\">0.90</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Multivariate analysis of the association between ever tobacco smoking and selected variables</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Variables</td><td align=\"center\">Crude ORs (95% CI)</td><td align=\"center\">Adjusted ORs (95% CI)*</td></tr></thead><tbody><tr><td align=\"left\"><italic>S/S </italic>+ <italic>S/L </italic>genotype</td><td align=\"center\">0.79 (0.50–1.25)</td><td align=\"center\">0.78 (0.46–1.33)</td></tr><tr><td align=\"left\">Age ≥ 50 years</td><td align=\"center\">3.13 (1.96–5.00)</td><td align=\"center\">4.03 (2.33–6.99)</td></tr><tr><td align=\"left\">Lower education (primary/vocational)</td><td align=\"center\">1.36 (0.82–2.24)</td><td align=\"center\">0.98 (0.54–1.79)</td></tr><tr><td align=\"left\">Psychiatric disorder +</td><td align=\"center\">3.26 (1.25–8.55)</td><td align=\"center\">2.90 (0.98–8.55)</td></tr><tr><td align=\"left\">Alcohol dependence +</td><td align=\"center\">8.73 (1.95–39.13)</td><td align=\"center\">10.23 (2.09–50.18)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Values for smoking characteristics by 5-<italic>HTTLPR </italic>genotype in ever smokers after excluding subjects with self-reported psychiatric disorders and/or alcohol dependence</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Characteristic</td><td align=\"center\" colspan=\"2\">Mean (SD)</td><td align=\"center\"><italic>P </italic>value</td></tr><tr><td/><td colspan=\"2\"><hr/></td><td/></tr><tr><td/><td align=\"center\"><italic>S/S</italic>+<italic>S/L</italic></td><td align=\"center\"><italic>L/L</italic></td><td/></tr></thead><tbody><tr><td align=\"left\">Duration of smoking</td><td align=\"center\">24.8 ± 11.0</td><td align=\"center\">28.0 ± 13.1</td><td align=\"center\">0.18</td></tr><tr><td align=\"left\">No. of cigarettes smoked daily</td><td align=\"center\">18.0 ± 9.2</td><td align=\"center\">17.1 ± 8.5</td><td align=\"center\">0.73</td></tr><tr><td align=\"left\">No of pack/years</td><td align=\"center\">22.9 ± 15.0</td><td align=\"center\">16.1 ± 17.4</td><td align=\"center\">0.73</td></tr><tr><td align=\"left\">Age of proceeding to regular smoking</td><td align=\"center\">20.6 ± 4.3</td><td align=\"center\">20.5 ± 4.7</td><td align=\"center\">0.76</td></tr><tr><td align=\"left\">FTND score*</td><td align=\"center\">5.1 ± 2.4</td><td align=\"center\">5.2 ± 2.0</td><td align=\"center\">0.79</td></tr><tr><td align=\"left\">Duration of the longest abstinence in quitting attempts*</td><td align=\"center\">413.9 ± 1482.3</td><td align=\"center\">265.9 ± 446.4</td><td align=\"center\">0.29</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><p>*only current daily smokers were analysed</p></table-wrap-foot>", "<table-wrap-foot><p>*Adjusted ORs are adjusted for all other items in the multivariate model</p></table-wrap-foot>", "<table-wrap-foot><p>*only current daily smokers were analysed</p></table-wrap-foot>" ]
[]
[]
[{"collab": ["World Health Organization"], "source": ["Guidelines for controlling and monitoring the tobacco epidemic"], "year": ["1998"], "publisher-name": ["Geneva, World Health Organization"], "fpage": ["76"], "lpage": ["10"]}, {"surname": ["Gilbert", "McClernon", "Gilbert", "Bollinger CT, Fagerstr\u00f6m KO"], "given-names": ["DG", "FJ", "BO"], "article-title": ["The psychology of the smokers"], "source": ["The tobacco epidemic Prog Respir Res [Basel]"], "year": ["1997"], "volume": ["28"], "fpage": ["132"], "lpage": ["150"]}]
{ "acronym": [], "definition": [] }
40
CC BY
no
2022-01-12 14:47:25
BMC Med Genet. 2008 Aug 8; 9:76
oa_package/1b/b6/PMC2529278.tar.gz
PMC2529279
18706099
[ "<title>Background</title>", "<p>Pancreatitis is generally believed to be a disease where pancreas is injured by enzymes that are normally secreted by the acinar cells. Chronic Pancreatitis (CP) is a continuing or relapsing inflammatory process of pancreas leading to exocrine and/or endocrine insufficiency. Tropical calcific pancreatitis (TCP) is a type of CP unique to developing countries in tropical region [##REF##14654569##1##]. An important feature of TCP is its consistent progression to diabetes, commonly known as fibro-calculous pancreatic diabetes (FCPD) [##REF##14654569##1##,##UREF##0##2##]. FCPD is thought to be a type of diabetes secondary to TCP, resulting from destruction of beta-cell mass in the pancreas [##REF##6358777##3##]. However, several studies have shown partial preservation of beta-cell mass [##REF##6358777##3##] and evidence of insulin resistance to a similar degree as seen in type 2 diabetes (T2D) patients [##REF##3220451##4##], suggesting that the diabetes in FCPD could be type T2D, while others have not found insulin resistance to be a major factor in FCPD [##REF##9109851##5##]. It was believed earlier that diabetes specific complications do not occur in FCPD [##REF##808121##6##], but prevalence of retinopathy, [##REF##3622214##7##] nephropathy and neuropathy [##REF##3751448##8##] in FCPD patients has been reported to be no different from matched group of patients with T2D. Similarly, although the diabetes is severe and insulin requiring in both FCPD and type 1 diabetes (T1D), FCPD patients rarely develop ketoacidosis, in contrast to the T1D patients, who are ketosis prone [##UREF##1##9##,##REF##4018450##10##].</p>", "<p>Mutations in the serine protease inhibitor, Kazal type 1 (<italic>SPINK1</italic>) [##REF##12011155##11##, ####REF##12360463##12##, ##REF##12187509##13##, ##REF##12120202##14##, ##REF##12360464##15####12360464##15##], cystic fibrosis transmembrane regulator (<italic>CFTR</italic>) [##REF##11151920##16##], cathepsin B (<italic>CTSB</italic>) [##REF##16492714##17##] and recently, chymotrypsin C (<italic>CTRC</italic>) [##REF##18059268##18##] genes have been identified to be associated with TCP but they mostly associate with pancreatic exocrine dysfunction. No study has yet investigated the genetic basis of diabetes in TCP and FCPD. Based on the suggestive linkage of T2D to chromosome 10q, a microsatellite, DG10S478, within intron 3 of transcription factor 7-like 2 (<italic>TCF7L2</italic>) gene was found to be strongly associated with T2D [##REF##16415884##19##]. An association of a variant of the gene, rs7903146 along with other SNPs in linkage disequilibrium with this polymorphism was first reported in Islandic, Danish and in the US cohort [##REF##16415884##19##]. Subsequently this association was replicated in other populations like Indian [##REF##17093941##20##,##REF##17697858##21##], French [##REF##17003360##22##], U.K [##REF##16936215##23##] and Finnish populations [##REF##16936217##24##], and these variants account for the highest T2D risk confirmed to date [##REF##17888129##25##]. <italic>TCF7L2 </italic>gene variants have also been proposed to play important role in T1D because of its effects on blood glucose homeostasis [##REF##15525634##26##]; however a recent study failed to find any association and age-of-onset effect of T1D with rs7903146 SNP in <italic>TCF7L2 </italic>gene [##REF##17683561##27##]. This suggested that a T2D mechanism mediated by polymorphisms in <italic>TCF7L2 </italic>does not participate in the etiology of T1D, thus susceptibility factors for T2D could be different from those involved in T1D. Hence, investigating a known susceptibility factor for T1D or T2D can help in understanding the type and mechanism of diabetes in FCPD patients.</p>", "<p>As the type of diabetes in FCPD is not clearly understood, we used association of <italic>TCF7L2 </italic>variants with T2D as a marker to decipher the type of diabetes in FCPD. Since there are suggestions that TCP is the pre-diabetic stage of FCPD, we also analyzed the association of <italic>TCF7L2 </italic>polymorphisms in TCP patients. We hypothesized that we would observe an association of variants in the <italic>TCF7L2 </italic>gene with FCPD, if diabetes in these patients is T2D. Since diabetes in TCP is also thought to be due to destruction of endocrine pancreatic cells secondary to destruction of exocrine pancreas, we investigated the interaction between the <italic>TCF7L2 </italic>variants and N34S <italic>SPINK1 </italic>and L26V <italic>CTSB </italic>mutations and explored whether presence of <italic>TCF7L2 </italic>variants in patients with these mutations predisposes them to FCPD.</p>" ]
[ "<title>Methods</title>", "<title>Patients and controls</title>", "<p>478 unrelated individuals (320 males and 158 females) were diagnosed as TCP (n = 286) or FCPD (n = 192) patients based on the established WHO criteria [##UREF##2##28##]. Of these, 333 patients were of Dravidian ethnicity and 145 belonged to Indo-European ethnicity. Six hundred and sixty one age matched individuals (332 males and 329 females) comprising of 259 Dravidians and 402 Indo-Europeans without any complaints and evidence of pancreatitis were included as controls [##REF##16492714##17##,##REF##17093941##20##]. Both patients and the controls filled a detailed questionnaire and signed a written informed consent for genetic analysis. The Institutional Ethics Committee of all the institutes approved the study following the Indian Council of Medical Research guidelines for research on human subjects.</p>", "<title>Genetic analysis</title>", "<p>Genomic DNA from all the patients and healthy volunteers were utilized for this study. Primers, amplifying segments of <italic>TCF7L2 </italic>gene harboring SNPs rs7903146 and rs12255372 were adopted from our earlier study [##REF##17093941##20##]. PCR products were purified and sequenced individually on both the strands using Big-dye terminator cycle sequencing ready kit (Applied Biosystems, Foster City, CA) on an ABI3730 Genetic Analyzer (Applied Biosystems Foster City, CA). In case of unclear sequence data, we repeated direct sequencing under various conditions until the genotype was determined correctly. Ten percent of the genotyping results were validated on tetra primer based analysis for the 2 SNPs [##REF##17093941##20##] and no discrepancy was observed.</p>", "<title>Statistical analysis</title>", "<p>The allele and genotype frequencies were calculated for the SNPs (table ##TAB##0##1##) in cohorts of both ethnicities separately as well as together and to analyze deviation from the Hardy-Weinberg equilibrium, observed and expected genotype frequencies were compared by Markov simulation based goodness of fit test [##REF##11791212##29##]. Chi-square test was used to analyze the statistical significance of the difference in allelic distribution of various polymorphisms in patients and controls (DeFinitte; <ext-link ext-link-type=\"uri\" xlink:href=\"http://ihg.gsf.de/cgi-bin/hw/hwa1.pl\"/>). For assessing the overall significance irrespective of ethnicities, the meta-analysis statistic was used and the forest plots were generated under the fixed effect model using Comprehensive Meta Analysis <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.Meta-Analysis.com\"/> software version 2.2.046 and the Q test was used to test for homogeneity of groupings [##UREF##3##30##]. The whole cohort was dichotomized initially based on the presence or absence of N34S <italic>SPINK1 </italic>and L26V <italic>CTSB </italic>mutations and then the two groups were subdivided into TCP and FCPD patients and distribution of <italic>TCF7L2 </italic>variants was compared between them. Unless indicated specifically, a p-value of 0.05 was considered significant in all the analyses. This study with random selection of patients and controls has 80% power to detect an effect with an OR as low as 1.3 at α = 0.05 and 95% power at the OR of 1.46, which was identified in our earlier study on T2D subjects [##REF##17093941##20##].</p>" ]
[ "<title>Results and discussion</title>", "<p>The two polymorphisms rs7903146 and rs12255372 in the <italic>TCF7L2 </italic>gene, reported to be most strongly associated with T2D, were analyzed in a cohort of TCP and FCPD patients and controls belonging to Dravidian and Indo-European ethnicities. It is believed by most workers in the field that FCPD is the logical end point of TCP and enough evidence exists to suggest that TCP is the pre-diabetic stage of FCPD [##REF##6358777##3##]. Thus, we also analyzed the association of <italic>TCF7L2 </italic>variants in the entire cohort irrespective of their diabetic status. In addition, clinical presentation is known to be variable for FCPD [##REF##14654569##1##,##UREF##0##2##]. Most of the patients present with pain abdomen and evidence of pancreatitis and subsequently develop diabetes at a later stage; a small proportion present with diabetes and are detected to have pancreatic stones and calcification on subsequent investigations [##REF##14654569##1##,##UREF##0##2##]. It may be surmised that additional diabetes susceptibility gene may account for the earlier phenotype of diabetes. Hence, an attempt was also made to dichotomize the cohort into TCP and FCPD to investigate whether FCPD patients have an additional risk due to <italic>TCF7L2 </italic>polymorphisms. We also investigated whether co-inheritance of <italic>TCF7L2 </italic>variants with the <italic>SPINK1 </italic>and <italic>CTSB </italic>mutations predisposes these patients to develop diabetes.</p>", "<p>Both the polymorphisms followed Hardy-Weinberg equilibrium (p &gt; 0.05) and on comparing the allele frequencies within the ethnic groups, Dravidian patients vs Dravidian controls and Indo-European patients vs Indo-European controls, no significant differences were seen (table ##TAB##0##1##), neither did the genotype relative risk differ significantly between patients and controls (table ##TAB##1##2##). A meta-analysis of all cases and control subjects from both ethnicities, showed similar results for both SNPs in <italic>TCF7L2 </italic>gene [(95%CI, 0.95–1.16; P = 0.63, Cochran's Q = 0.0092, P = 0.92 for rs7903146) and (95%CI, 1.01–1.29; P = 0.92, Cochran's Q = 0.0482, P = 0.83 for rs12255372)] (fig ##FIG##0##1##). In order to explore the possibility of association of <italic>TCF7L2 </italic>variants with FCPD, the analysis was carried out in TCP patients with diabetes (FCPD) and those without diabetes separately. Allele and genotypic frequencies did not differ significantly, between TCP patients and controls, FCPD patients and controls and between TCP and FCPD patients, suggesting lack of statistically significant association of <italic>TCF7L2 </italic>polymorphisms with FCPD (table ##TAB##2##3##).</p>", "<p>Association analysis of the total cohort after dichotomization based on N34S <italic>SPINK1 </italic>and L26V <italic>CTSB </italic>mutation status showed comparable allele and genotype frequencies for rs7903146 in both groups, indicating that co-existence of these variants does not increase the risk of developing diabetes in these patients (table ##TAB##3##4##). However, the minor allele frequency for rs7903146 was different between TCP and FCPD patients carrying the N34S <italic>SPINK1 </italic>variant but did not reach statistical significance (OR = 1.59, 95% CI = 0.93–2.70, P = 0.09). Interestingly, similar analysis using L26V <italic>CTSB </italic>variant showed a statistically significant association between TCP and FCPD patients carrying the mutant allele compared to those without the variant (OR = 1.69, 95% CI = 1.11–2.56, P = 0.013) (table ##TAB##3##4##). Similar results were obtained on analysis of the rs12255372 variant in <italic>TCF7L2 </italic>gene (data not presented). This suggests that co-existence of <italic>TCF7L2 </italic>variants and the variants predicting susceptibility to exocrine damage may interact to determine the onset of diabetes in TCP patients. However, this may need to be replicated in larger sample size since there is a possibility of a chance association due to small sample size.</p>", "<p>We and others have earlier replicated the strong association of <italic>TCF7L2 </italic>variants with T2D in Indian population and provided evidence of its likely role in the pathogenesis of T2D by influencing both insulin secretion and insulin resistance [##REF##17093941##20##]. According to accelerator hypothesis, T1D and T2D may share a common etiology of hyperglycemia-induced beta cell damage but T1D may have the added effects of autoimmunity [##REF##11508279##31##]. However, the lack of association of <italic>TCF7L2 </italic>with T1D, as shown by Field et al., does not support the model of a shared major causal pathway in T2D and T1D [##REF##17063324##32##]. Hence the genes that determine susceptibility to T1D must be different from the susceptibility genes of T2D. This has important implications for diabetes in TCP since overlapping features of T1D and T2D are observed in FCPD. As the overall evidence for association of <italic>TCF7L2 </italic>gene variants exceeds genome-wide significance criteria (10<sup>-5</sup>) and clearly establishes <italic>TCF7L2 </italic>as a T2D susceptibility gene of substantial importance in majority of populations world-wide [##REF##16936216##33##] including Indian population [##REF##17093941##20##], it is less likely that T2D may have a susceptibility factor stronger than <italic>TCF7L2</italic>. A lack of association of <italic>TCF7L2 </italic>with TCP or FCPD observed in our study, may suggest a role for genes other than <italic>TCF7L2 </italic>to be predictive of susceptibility to T2D. Since there is debate about the type of diabetes in TCP and FCPD, the lack of association with <italic>TCF7L2</italic>, the gene most strongly associated with T2D may suggest that the diabetes in TCP patients does not have similar features as T2D.</p>" ]
[ "<title>Results and discussion</title>", "<p>The two polymorphisms rs7903146 and rs12255372 in the <italic>TCF7L2 </italic>gene, reported to be most strongly associated with T2D, were analyzed in a cohort of TCP and FCPD patients and controls belonging to Dravidian and Indo-European ethnicities. It is believed by most workers in the field that FCPD is the logical end point of TCP and enough evidence exists to suggest that TCP is the pre-diabetic stage of FCPD [##REF##6358777##3##]. Thus, we also analyzed the association of <italic>TCF7L2 </italic>variants in the entire cohort irrespective of their diabetic status. In addition, clinical presentation is known to be variable for FCPD [##REF##14654569##1##,##UREF##0##2##]. Most of the patients present with pain abdomen and evidence of pancreatitis and subsequently develop diabetes at a later stage; a small proportion present with diabetes and are detected to have pancreatic stones and calcification on subsequent investigations [##REF##14654569##1##,##UREF##0##2##]. It may be surmised that additional diabetes susceptibility gene may account for the earlier phenotype of diabetes. Hence, an attempt was also made to dichotomize the cohort into TCP and FCPD to investigate whether FCPD patients have an additional risk due to <italic>TCF7L2 </italic>polymorphisms. We also investigated whether co-inheritance of <italic>TCF7L2 </italic>variants with the <italic>SPINK1 </italic>and <italic>CTSB </italic>mutations predisposes these patients to develop diabetes.</p>", "<p>Both the polymorphisms followed Hardy-Weinberg equilibrium (p &gt; 0.05) and on comparing the allele frequencies within the ethnic groups, Dravidian patients vs Dravidian controls and Indo-European patients vs Indo-European controls, no significant differences were seen (table ##TAB##0##1##), neither did the genotype relative risk differ significantly between patients and controls (table ##TAB##1##2##). A meta-analysis of all cases and control subjects from both ethnicities, showed similar results for both SNPs in <italic>TCF7L2 </italic>gene [(95%CI, 0.95–1.16; P = 0.63, Cochran's Q = 0.0092, P = 0.92 for rs7903146) and (95%CI, 1.01–1.29; P = 0.92, Cochran's Q = 0.0482, P = 0.83 for rs12255372)] (fig ##FIG##0##1##). In order to explore the possibility of association of <italic>TCF7L2 </italic>variants with FCPD, the analysis was carried out in TCP patients with diabetes (FCPD) and those without diabetes separately. Allele and genotypic frequencies did not differ significantly, between TCP patients and controls, FCPD patients and controls and between TCP and FCPD patients, suggesting lack of statistically significant association of <italic>TCF7L2 </italic>polymorphisms with FCPD (table ##TAB##2##3##).</p>", "<p>Association analysis of the total cohort after dichotomization based on N34S <italic>SPINK1 </italic>and L26V <italic>CTSB </italic>mutation status showed comparable allele and genotype frequencies for rs7903146 in both groups, indicating that co-existence of these variants does not increase the risk of developing diabetes in these patients (table ##TAB##3##4##). However, the minor allele frequency for rs7903146 was different between TCP and FCPD patients carrying the N34S <italic>SPINK1 </italic>variant but did not reach statistical significance (OR = 1.59, 95% CI = 0.93–2.70, P = 0.09). Interestingly, similar analysis using L26V <italic>CTSB </italic>variant showed a statistically significant association between TCP and FCPD patients carrying the mutant allele compared to those without the variant (OR = 1.69, 95% CI = 1.11–2.56, P = 0.013) (table ##TAB##3##4##). Similar results were obtained on analysis of the rs12255372 variant in <italic>TCF7L2 </italic>gene (data not presented). This suggests that co-existence of <italic>TCF7L2 </italic>variants and the variants predicting susceptibility to exocrine damage may interact to determine the onset of diabetes in TCP patients. However, this may need to be replicated in larger sample size since there is a possibility of a chance association due to small sample size.</p>", "<p>We and others have earlier replicated the strong association of <italic>TCF7L2 </italic>variants with T2D in Indian population and provided evidence of its likely role in the pathogenesis of T2D by influencing both insulin secretion and insulin resistance [##REF##17093941##20##]. According to accelerator hypothesis, T1D and T2D may share a common etiology of hyperglycemia-induced beta cell damage but T1D may have the added effects of autoimmunity [##REF##11508279##31##]. However, the lack of association of <italic>TCF7L2 </italic>with T1D, as shown by Field et al., does not support the model of a shared major causal pathway in T2D and T1D [##REF##17063324##32##]. Hence the genes that determine susceptibility to T1D must be different from the susceptibility genes of T2D. This has important implications for diabetes in TCP since overlapping features of T1D and T2D are observed in FCPD. As the overall evidence for association of <italic>TCF7L2 </italic>gene variants exceeds genome-wide significance criteria (10<sup>-5</sup>) and clearly establishes <italic>TCF7L2 </italic>as a T2D susceptibility gene of substantial importance in majority of populations world-wide [##REF##16936216##33##] including Indian population [##REF##17093941##20##], it is less likely that T2D may have a susceptibility factor stronger than <italic>TCF7L2</italic>. A lack of association of <italic>TCF7L2 </italic>with TCP or FCPD observed in our study, may suggest a role for genes other than <italic>TCF7L2 </italic>to be predictive of susceptibility to T2D. Since there is debate about the type of diabetes in TCP and FCPD, the lack of association with <italic>TCF7L2</italic>, the gene most strongly associated with T2D may suggest that the diabetes in TCP patients does not have similar features as T2D.</p>" ]
[ "<title>Conclusion</title>", "<p>As <italic>TCF7L2 </italic>is a major susceptibility gene for T2D, a lack of association of <italic>TCF7L2 </italic>variants with TCP or FCPD observed in our study suggests that T2D associated <italic>TCF7L2 </italic>variants are not associated with diabetes in TCP or the diabetes in TCP patients may not be similar to T2D. Thus, although the variations in <italic>TCF7L2 </italic>increase the risk for T2D and may affect insulin secretion, they do not alter susceptibility to FCPD, the diabetes in TCP patients. However, co-inheritance of the <italic>TCF7L2 </italic>variants with the pancreatitis associated susceptibility variants in <italic>SPINK1 </italic>and <italic>CTSB </italic>genes may predict the development of diabetes in these patients, but these observations need to be confirmed independently.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Tropical calcific pancreatitis (TCP) is a type of chronic pancreatitis unique to developing countries in tropical regions and one of its important features is invariable progression to diabetes, a condition called fibro-calculous pancreatic diabetes (FCPD), but the nature of diabetes in TCP is controversial. We analysed the recently reported type 2 diabetes (T2D) associated polymorphisms in the <italic>TCF7L2 </italic>gene using a case-control approach, under the hypothesis that <italic>TCF7L2 </italic>variants should show similar association if diabetes in FCPD is similar to T2D. We also investigated the interaction between the <italic>TCF7L2 </italic>variants and N34S <italic>SPINK1 </italic>and L26V <italic>CTSB </italic>mutations, since they are strong predictors of risk for TCP.</p>", "<title>Methods</title>", "<p>Two polymorphisms rs7903146 and rs12255372 in the <italic>TCF7L2 </italic>gene were analyzed by direct sequencing in 478 well-characterized TCP patients and 661 healthy controls of Dravidian and Indo-European ethnicities. Their association with TCP with diabetes (FCPD) and without diabetes was tested in both populations independently using chi-square test. Finally, a meta analysis was performed on all the cases and controls for assessing the overall significance irrespective of ethnicity. We dichotomized the whole cohort based on the presence or absence of N34S <italic>SPINK1 </italic>and L26V <italic>CTSB </italic>mutations and further subdivided them into TCP and FCPD patients and compared the distribution of <italic>TCF7L2 </italic>variants between them.</p>", "<title>Results</title>", "<p>The allelic and genotypic frequencies for both <italic>TCF7L2 </italic>polymorphisms, did not differ significantly between TCP patients and controls belonging to either of the ethnic groups or taken together. No statistically significant association of the SNPs was observed with TCP or FCPD or between carriers and non-carriers of N34S <italic>SPINK1 </italic>and L26V <italic>CTSB </italic>mutations. The minor allele frequency for rs7903146 was different between TCP and FCPD patients carrying the N34S <italic>SPINK1 </italic>variant but did not reach statistical significance (OR = 1.59, 95% CI = 0.93–2.70, P = 0.09), while, <italic>TCF7L2</italic>variant showed a statistically significant association between TCP and FCPD patients carrying the 26V allele (OR = 1.69, 95% CI = 1.11–2.56, P = 0.013).</p>", "<title>Conclusion</title>", "<p>Type 2 diabetes associated <italic>TCF7L2 </italic>variants are not associated with diabetes in TCP. Since, <italic>TCF7L2 </italic>is a major susceptibility gene for T2D, it may be hypothesized that the diabetes in TCP patients may not be similar to T2D. Our data also suggests that co-existence of <italic>TCF7L2 </italic>variants and the <italic>SPINK1 </italic>and <italic>CTSB </italic>mutations, that predict susceptibility to exocrine damage, may interact to determine the onset of diabetes in TCP patients.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>SM did all the genotyping, statistical analysis and wrote the first draft of the manuscript. SB and SP assisted in the genotyping and statistical analysis whereas DNR, GVR, SPS and VT were involved in the recruitment of the patients and controls. GRC conceptualized the study, supervised the results and finalized the manuscript. All the authors have gone through the manuscript and have consented to the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2350/9/80/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>The authors express their gratitude to all the patients and the normal individuals for voluntarily participating in the study and especially for giving informed consent for genetic studies. The help of Dr A Ramakrishna, Asian Institute of Gastroenterology, Hyderabad in recruitment of patients and collection of blood samples and Mr Charles J Spurgeon, CCMB, Hyderabad in genotyping is gratefully acknowledged. The study was conducted with the financial support of Council of Scientific and Industrial Research, Ministry of Science and Technology, Government of India (NWP0032).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Meta-analysis for association of TCF7L2 variants with TCP</bold>. Forest plot showing results of meta analysis. Odds ratio for each study is represented by a block bounded by its confidence interval; Combined effect for the two studies has been calculated using fixed effect model; A, rs7903146; B, rs12255372.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Allelic and genotypic frequencies for the <italic>TCF7L2 </italic>variants in TCP patients and controls of different ethnic groups</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">SNP (NCBI 36.2<sup>&amp;</sup>)</td><td align=\"center\">Allele</td><td align=\"center\" colspan=\"2\">Dravidian</td><td align=\"center\" colspan=\"2\">Indo-Europeans</td><td align=\"center\" colspan=\"2\">Total</td><td align=\"center\">Genotype</td><td align=\"left\" colspan=\"2\">Dravidian<sup>@</sup></td><td align=\"left\" colspan=\"2\">Indo-Europeans<sup>@</sup></td><td align=\"left\" colspan=\"2\">Total<sup>@</sup></td></tr><tr><td/><td/><td align=\"center\">Patients</td><td align=\"center\">Controls</td><td align=\"center\">Patients</td><td align=\"center\">Controls</td><td align=\"center\">Patients</td><td align=\"center\">Controls</td><td/><td align=\"left\">Patients</td><td align=\"left\">Controls</td><td align=\"left\">Patients</td><td align=\"left\">Controls</td><td align=\"left\">Patients</td><td align=\"left\">Controls</td></tr><tr><td/><td/><td align=\"center\">n = 333</td><td align=\"center\">n = 259</td><td align=\"center\">n = 145</td><td align=\"center\">n = 402</td><td align=\"center\">n = 478</td><td align=\"center\">n = 661</td><td/><td align=\"left\">n = 333</td><td align=\"left\">n = 259</td><td align=\"left\">n = 145</td><td align=\"left\">n = 402</td><td align=\"left\">n = 478</td><td align=\"left\">n = 661</td></tr></thead><tbody><tr><td align=\"center\">rs7903146 (114748339)</td><td align=\"center\">C</td><td align=\"center\">0.71</td><td align=\"center\">0.70</td><td align=\"center\">0.72</td><td align=\"center\">0.71</td><td align=\"center\">0.73</td><td align=\"center\">0.71</td><td align=\"center\">CC</td><td align=\"left\">175 (52.6)</td><td align=\"left\">130 (50.2)</td><td align=\"left\">78 (53.8)</td><td align=\"left\">207 (51.5)</td><td align=\"left\">253 (53.0)</td><td align=\"left\">337 (51.0)</td></tr><tr><td/><td/><td/><td/><td/><td/><td/><td/><td align=\"center\">CT</td><td align=\"left\">126 (37.8)</td><td align=\"left\">104 (40.2)</td><td align=\"left\">53 (36.6)</td><td align=\"left\">160 (39.8)</td><td align=\"left\">179 (37.3)</td><td align=\"left\">264 (39.9)</td></tr><tr><td/><td align=\"center\">T</td><td align=\"center\">0.29</td><td align=\"center\">0.30</td><td align=\"center\">0.28</td><td align=\"center\">0.29</td><td align=\"center\">0.27</td><td align=\"center\">0.29</td><td align=\"center\">TT</td><td align=\"left\">32 (9.6)</td><td align=\"left\">25 (9.7)</td><td align=\"left\">14 (9.7)</td><td align=\"left\">35 (8.7)</td><td align=\"left\">46 (9.6)</td><td align=\"left\">60 (9.1)</td></tr><tr><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td/><td/><td align=\"center\">n = 332</td><td align=\"center\">n = 180</td><td align=\"center\">n = 144</td><td align=\"center\">n = 402</td><td align=\"center\">n = 476</td><td align=\"center\">n = 582</td><td/><td align=\"left\">n = 332</td><td align=\"left\">n = 180</td><td align=\"left\">n = 144</td><td align=\"left\">n = 402</td><td align=\"left\">n = 476</td><td align=\"left\">n = 582</td></tr><tr><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"center\">rs12255372 (114798892)</td><td align=\"center\">G</td><td align=\"center\">0.77</td><td align=\"center\">0.78</td><td align=\"center\">0.78</td><td align=\"center\">0.78</td><td align=\"center\">0.77</td><td align=\"center\">0.78</td><td align=\"center\">GG</td><td align=\"left\">201 (60.5)</td><td align=\"left\">108 (60.0)</td><td align=\"left\">88 (61.1)</td><td align=\"left\">244 (60.7)</td><td align=\"left\">289 (60.7)</td><td align=\"left\">352 (60.5)</td></tr><tr><td/><td/><td/><td/><td/><td/><td/><td/><td align=\"center\">GT</td><td align=\"left\">110 (33.1)</td><td align=\"left\">64 (35.6)</td><td align=\"left\">48 (33.3)</td><td align=\"left\">135 (33.6)</td><td align=\"left\">158 (33.2)</td><td align=\"left\">199 (34.2)</td></tr><tr><td/><td align=\"center\">T</td><td align=\"center\">0.23</td><td align=\"center\">0.22</td><td align=\"center\">0.22</td><td align=\"center\">0.22</td><td align=\"center\">0.23</td><td align=\"center\">0.22</td><td align=\"center\">TT</td><td align=\"left\">21 (6.3)</td><td align=\"left\">8 (4.4)</td><td align=\"left\">8 (5.6)</td><td align=\"left\">23 (5.7)</td><td align=\"left\">29 (6.1)</td><td align=\"left\">31 (5.3)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Estimates of the genotype and allele relative risks for the <italic>TCF7L2 </italic>variants in the cases and controls based on ethnicity</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">SNP*</td><td align=\"left\">Het OR (95% CI)</td><td align=\"left\">P</td><td align=\"left\">Hom OR (95% CI)</td><td align=\"left\">P</td><td align=\"left\"><sup>&amp;</sup>OR (95% CI)</td><td align=\"left\">P</td></tr></thead><tbody><tr><td align=\"left\">All cases vs all controls</td><td align=\"left\">rs7903146</td><td align=\"left\">0.90 (0.70–1.15)</td><td align=\"left\">0.40</td><td align=\"left\">1.02 (0.67–1.55)</td><td align=\"left\">0.92</td><td align=\"left\">0.96 (0.80–1.16)</td><td align=\"left\">0.70</td></tr><tr><td/><td align=\"left\">rs12255372</td><td align=\"left\">1.03 (0.80–1.34)</td><td align=\"left\">0.80</td><td align=\"left\">0.88 (0.52–1.49)</td><td align=\"left\">0.62</td><td align=\"left\">0.98 (0.80–1.21)</td><td align=\"left\">0.88</td></tr><tr><td align=\"left\">DR cases vs DR controls</td><td align=\"left\">rs7903146</td><td align=\"left\">0.90 (0.64–1.27)</td><td align=\"left\">0.55</td><td align=\"left\">0.95 (0.54–1.68)</td><td align=\"left\">0.86</td><td align=\"left\">0.94 (0.73–1.21)</td><td align=\"left\">0.65</td></tr><tr><td/><td align=\"left\">rs12255372</td><td align=\"left\">0.92 (0.63–1.36)</td><td align=\"left\">0.69</td><td align=\"left\">1.41 (0.60–3.30)</td><td align=\"left\">0.42</td><td align=\"left\">1.04 (0.76–1.41)</td><td align=\"left\">0.81</td></tr><tr><td align=\"left\">IE cases vs IE controls</td><td align=\"left\">rs7903146</td><td align=\"left\">0.86 (0.57–1.30)</td><td align=\"left\">0.48</td><td align=\"left\">1.06 (0.54–2.08)</td><td align=\"left\">0.86</td><td align=\"left\">0.96 (0.71–1.29)</td><td align=\"left\">0.79</td></tr><tr><td/><td align=\"left\">rs12255372</td><td align=\"left\">0.99 (0.65–1.48)</td><td align=\"left\">0.95</td><td align=\"left\">0.96 (0.42–2.23)</td><td align=\"left\">0.93</td><td align=\"left\">0.98 (0.71–1.36)</td><td align=\"left\">0.92</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Estimates of the genotype and allele relative risks for the <italic>TCF7L2 </italic>variants in the cases and controls based on clinical diagnosis</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\">SNP*</td><td align=\"center\">Het OR (95% CI)</td><td align=\"center\">P</td><td align=\"center\">Hom OR (95% CI)</td><td align=\"center\">P</td><td align=\"center\"><sup>&amp;</sup>OR (95% CI)</td><td align=\"center\">P</td></tr></thead><tbody><tr><td align=\"center\">TCP (n = 286) vs Controls</td><td align=\"center\">rs7903146</td><td align=\"center\">0.95 (0.71–1.28)</td><td align=\"center\">0.74</td><td align=\"center\">0.98 (0.59–1.61)</td><td align=\"center\">0.94</td><td align=\"center\">0.97 (0.78–1.21)</td><td align=\"center\">0.81</td></tr><tr><td/><td align=\"center\">rs12255372</td><td align=\"center\">0.92 (0.68–1.26)</td><td align=\"center\">0.62</td><td align=\"center\">1.31 (0.72–2.36)</td><td align=\"center\">0.38</td><td align=\"center\">1.03 (0.81–1.31)</td><td align=\"center\">0.79</td></tr><tr><td align=\"center\">FCPD (n = 192) vs Controls</td><td align=\"center\">rs7903146</td><td align=\"center\">0.82 (0.58–1.16)</td><td align=\"center\">0.27</td><td align=\"center\">1.08 (0.62–1.87)</td><td align=\"center\">0.78</td><td align=\"center\">0.95 (0.74–1.22)</td><td align=\"center\">0.69</td></tr><tr><td/><td align=\"center\">rs12255372</td><td align=\"center\">1.03 (0.73–1.46)</td><td align=\"center\">0.86</td><td align=\"center\">0.89 (0.41–1.92)</td><td align=\"center\">0.76</td><td align=\"center\">0.99 (0.75–1.31)</td><td align=\"center\">0.94</td></tr><tr><td align=\"center\">TCP vs FCPD</td><td align=\"center\">rs7903146</td><td align=\"center\">0.86 (0.58–1.28)</td><td align=\"center\">0.47</td><td align=\"center\">1.10 (0.58–2.08)</td><td align=\"center\">0.76</td><td align=\"center\">0.98 (0.73–1.30)</td><td align=\"center\">0.87</td></tr><tr><td/><td align=\"center\">rs12255372</td><td align=\"center\">1.11 (0.75–1.65)</td><td align=\"center\">0.59</td><td align=\"center\">0.68 (0.30–1.55)</td><td align=\"center\">0.36</td><td align=\"center\">0.96 (0.70–1.31)</td><td align=\"center\">0.79</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Association of <italic>TCF7L2 </italic>variant rs7903146 on dichotomization of the patient cohort based on N34S <italic>SPINK1 </italic>and L26V <italic>CTSB </italic>mutation status</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\"><bold>N34S <italic>SPINK1</italic></bold></td><td/><td/><td align=\"center\" colspan=\"2\"><bold>L26V <italic>CTSB</italic></bold></td><td/><td/></tr><tr><td/><td colspan=\"2\"><hr/></td><td/><td/><td colspan=\"2\"><hr/></td><td/><td/></tr><tr><td/><td align=\"center\"><bold>Wild</bold></td><td align=\"center\"><bold>Mutant</bold></td><td/><td/><td align=\"center\"><bold>Wild</bold></td><td align=\"center\"><bold>Mutant</bold></td><td/><td/></tr><tr><td align=\"center\"><bold>rs7903146</bold></td><td align=\"center\"><bold>MAF</bold></td><td align=\"center\"><bold>MAF</bold></td><td align=\"center\"><bold>OR (95% CI)</bold></td><td align=\"center\"><bold>P</bold></td><td align=\"center\"><bold>MAF</bold></td><td align=\"center\"><bold>MAF</bold></td><td align=\"center\"><bold>OR (95% CI)</bold></td><td align=\"center\"><bold>P</bold></td></tr></thead><tbody><tr><td align=\"center\"><bold>Total Cohort</bold></td><td align=\"center\">(n = 307)</td><td align=\"center\">(n = 148)</td><td/><td/><td align=\"center\">(n = 165)</td><td align=\"center\">(n = 236)</td><td/><td/></tr><tr><td/><td align=\"center\">0.29</td><td align=\"center\">0.28</td><td align=\"center\">0.95 (0.70–1.30)</td><td align=\"center\">0.75</td><td align=\"center\">0.28</td><td align=\"center\">0.28</td><td align=\"center\">1.01 (0.74–1.38)</td><td align=\"center\">0.95</td></tr><tr><td align=\"center\"><bold>FCPD</bold></td><td align=\"center\">(n = 129)</td><td align=\"center\">(n = 49)</td><td/><td/><td align=\"center\">(n = 65)</td><td align=\"center\">(n = 78)</td><td/><td/></tr><tr><td/><td align=\"center\">0.31</td><td align=\"center\">0.35</td><td align=\"center\">1.13 (0.69–1.85)</td><td align=\"center\">0.63</td><td align=\"center\">0.28</td><td align=\"center\">0.36</td><td align=\"center\">1.42 (0.86–2.36)</td><td align=\"center\">0.17</td></tr><tr><td align=\"center\"><bold>TCP</bold></td><td align=\"center\">(n = 178)</td><td align=\"center\">(n = 99)</td><td/><td/><td align=\"center\">(n = 100)</td><td align=\"center\">(n = 158)</td><td/><td/></tr><tr><td/><td align=\"center\">0.27</td><td align=\"center\">0.24</td><td align=\"center\">0.87 (0.58–1.29)</td><td align=\"center\">0.48</td><td align=\"center\">0.28</td><td align=\"center\">0.24</td><td align=\"center\">0.83 (0.55–1.24)</td><td align=\"center\">0.36</td></tr><tr><td align=\"center\"><bold>FCPD vs TCP</bold></td><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"center\">OR</td><td align=\"center\">1.22 (0.85–1.73)</td><td align=\"center\">1.59 (0.93–2.70)</td><td/><td/><td align=\"center\">0.98 (0.60–1.61)</td><td align=\"center\">1.69 (1.11–2.56)</td><td/><td/></tr><tr><td align=\"center\">P</td><td align=\"center\">0.27</td><td align=\"center\">0.09</td><td/><td/><td align=\"center\">0.95</td><td align=\"center\">0.013*</td><td/><td/></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>SNP, single nucleotide polymorphism; n, number of individuals; <sup>&amp;</sup>, Chromosome position according to National Centre for Biotechnology Information (NCBI), Build 36.2, contig accession number NT 030059.12; <sup>@</sup>, values in the parentheses indicate percentage genotype frequency</p></table-wrap-foot>", "<table-wrap-foot><p>SNP, single nucleotide polymorphism; *baseline genotype at rs7903146-CC, rs12255372-GG; Het OR &amp; Hom OR, genotype relative risk (GRR) for heterozygotes and homozygotes respectively (GRR was calculated by comparing with the baseline genotype); <sup>&amp;</sup>, allelic OR; P, P value; DR, Dravidians; IE, Indo-European</p></table-wrap-foot>", "<table-wrap-foot><p>SNP, single nucleotide polymorphism; TCP, tropical calcific pancreatitis; FCPD, fibro-calculous pancreatic diabetes; *baseline genotype at rs7903146-CC, rs12255372-GG; Het OR &amp; Hom OR, genotype relative risk (GRR) for heterozygotes and homozygotes respectively (GRR was calculated by comparing with the baseline genotype); <sup>&amp;</sup>, allelic OR; P, P value</p></table-wrap-foot>", "<table-wrap-foot><p>TCP, tropical calcific pancreatitis; FCPD, fibro-calculous pancreatic diabetes; MAF, Minor allele frequency; OR, odds ratio; CI, confidence interval; P, p value;</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1471-2350-9-80-1\"/>" ]
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[{"surname": ["Reddy"], "given-names": ["DN"], "article-title": ["Tropical pancreatitis. The Indian Experience"], "source": ["Proceedings of the 8th World Congress of the International Gastro-Surgical Club, 15\u201318 April Strasbourg, France"], "year": ["1998"], "fpage": ["249"], "lpage": ["253"]}, {"surname": ["Yajnik", "Shelgikar", "Naik", "Kanitkar", "Orskov", "Alberti", "Hockaday"], "given-names": ["CS", "KM", "SS", "SV", "H", "KG", "TD"], "article-title": ["The ketosis resistance in Fibrocalculous Pancreatic Diabetes 1. Clinical observations and endocrine metabolic measurements during oral glucose tolerance test"], "source": ["Diab Res Clin Pract"], "year": ["1992"], "volume": ["15"], "fpage": ["145"], "lpage": ["56"], "pub-id": ["10.1016/0168-8227(92)90019-N"]}, {"collab": ["WHO Study Group Report on Diabetes Mellitus"], "source": ["WHO technical report series 727"], "year": ["1985"], "publisher-name": ["Geneva: WHO"]}, {"surname": ["Cochran"], "given-names": ["WG"], "article-title": ["Some methods for strengthening the common \u03c72 tests"], "source": ["Biometrics"], "year": ["1954"], "volume": ["10"], "fpage": ["417"], "lpage": ["451"], "pub-id": ["10.2307/3001616"]}]
{ "acronym": [], "definition": [] }
33
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no
2022-01-12 14:47:25
BMC Med Genet. 2008 Aug 16; 9:80
oa_package/57/29/PMC2529279.tar.gz
PMC2529280
18700956
[ "<title>Background</title>", "<p>Canine cutaneous mast cell tumor (MCT) is a common neoplastic disease in dogs, accounting for 7–21% of all cutaneous neoplasms [##REF##4922146##1##, ####REF##231964##2##, ##REF##4735678##3##, ##REF##3115242##4####3115242##4##]. Canine MCTs have variable biologic behaviors, ranging from solitary benign masses that can be cured with surgery alone to systemic and potentially fatal metastatic disease [##REF##4199921##5##, ####REF##15663212##6##, ##REF##6435301##7##, ##UREF##0##8##, ##REF##12243462##9####12243462##9##]. Currently, surgical excision is the primary treatment modality for canine MCT. In the event of incomplete surgical excision or a non-resectable tumor, radiation therapy (RT) may be used as an adjunct local therapy, and in the face of multicentric, metastatic, or aggressive MCT, postoperative chemotherapy is commonly employed. Canine mast cell tumors have variable response rates to different chemotherapy protocols, and, as with any drug, there is potential for the development of serious adverse events as a result of chemotherapy administration[##REF##15663212##6##,##UREF##0##8##]. Therefore, it is critical to identify patients that will benefit most from such treatments and to identify prognostic markers that are associated with specific treatment outcomes.</p>", "<p>Previously, our laboratory has evaluated the prognostic significance of several markers for canine MCT patients treated with surgery alone. In these studies we found that KIT staining patterns[##REF##15232137##10##], <italic>c-KIT </italic>internal tandem duplication (ITD) mutations[##REF##16611403##11##], and cellular proliferation as measured by Ki67 immuno-staining and AgNOR histochemical staining[##REF##17491070##12##] are significantly associated with the progression of canine MCT when treated with surgery alone. Although the results of these studies demonstrate the utility of these markers as diagnostic and prognostic tools for MCT treated with surgery alone, they do not provide information regarding their association with the outcome of patients treated with postoperative RT or chemotherapy.</p>", "<p>Combination chemotherapy with vinblastine and prednisone has been previously suggested to be an efficacious postoperative therapy for a subset of MCT patients[##REF##10499735##13##]; however, no clear determinants have been identified to discriminate between MCTs that are likely to benefit from vinblastine and prednisone and those that are not. Therefore, the primary goal of this study was to evaluate the prognostic significance of histologic grade; <italic>c-KIT </italic>ITD mutation; KIT staining patterns; and the proliferation markers Ki67 and AgNORs for dogs treated with vinblastine and prednisone following surgery +/- RT. Additionally, in order to better determine the efficacy of postoperative vinblastine and prednisone, an additional goal of this study was to compare the outcomes of dogs postoperatively treated with vinblastine and prednisone +/- RT to those treated with surgery alone, when stratified based on identified prognostic markers. The results of this paper show that histologic grade; <italic>c-KIT </italic>ITD mutation; KIT staining patterns; and the proliferation markers Ki67 and AgNORs are prognostically significant for dogs with MCTs treated with vinblastine and prednisone following surgery +/- RT. Additionally, this study strongly suggests that dogs with grade 3 MCTs benefit from post-operative chemotherapy treatment when compared to treatment with surgery alone.</p>" ]
[ "<title>Methods</title>", "<p>Twenty-eight canine cutaneous MCTs from 28 dogs included in this study were identified as part of a larger retrospective study[##REF##10499735##13##] with the goals of: 1. evaluating the combination of vinblastine and prednisone as postoperative chemotherapy for canine MCT following surgical excision +/- RT; and 2. identifying clinical prognostic factors associated with this treatment. All cases were treated at the University of Wisconsin – Madison Veterinary Medical Teaching Hospital and were included in this study based on the following inclusion criteria: 1. absence of severe concurrent disease; 2. complete staging; 3. no concurrent systemic antineoplastic therapy other then prednisone and vinblastine; 4. absence of measurable disease following surgery; 5. confirmed histologic diagnosis of canine cutaneous MCT; 6. adequate tissues available for <italic>c-KIT </italic>polymerase chain reaction and immunohistochemistry. Prednisone was administered at an initial dose of 2 mg/kg PO daily, and tapered over 12–26 weeks. Vinblastine was administered at 2 mg/m<sup>2 </sup>as a rapid intravenous bolus weekly for 4 weeks, followed by 4 biweekly treatments. One dog was given a total of 16 vinblastine treatments, as treatment was re-initiated at the time of disease recurrence. Tumors were considered to have been treated with \"adequate local therapy\" when tumors were completely excised with no evidence of neoplastic mast cells at the surgical margins or when surgical excision was performed with subsequent local RT. According to these standards, adequate local therapy was obtained in 24 of 28 dogs. Four dogs had histologic evidence of microscopic disease at the surgical site at the time that chemotherapy was initiated. Three of these dogs had histologic grade 2 MCT and one dog had grade 3 MCT. Postoperative RT was performed in 14 patients, and regional lymph nodes were irradiated in 10 of these patients. Eleven dogs received 4 once-weekly, 8 Gray radiation treatments; one dog received 4 once weekly, 6.5 Gray treatments; and 2 dogs received 15 daily (M-F) treatments of 3.2 Gray each. Histologic grade of each tumor was confirmed according to the Patnaik histologic grading system for canine cutaneous MCT by a single pathologist[##REF##6435301##7##]. Complete clinical staging was performed in all patients, and included physical examination, complete blood count, serum biochemistry profile, abdominal ultrasound, and regional lymph node palpation with or without fine needle aspiration cytology. Lymph nodes were only considered positive for lymph node metastasis if they contained clusters or sheets of mast cells; scattered individual mast cells were not sufficient to diagnose lymph node metastasis. A control population of 56 dogs with canine MCT treated with surgery alone, which were evaluated as part of a previous study[##REF##17491070##12##], were used to compare outcomes between dogs treated with surgery alone and those treated with surgery and postoperative chemotherapy. These cases were submitted as routine biopsies to the Diagnostic Center for Population and Animal Health from multiple referring veterinarians; therefore, clinical staging was not standardized among these cases. Of these 56 dogs, 8 dogs had histologic grade 1 MCT, 41 dogs had grade 2 MCT, and 7 dogs had grade 3 MCT. Nine of these dogs had internal tandem duplication <italic>c-KIT </italic>mutations.</p>", "<p>Immunohistochemical staining, AgNOR staining; evaluation of KIT staining patterns, evaluation of proliferation indices as measured by Ki67 labeling and AgNOR staining, and analysis of <italic>c-KIT </italic>mutations were performed as previously described[##REF##17491070##12##] In brief, 3 patterns of KIT protein localization were identified: 1. KIT pattern I, which consisted of a predominately peri-membrane pattern of KIT protein localization with minimal cytoplasmic KIT protein localization; 2. KIT pattern II, which consisted of focal to stippled cytoplasmic KIT protein localization; and 3. KIT pattern III, which consisted of diffuse KIT cytoplasmic KIT protein localization. Each MCT was classified based on the highest staining pattern present in at least 10% (estimated based on 100 neoplastic cells in a high power field) of the neoplastic cell population or being present in large clusters. For Ki67 immunohistochemical staining evaluations, areas with the highest proportion of immuno-positive neoplastic mast cells were identified at 100× magnification using an American Optical light microscope. The number of immuno-positive cells present in a 10 mm × 10 mm grid area was counted using a 1 cm<sup>2 </sup>10 × 10 grid reticle at 400× magnification. The number of immuno-positive cells per grid area was evaluated in 5 high powered fields and subsequently averaged in order to obtain the growth fraction. In order to determine the average AgNOR count/cell in each tumor, AgNORs were counted in 100 randomly selected neoplastic mast cells throughout the tumor at 1000× magnification. Individual AgNORs were resolved by focusing up and down while counting within individual nuclei. Average AgNOR counts/cell was determined based on averaging these counts.</p>", "<title>Statistical Analysis</title>", "<p>This study used two different approaches to analysis: log-rank models(SAS PROC LIFETEST) to describe associations between risk factors and the occurrence (yes/no) of MCT outcomes, and the Cox proportional hazards models (SAS PROC PHREG)<sup>b </sup>for survival analysis to describe the relationships between risk factors and the time to the occurrence of different MCT outcomes (time-to-event). Survival analysis produces point estimates of the hazard ratio (risk ratio) for risk factors in the model. The MCT outcomes used in this study included disease-free interval and overall survival time. Median times for disease-free interval (DFI) and overall survival (OS) times were reported as the time from treatment initiation to event or last follow-up. Disease-free interval was defined as the time until local recurrence, development of metastatic disease, or development of a new mast cell tumor.</p>", "<title>Univariable Analyses</title>", "<p>Before developing multivariable models, each risk factor was evaluated for its association with MCT outcomes. Both univariable log-rank (for occurrence) and proportional hazards (for time to event) models were developed for each risk factor for each outcome, and the level of association was assessed through the risk factor's p-value in the model. Risk factors with p less than or equal to 0.20 were considered for inclusion in the multivariable models.</p>", "<title>Multivariable Survival Analysis Models</title>", "<p>Multivariable proportional hazards regression models were developed for survival analysis of different outcomes associated with MCT, DFI and OS. As for the multivariable log-rank models, animal signalment (age, sex, weight) was included in the multivariable models to account for their effects on model outcomes. The effects of risk factors on days to events were reported as p-values. The cut off for significance of the multivariate analysis was set a p &lt; 0.05.</p>" ]
[ "<title>Results</title>", "<p>Twenty-eight MCTs dogs postoperatively treated with vinblastine and prednisone +/- RT were included in this study. Fourteen dogs were female and 14 were male. Dogs ranged from 2 to 14 years of age (mean = 9.29 years) and represented 16 breeds including mixed breed dogs (9), Labrador retrievers (4), Gordon setters (2), and 13 other breeds which were represented by single animals. Ten tumors were histologic grade 3 MCT and 18 were grade 2. Four MCTs, all of which were histologic grade 3 tumors, had ITD <italic>c-KIT </italic>mutations. Two MCTs had pattern 1 KIT protein expression, 6 MCTs had KIT pattern 3, and 20 MCTs had KIT pattern 2.</p>", "<p>Results of multivariable analyses are presented in Table ##TAB##0##1##. According to multivariable analyses, dogs with histologic grade 3 MCT and dogs with ITD <italic>c-KIT </italic>mutations had significantly shorter disease-free intervals (DFI) (p = 0.0220 and p = 0.0413, respectively) and overall survival times (OS) (p = 0.010 and p = 0.0112, respectively) compared to dogs with grade 2 MCT and dogs lacking <italic>c-KIT </italic>mutations, respectively (Figures ##FIG##0##1## and ##FIG##1##2##). Tumors with KIT staining pattern 3 were significantly associated with decreased DFI and OS compared to tumors with KIT pattern 2 (p = 0.0022 and p = 0.0049, respectively; Figure ##FIG##2##3##). Increased Ki67 expression and AgNOR values were associated with decreased DFI (p = 0.0386 and p = 0.0168, respectively) and decreased OS (p = 0.0097 and p = 0.0066, respectively) [##REF##17491070##12##].</p>", "<p>Although previous studies have described patient survival following postoperative treatment with vinblastine and prednisone[##REF##10499735##13##], no studies have compared the outcome of treatment with vinblastine and prednisone to that of surgery alone in a similar group of dogs. In order to retrospectively evaluate the efficacy of postoperative vinblastine and prednisone, survival curves representing DFI and OS times were compared between dogs treated in this study and dogs treated with surgery alone that were evaluated as part of previous studies[##REF##15232137##10##, ####REF##16611403##11##, ##REF##17491070##12####17491070##12##,##REF##16050909##14##]. As a means of ensuring that similar populations of animals were compared, comparisons were made between groups of animals that had similar pre-treatment prognoses based on histologic grade, KIT staining pattern, and the presence of ITD <italic>c-KIT </italic>mutations.</p>", "<p>According to multivariable analyses, dogs with histologic grade III MCT treated with surgery alone had significantly shorter DFI (1 month vs. 7 months, p = 0.0082) and OS times (1 month vs. 9.2 months, p = 0.0427) compared to dogs treated with vinblastine and prednisone (Figure ##FIG##3##4##). Dogs with KIT pattern 2 MCT treated with surgery alone had significantly shorter OS times compared to those treated with vinblastine and prednisone (16.6 months vs. 19 months, p = 0.0362). Mast cell tumors with c-<italic>KIT </italic>mutations treated with vinblastine and prednisone tended to have increased DFI (1 month vs. 6.5 months) and OS times (1 month vs. 8.9 months); however, these associations were not statistically significant (Figure ##FIG##4##5##).</p>" ]
[ "<title>Discussion</title>", "<p>In this study, treatment with vinblastine and prednisone following surgery +/- RT benefited dogs with grade III MCT over treatment with surgery alone. The results of this study further validate the use of postoperative vinblastine and prednisone for histologic grade III MCT. Additionally, dogs with <italic>c-KIT </italic>mutations that were treated with vinblastine and prednisone tended to have longer DFI and OS times as compared to those treated with surgery alone, although this association was not statistically significant. This lack of statistical significance might be due to the relatively low number of cases with <italic>c-KIT </italic>mutations evaluated or the similar relative frequency of DFI and OS after 5 and 10 months, respectively. Additionally, 2/9 dogs with <italic>c-KIT </italic>mutations treated with surgery alone survived greater than 20 months. However, one mutation was confined to intron 11; therefore the biologic significance of this mutation is unknown[##REF##16611403##11##]. The difference in median OS times between MCTs with <italic>c-KIT </italic>mutations treated with local therapy alone and those treated with vinblastine and prednisone suggests that MCTs with <italic>c-KIT </italic>mutations also might benefit from postoperative chemotherapy with vinblastine and prednisone.</p>", "<p>In a previous study, we showed MCTs treated with surgery alone with more than 23 Ki67-positive cells per grid area or a Ki67 × AgNOR (Ag67) value of more than 53 per grid area were associated with decreased patient survival[##REF##17491070##12##]; however, in this study, these cut-off values did not discriminate patient survival. Interestingly, in this study only 2/14 dogs with Ki67 indices &lt; 23 died by the end of this study, while 8/14 dogs with Ki67 indices &gt; 23 died. Similarly only 1/14 dogs with Ag67 indices &lt; 53 died, while 9/14 dogs with Ag67 indices &gt; 53 died by the end of this study. The lack of statistical significance in this study is likely due to variations in the patient populations of these studies and the small population size in this study. Our previous study included 8 dogs with histologic grade 1 MCT, while this study only included grade 2 and grade 3 MCTs. Additionally, all cases included in this study received chemotherapy, suggesting that as a whole, this study was biased towards more aggressive MCT.</p>", "<p>In this study, histologic grade, the presence of <italic>c-KIT </italic>mutations, KIT staining patterns, and proliferation markers, Ki67 and AgNORs, were associated with the prognosis of MCT postoperatively treated with vinblastine and prednisone. Compared with grade 2 MCTs, histologic grade 3 MCTs were significantly associated with decreased DFI and OS times. Additionally, dogs with grade 3 MCT significantly benefited from treatment with vinblastine and prednisone. These results support the use of histologic grading for the prognostication of canine MCT; however, interobserver variations and the unpredictable behavior of grade 2 MCT continue to limit the utility of histologic evaluation as the sole predictor of outcome [##UREF##1##15##, ####REF##15945380##16##, ##REF##16475514##17##, ##REF##11318363##18##, ##REF##11804319##19####11804319##19##]. Therefore, in the face of intermediate grade MCTs, histologic grade should be complemented with additional prognostic markers, such as KIT immunohistochemistry, <italic>c-KIT </italic>mutation evaluation, and proliferation markers using Ki67 and Ag67 cut-off values of 23 and 53, respectively. No single marker can successfully identify all MCTs that will result in death; therefore, a panel of markers should be used in order to evaluate the entire spectrum of the disease.</p>", "<p>Major limitations of this study include the levels of historical inaccuracy associated with any retrospective study, small samples sizes in some subgroups, and variations in treatment and staging; most notably the fact that 50% of the chemotherapy treated patients received RT. However, dogs treated with chemotherapy and radiation therapy did not have significantly different times to treatment failure or overall survival times as compared to those treated without radiation therapy (log rank p-value = 0.223 and p = 0.314, respectively). An additional limitation of this study was that many of the control treated animals were treated in primary care private practices, while those treated with chemotherapy were treated at a single referral practice. Differences in care between clinicians and hospitals might further affect treatment outcomes; however, data available from for this study do not allow us to evaluate this effect.</p>" ]
[ "<title>Conclusion</title>", "<p>The results of this study add further credence supporting the use of histologic grade, <italic>c-KIT </italic>mutations, and KIT protein localization for the prognostication of canine MCTs. Additionally, these results further support that hypothesis that <italic>c-KIT </italic>mutations and intracellular localization of the KIT protein both contribute to the progression of canine MCT. Finally, although previous studies have evaluated the use of vinblastine and prednisone for the treatment of high-risk MCTs, this is the first study to compare the outcome of animals treated with adjuvant vinblastine and prednisone +/- RT to a prognostically matched group of animals treated with surgery alone. Although retrospective studies provide critical, relevant information regarding the utility of treatments and prognostic markers, controlled, blinded prospective studies remain the gold standard for evaluating therapeutics and prognostic markers and the data presented here need to be corroborated in a controlled, prospective study in order to truly define their prognostic significance. In the future, prospective evaluations of new therapeutics should not only be focused on the safety and efficacy of the therapy, but also on identifying biomarkers including histopathology, clinical pathology, and molecular biology-based markers associated with outcome.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Canine cutaneous mast cell tumor (MCT) is a common neoplastic disease associated with a variable biologic behavior. Surgery remains the primary treatment for canine MCT; however, radiation therapy (RT) and chemotherapy are commonly used to treat aggressive MCT. The goals of this study were to evaluate the prognostic utility of histologic grade, <italic>c-KIT </italic>mutations, KIT staining patterns, and the proliferation markers Ki67 and AgNORs in dogs postoperatively treated with vinblastine and prednisone +/- RT, and to compare the outcome of dogs treated with post-operative chemotherapy +/- RT to that of a prognostically matched group treated with surgery alone. Associations between prognostic markers and survival were evaluated. Disease-free intervals (DFI) and overall survival times (OS) of dogs with similar pretreatment prognostic indices postoperatively treated with chemotherapy were compared to dogs treated with surgery alone.</p>", "<title>Results</title>", "<p>Histologic grade 3 MCTs, MCTs with c-<italic>KIT </italic>mutations, MCTs with increased cytoplasmic KIT, and MCTs with increased Ki67 and AgNOR values were associated with decreased DFI and OS. Dogs with histologic grade 3 MCT had significantly increased DFI and OS when treated with chemotherapy vs. surgery alone. Although not statistically significant due to small sample sizes, MCTs with <italic>c-KIT </italic>mutations had increased DFI and OS when treated with chemotherapy vs. surgery alone.</p>", "<title>Conclusion and clinical importance</title>", "<p>This study confirms the prognostic value of histologic grade, c-<italic>KIT </italic>mutations, KIT staining patterns, and proliferation analyses for canine MCT. Additionally, the results of this study further define the benefit of postoperative vinblastine and prednisone for histologic grade 3 MCTs.</p>" ]
[ "<title>Abbreviations</title>", "<p>Disease-free intervals: DFI; mast cell tumor: MCT; overall survival times: OS; radiation therapy: RT.</p>", "<title>Authors' contributions</title>", "<p>JDW coordinated the study, performed histologic and immunohistologic evaluations, performed polymerase chain reactions, and drafted the manuscript. VY–G and MK designed the study and oversaw all post-clinical aspects of the study. EH performed all statistical analyses. DHT collected all clinical data. All authors helped interpret results and have read and approved the final manuscript.</p>" ]
[ "<title>Acknowledgements</title>", "<p>This work was partially funded by a Companion Animal Fund Grant from Michigan State University College of Veterinary Medicine. Joshua Webster was supported by NIH T-32 post-doctoral training grant RR17189. An abstract of this work has been submitted for the 58<sup>th </sup>American College of Veterinary Pathologists Annual Meeting in November 2007.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Survival curves of dogs with histologic grade 2 and 3 MCTs</bold>. Kaplan-Meier survival curves comparing disease-free interval (A) and overall survival times (B) of dogs with histologic grade 2 and 3 MCTs.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Survival curves of dogs with and without <italic>c-KIT </italic>mutations</bold>. Kaplan-Meier survival curves comparing disease-free interval (A) and overall survival times (B) of dogs with and without <italic>c-KIT </italic>mutations.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Survival curves of dogs with different KIT staining patterns</bold>. Kaplan-Meier survival curves comparing disease-free interval (A) and overall survival times (B) of dogs with MCTs with different KIT staining patterns.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Survival curves of dogs with grade 3 MCTs treated with surgery alone or with post-operative chemotherapy</bold>. Kaplan-Meier survival curves comparing disease-free interval (A) and overall survival times (B) of dogs with histologic grade 3 MCT treated with surgery alone or with vinblastine and prednisone in addition to surgery.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Survival curves of dogs with <italic>c-KIT </italic>mutations treated with surgery alone or with post-operative chemotherapy</bold>. Kaplan-Meier survival curves comparing disease-free intervals (A) and overall survival times (B) of dogs with <italic>c-KIT </italic>mutations treated with surgery alone or with vinblastine and prednisone in addition to surgery.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Multivariate analyses of associations between prognostic factors and patient outcome.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Variable</bold></td><td align=\"center\"><bold>N</bold></td><td align=\"center\"><bold>Median DFI*</bold></td><td align=\"center\"><bold>DFI p-value</bold></td><td align=\"center\"><bold>Median OS time<sup><bold># </bold></sup></bold></td><td align=\"center\"><bold>OS time p-value</bold></td></tr><tr><td/><td/><td align=\"center\"><bold>(months)</bold></td><td/><td align=\"center\"><bold>(months)</bold></td><td/></tr></thead><tbody><tr><td align=\"center\">Histologic grade</td><td/><td/><td/><td/><td/></tr><tr><td align=\"center\"><italic>Grade 2</italic></td><td align=\"center\">18</td><td align=\"center\">14.1</td><td align=\"center\">0.022</td><td align=\"center\">21.5</td><td align=\"center\">0.01</td></tr><tr><td align=\"center\"><italic>Grade 3</italic></td><td align=\"center\">10</td><td align=\"center\">7</td><td/><td align=\"center\">9.2</td><td/></tr><tr><td align=\"left\">KIT staining pattern<sup>+</sup></td><td/><td/><td/><td/><td/></tr><tr><td align=\"center\"><italic>Pattern 2</italic></td><td align=\"center\">20</td><td align=\"center\">12.7</td><td align=\"center\">0.0022</td><td align=\"center\">19</td><td align=\"center\">0.0049</td></tr><tr><td align=\"center\"><italic>Pattern 3</italic></td><td align=\"center\">6</td><td align=\"center\">6.7</td><td/><td align=\"center\">10.8</td><td/></tr><tr><td align=\"center\"><italic>c-KIT </italic>mutation</td><td/><td/><td/><td/><td/></tr><tr><td align=\"center\"><italic>No</italic></td><td align=\"center\">24</td><td align=\"center\">11</td><td align=\"center\">0.0413</td><td align=\"center\">17.4</td><td align=\"center\">0.0112</td></tr><tr><td align=\"center\"><italic>Yes</italic></td><td align=\"center\">4</td><td align=\"center\">6.5</td><td/><td align=\"center\">8.9</td><td/></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>*DFI: disease-free interval.</p><p><sup>#</sup>OS: overall survival.</p><p><sup>+</sup>Two KIT pattern 1 MCTs were excluded from statistical analyses.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1746-6148-4-32-1\"/>", "<graphic xlink:href=\"1746-6148-4-32-2\"/>", "<graphic xlink:href=\"1746-6148-4-32-3\"/>", "<graphic xlink:href=\"1746-6148-4-32-4\"/>", "<graphic xlink:href=\"1746-6148-4-32-5\"/>" ]
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[{"surname": ["Thamm", "Vail", "Withrow SJ, MacEwen EG"], "given-names": ["DH", "DM"], "article-title": ["Mast cell tumors"], "source": ["Small Animal Clinical Oncology"], "year": ["2001"], "edition": ["3"], "publisher-name": ["Philadelphia, PA , W.B. Saunders"], "fpage": ["261"], "lpage": ["282"]}, {"surname": ["Kiupel", "Webster", "Bailey", "Best", "DeLay", "Detrisac", "Gamble", "Ginn", "Goldschmidt", "Hendrick", "Howerth", "Janovitz", "Lenz", "Lipscomb", "Miller", "Misdorp", "Moroff", "Neyens", "O'Toole", "Ramos-Vara", "Scase", "Schulman", "Smith", "Snyder", "Southorn", "Stedman", "Steficek", "Stromberg", "Valli", "Weisbrode", "Yager", "Kaneene"], "given-names": ["M", "JD", "KL", "S", "J", "CJ", "D", "PE", "MH", "MJ", "EW", "EB", "SD", "T", "E", "W", "S", "I", "D", "J", "TJ", "Y", "K", "P", "E", "NL", "BA", "PC", "VE", "SE", "J", "JB"], "article-title": ["Microscopic grading of canine cutaneous mast cell tumors: a multi-institutional review"], "source": ["Vet Pathol"], "year": ["2004"], "volume": ["41"], "fpage": ["576"], "pub-id": ["10.1354/vp.41-4-371"]}]
{ "acronym": [], "definition": [] }
19
CC BY
no
2022-01-12 14:47:25
BMC Vet Res. 2008 Aug 13; 4:32
oa_package/a6/cd/PMC2529280.tar.gz
PMC2529281
18684319
[]
[ "<title>Methods</title>", "<title>Reporter Vectors and DNA constructs</title>", "<p>Reporter vectors containing the putative miRNA target sites from the <italic>APP 3'</italic>UTR, were synthesized with double-stranded oligos perfectly complementary to putative miRNA target sites and oligos in which the seed regions were mutated. The mir-106a target oligos had the sequence (seed region bolded):</p>", "<p>5' CTAGTAATCCCTGTTCATTGTAA<bold>GCACTTT</bold>TGCTCAGCA 3'</p>", "<p>3' ATTAGGGACAAGTAACATT<bold>CGTGAAA</bold>ACGAGTCGTTCGA 5'</p>", "<p>The mutant mir-106a target oligos had nucleotides three through six of the seed region mutated (italicized):</p>", "<p>5' CTAGTAATCCCTGTTCATTGTAA<bold>GC<italic>GTCC</italic>T</bold>TGCTCAGCA 3'</p>", "<p>3' ATTAGGGACAAGTAACATT<bold>CG<italic>CAGG</italic>A</bold>ACGAGTCGTTCGA 5'</p>", "<p>We utilized established methods [##REF##15741182##21##] to clone these synthetic versions of putative miRNA target sites into a luciferase reporter gene (pMIR-REPORT; Ambion).</p>", "<title>Cells and Cell Culture</title>", "<p>Naïve human embryonic kidney (HEK)-293 cells were purchased from ATCC. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine,100 units/ml penicillin and 100 μg/ml streptomycin.</p>", "<title>Transfections and Luciferase Assays</title>", "<p>10,000 Naïve HEK-293 were plated in 24 well plates. The next day, cells were transfected with a miRNA overexpression vector [##REF##18193036##14##], reporter vectors bearing either the miRNA target sequence or the miRNA seed region mutant target sequence, and one tenth of the molar volume of pRL-SV40, a Renilla Luciferase control vector. We utilized Arrest-In transfection reagent (Open Biosystems Inc.); any differences in transfection efficiency were accounted for by measuring Renilla luciferase activity. 48 hours post-transfection, cell were lysed using 100 μL of GLB (Glo Lysis Buffer, Promega). Firefly and Renilla luciferase activities were measured using a dual luciferase reporter assay kit (Promega), per the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.</p>", "<title>Western Blot Analysis</title>", "<p>200,000 Naïve 293 cells were plated in 6 well plates. The next day, cells were transfected with a miRNA overexpression vector. Using previously described methods [##REF##17718916##22##], quantitative Western blots were performed using equal amounts of total protein.</p>", "<title>Antibodies</title>", "<p>A polyclonal antibody specific for the C-terminus of human APP (A8717; Sigma Aldrich, Inc) and a monoclonal antibody specific for human β-Actin (A5441; Sigma Aldrich) were used as primary antibodies. Secondary antibodies were HRP-conjugated goat anti-rabbit (GE Healthcare) and HRP-conjugated goat anti-mouse (GE Healthcare).</p>", "<title>RNA extraction and Quantitative PCR</title>", "<p>48 hours post-transfection, cells were washed with cold PBS and total RNA was isolated using RNeasy Mini Kit (Qiagen Inc.). To quantify APP mRNA levels, cDNA was synthesized using total RNA, N6 random primers and SuperScript II Reverse Transcriptase (Invitrogen). cDNA was then diluted 1:15 using RNase free water and mixed with APP or GAPDH primer/probe sets (Applied Biosystems, Inc.; APP Catalog # Hs00169098_m1; GAPDH Catalog # Hs99999905_m1), 2× PCR Universal Master Mix (Applied Biosystems, Inc.) and amplified using an ABI 7500 Real Time PCR system following the manufacturer's directions. GAPDH was used as an internal control. To determine differences in <italic>APP </italic>mRNA levels, we utilized the ΔΔCt method.</p>", "<p>To quantify miRNA levels, cDNA was reverse transcribed from total RNA samples using specific miRNA primers from the TaqMan MicroRNA Assays and reagents from the Taq Man MicroRNA Reverse Transcription kit (Applied Biosystems). The resulting cDNA was amplified by PCR using TaqMan MicroRNA Assay primers with the TaqMan Universal PCR Master Mix and analyzed with a 7500 ABI PRISM Sequence Detector System (Applied Biosystems) according to the manufacturer's instructions. The relative levels of miRNA expression were calculated from the relevant signals by the ΔΔCt method by normalization to the signal of RNU44 [##REF##18270258##23##].</p>", "<title>Statistical Analysis</title>", "<p>Values in the text and figures are presented as means ± standard deviations of experiments carried out in triplicate, at least. Each experiment was carried three times. Equal variance or separate variance two-sample student's t-test were used, as appropriate, to compare two groups. Where appropriate, Bonferroni analysis was used to correct for multiple comparisons within a single experiment.</p>" ]
[ "<title>Results</title>", "<p>Accumulating evidence suggests that increased expression of the amyloid precursor protein gene (<italic>APP</italic>) increases Alzheimer's disease (AD) risk. The resulting increase in APP protein levels results in increased Aβ levels, leading to synaptic dysfunction, neurodegeneration and, eventually, cognitive decline.</p>", "<p>APP levels can be regulated at the genomic, transcriptional or translational level. At the genomic level, Down's Syndrome (Trisomy 21) patients have three copies of the <italic>APP </italic>gene and develop AD symptoms early in life [##REF##2566117##1##]. Similarly, duplication of the <italic>APP </italic>locus, in the absence of a full trisomy 21, also leads to early-onset AD [##REF##16921174##2##]. Dysregulation of <italic>APP </italic>transcription can also increase the risk of AD. Genetic variants in the <italic>APP </italic>promoter increase <italic>APP </italic>transcription by ~2–3 fold and have been reported to increase AD risk [##REF##16931535##3##]. Growth factors have been reported to control <italic>APP </italic>mRNA half-life [##REF##10617105##4##]. These growth factors effects are dependent on a 29 bp sequence in the <italic>APP </italic>3' UTR [##REF##10617105##4##,##REF##9649628##5##]. APP translation is also regulated; for example, IL-1 can induce an increase in APP translation [##REF##10037734##6##]. IL-1 is a pro-inflammatory cytokine and genetic variants have been linked to increased AD risk [##REF##10959036##7##,##REF##17192785##8##]. Taken together, these findings provide strong evidence that increased APP levels increase AD risk.</p>", "<p>MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression post-transcriptionally. Complementary binding between miRNAs and sequences within the 3' UTR of target genes results in repression of target gene expression by translational inhibition or mRNA degradation [##REF##16314451##9##]. Approximately 700 miRNA genes are encoded in the human genome and recent evidence demonstrates that some miRNAs are differentially expressed in AD patients compared to age-matched controls [##REF##17314675##10##]. These differences in miRNA expression may play an important role in AD pathogenesis. In an attempt to address this possibility, we test the hypothesis that miRNAs can regulate APP levels.</p>", "<p>Bioinformatic analysis predicts that the 3' UTR of human <italic>APP </italic>contains 28 unique miRNA target sites [##UREF##0##11##,##UREF##1##12##]. To experimentally confirm that APP levels can be regulated by miRNAs, we chose to initially study miRNA hsa-mir-106a (mir-106a; Figure ##FIG##0##1A##) since (i) the putative target site in the <italic>APP </italic>3'UTR is 100% complementary to the seed region of the miRNA, (ii) it has a large free energy of seed region binding, and (iii) it is expressed in human brain [##REF##17565689##13##]. To determine if the putative mir-106a target site in the <italic>APP </italic>3'UTR is capable of regulating gene expression, we cloned it into the 3' UTR of firefly luciferase. We co-transfected this reporter into naïve HEK-293 cells along with a mir-106a over-expression vector [##REF##18193036##14##] and measured luciferase activity (Figure ##FIG##0##1B##). We observed a significant ~50% decrease (p &lt; 0.0001) in luciferase activity when the putative mir-106a target site was included in the reporter compared to either a reporter lacking the putative target site or reporter carrying a seed-region mutant of the putative mir-106a target site. To determine if this effect was simply due to over-expressing miRNAs, we repeated the experiment while over-expressing mir-373, a miRNA not predicted to target the <italic>APP </italic>3'UTR. We observed no change in luciferase activity. Another miRNA, mir-520c, shares the same seed region target sequence as mir-106a but is not expressed in human brain (Figure ##FIG##0##1A##) [##REF##17565689##13##]. Therefore we tested mir-520c, we observed that mir-520c over-expression significantly decreased luciferase activity when the putative mir-106a target site was included in the reporter compared to either a reporter lacking the putative target site or a reporter carrying a seed-region mutant of the putative mir-106a target site (Figure ##FIG##0##1B##). We repeated these experiments in the human neuroblastoma cell line SH-SY5Y and observed similar results (data not shown). To confirm that the miRNAs were being over-expressed, we utilized RT-QPCR to quantify miRNA levels and observed significant increases in both mir106a and mir-520c (Table ##TAB##0##1##) levels (p &lt; 0.0001).</p>", "<p>Having demonstrated that over-expression of mir-106a or mir-520c was capable of repressing reporter gene expression via interaction with its putative target site, we investigated whether over-expression of these miRNAs could decrease endogenous APP levels in human cell lines. We transfected naïve HEK-293 with mir-106a and mir-125b over-expression vectors and then performed quantitative Western blot analysis to determine APP steady state levels. We utilized mir-125b as a negative control since it is not predicted to target APP but has increased expression in AD brain [##REF##17314675##10##]. We observed that mir-106a over-expression significantly decreased APP levels (Figure ##FIG##1##2A##). Both APP isoforms expressed in this cell line, APP<sub>770 </sub>and APP<sub>751</sub>, were significantly and similarly affected. Mir-106a over-expression reduced APP levels by ~50% (p &lt; 0.01) compared to cells transfected with the empty vector (Figure ##FIG##1##2C##). Over-expression of mir-125b had no significant effect on APP levels. Over-expression of mir-520c had a similar effect on APP levels as mir-106a (Figure ##FIG##1##2B## and ##FIG##1##2D##).</p>", "<p>Most human miRNAs repress gene expression by inhibiting translation and do not affect target gene mRNA levels [##REF##16503132##15##,##REF##17507929##16##]. This seems to be the case in our experimental setting. We utilized RT-QPCR to determine if miRNA over-expression resulted in decreased <italic>APP </italic>mRNA levels. Over-expression of mir-106a or mir-520c had no effect on <italic>APP </italic>mRNA levels (Figure ##FIG##1##2E##). Mir-106a and mir-520c, therefore, appear to inhibit translation of the <italic>APP </italic>transcript.</p>", "<p>Our results are the first to experimentally demonstrate that human APP levels can be regulated by miRNAs. In 2004, it was predicted that APP levels could be regulated by miRNAs [##UREF##1##12##]; recently it was shown that expression of the <italic>C. elegans </italic>orthologue of <italic>APP</italic>, <italic>APL-1</italic>, is regulated by developmentally-timed miRNAs [##REF##18262516##17##].</p>", "<p>In human neurons the APP<sub>695 </sub>isoform is the predominant expressed isoform. All APP isoforms (APP<sub>695</sub>, APP<sub>751 </sub>and APP<sub>770</sub>) share the same 3' UTR [##REF##16672170##18##] therefore we expect that the mir-106a mediated regulation of APP levels that we observe in the HEK-293 cell line should also occur in neurons given that mir-106a is expressed in the brain. We do not expect mir-520c mediated APP regulation to occur in neurons since this miRNA is not expressed in brain. It is important to test if miRNA regulation is a normal aspect of APP metabolism in neurons. If so, it will be important to determine whether AD pathogenesis is affected by alterations in miRNA function and/or expression. It is possible that aging- or environment-induced changes in miRNA expression, and/or sequence variation in miRNAs or their targets, contribute to increased APP levels and increased AD risk. Recently, it was demonstrated that expression of the β-secretase BACE can be regulated by miR-29a/b-1 and mir-107; furthermore, increased BACE levels correlated with decreased miR-29a/b-1 and mir-107 levels in AD patients [##REF##18434550##19##,##REF##18234899##20##].</p>", "<p>Regardless of the biological roles of miRNA in APP metabolism, therapeutics based on miRNA-induced decrease in APP levels would offer a treatment targeting the underlying pathophysiology of the disease. In the near future, substantial progress will be made in understanding the role of miRNAs in AD pathogenesis and in therapeutic approaches to treating AD.</p>" ]
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[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>A number of studies have shown that increased APP levels, resulting from either a genomic locus duplication or alteration in <italic>APP </italic>regulatory sequences, can lead to development of early-onset dementias, including Alzheimer's disease (AD). Therefore, understanding how APP levels are regulated could provide valuable insight into the genetic basis of AD and illuminate novel therapeutic avenues for AD. Here we test the hypothesis that APP protein levels can be regulated by miRNAs, evolutionarily conserved small noncoding RNA molecules that play an important role in regulating gene expression. Utilizing human cell lines, we demonstrate that miRNAs hsa-mir-106a and hsa-mir-520c bind to their predicted target sequences in the <italic>APP </italic>3'UTR and negatively regulate reporter gene expression. Over-expression of these miRNAs, but not control miRNAs, results in translational repression of <italic>APP </italic>mRNA and significantly reduces APP protein levels. These results are the first to demonstrate that levels of human APP can be regulated by miRNAs.</p>" ]
[ "<title>Competing interests</title>", "<p>AJS declares that he is a share holder in TorreyPines Therapeutics. The remaining authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>All authors have read and approved the final manuscript. NP designed the experiment, acquired, analyzed and interpreted the data and drafted the manuscript. DH and NM cloned the target and mutant sequences in the reporter vectors. SA helped to draft and edit the manuscript. JTR contributed towards experimental design. QH contributed towards experimental design and provided miRNA over-expression vectors.JCL performed the statistical analysis. AJS oversaw the experimental design, data analysis, data interpretation, and drafting/editing the manuscript.</p>" ]
[ "<title>Acknowledgements</title>", "<p>We would like to thank members of Saunders lab and Trinna Cuellar for comments on the manuscript. We would also like to thank Trinna Cuellar and Michael T. McManus for helpful discussions (RO3-DA022201). This work was funded by the NIH (AJS: R21NS48227), Commonwealth of Pennsylvania (AJS), and Drexel University (AJS).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>mir-106a target sequence regulates reporter gene expression</bold>. <bold>(A) </bold>Predicted mir-106a and mir-520c target sites in the 3'UTR of APP. <bold>(B) </bold>Over-expression of mir-106a or mir-520c, but not mir-373, significantly reduced luciferase expression (p = 0.0006) controlled by the putative mir-106a <italic>APP 3'</italic>UTR target sequence. This reduction is not observed when a seed region mutant of mir-106a (106a*) is utilized. For all experiments, three independent trials were performed. Error bars represent standard deviation. *<italic>p </italic>&lt; 0.05; **<italic>p </italic>&lt; 0.01, compared to the appropriate control.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>mir-106a and mir-520c can regulate APP levels post-transcriptionally</bold>. APP<sub>770 </sub>and APP<sub>751 </sub>levels are reduced in cells over-expressing <bold>(A) </bold>mir-106a compared to cells expressing either mir-125b or the empty vector and <bold>(B) </bold>mir-520c compared to cells expressing the empty vector. <bold>(C) </bold>Quantification of these Western blot results reveals that mir-106a over-expression significantly decreases APP levels (p &lt; 0.01) compared to cells expressing mir-125b or cells transfected with the empty vector. <bold>(D) </bold>Quantification of these Western blot results reveals that mir-520c over-expression significantly decreases APP levels (p &lt; 0.01) compared to cells transfected with the empty vector. <bold>(E) </bold>QPCR results show that <italic>APP </italic>mRNA levels are not altered by mir-106a or mir-520c over-expression. For all experiments, three independent trials were performed. Error bars represent standard deviation. *<italic>p </italic>&lt; 0.05; **<italic>p </italic>&lt; 0.01, compared to the appropriate control.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Relative miRNA levels</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td/><td align=\"center\"><bold>Fold Change 2<sup>-ΔΔCt</sup></bold></td><td align=\"center\"><bold>p-value</bold></td></tr><tr><td/><td colspan=\"2\"><hr/></td></tr><tr><td align=\"center\"><bold>Vector</bold></td><td align=\"center\">1</td><td/></tr><tr><td align=\"center\"><bold>mir-106a</bold></td><td align=\"center\">30.1 ± 1.2</td><td align=\"center\">&lt; 0.0001</td></tr><tr><td align=\"center\"><bold>mir-520c</bold></td><td align=\"center\">1964.6 ± 1.1</td><td align=\"center\">&lt; 0.0001</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>QPCR results demonstrate a significant increase in mir-106a and mir-520c levels in response to over-expression compared to cells transfected with an empty vector.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1750-1326-3-10-1\"/>", "<graphic xlink:href=\"1750-1326-3-10-2\"/>" ]
[]
[{"article-title": ["Mirbas"]}, {"surname": ["John", "Enright", "Aravin", "Tuschl", "Sander", "Marks"], "given-names": ["B", "AJ", "A", "T", "C", "DS"], "article-title": ["Human microRNA targets"], "source": ["LoS Biol"], "year": ["2004"], "volume": ["2"], "fpage": ["e363"]}]
{ "acronym": [], "definition": [] }
23
CC BY
no
2022-01-12 14:47:25
Mol Neurodegener. 2008 Aug 6; 3:10
oa_package/16/91/PMC2529281.tar.gz
PMC2529282
18702829
[ "<title>Background</title>", "<p>The chemist and pharmacologist Fritz Hauschild developed the sympathomimetic agent Pervitin (metamphetamin) in the 1930s [##REF##15997840##1##]. Methamphetamine, a derivative of amphetamine, was widely prescribed in the 1950s and 1960s as a medication for depression and obesity, reaching a peak of 31 million prescriptions in the United States in 1967 [##REF##10908000##2##]. Now widely available in many nations as a street drug in either base, powder or crystalline ('crystal meth' or 'ice') forms, this central nervous system stimulant can be injected or ingested nasally or orally. The most frequently reported positive effects are feeling more awake or alert, increased energy and sociability, and aphrodisiac qualities, while negative effects reported by users include 'comedown', paranoia and inability to sleep [##UREF##0##3##]. A study of regular methamphetamine users in Sydney found that most preferred base and ice because they 'provided a more intense and longer lasting high' [##REF##16298838##4##].</p>", "<p>In a poignant reversal of the original application of methamphetamine as an antidepressant treatment, rates of depression diagnosis and prescription of antidepressants are significantly higher among Sydney methamphetamine users than the general population [##REF##16298838##4##]. While there is little evidence that depressed people are more likely to use crystal meth [##REF##15738315##5##], dependence on and recent use of crystal meth has been associated with reported symptoms of depression in multiple studies [eg. [##REF##11083165##6##]]. Methamphetamine withdrawal symptoms, including fatigue, anhedonia, depressed mood, paranoia and hypersomnia, are common to depression and are often severe enough to precipitate relapse [##REF##15738315##5##]. These effects are reported to be more severe with crystal meth than other forms of methamphetamine [##REF##16298838##4##].</p>", "<p>The behavioural data indicate that crystal meth is becoming an increasingly normative part of the Australian gay scene, associated mostly with dance parties but also with sexual activity in other settings for some users [##UREF##0##3##]. Importantly, the research indicates that most of the gay men who use crystal meth regularly manage this use with few problems [##UREF##1##7##]. However, there is still a considerable minority of men who report dependent levels of crystal meth use [##UREF##1##7##]. Just over 20% of gay men participating in the 2007 Sydney Gay Community Periodic Survey reported using crystal meth in the previous six months [##UREF##2##8##]. This had increased significantly from around 12% in 2002. Amongst those who used crystal meth in the six months prior to the 2007 survey, nearly half used it at least monthly [##UREF##2##8##]. In people living with HIV/AIDS (of which the great majority in Australia are gay men [##UREF##3##9##]), depression has been shown to be associated with a history of illicit drug use [##REF##12534660##10##], and other studies have suggested that co-morbid depression and drug use are associated with decreased HIV treatment uptake, treatment adherence problems and virological suppression [##REF##16706551##11##]. In sum, this literature suggests that while some gay men and some HIV positive men may be able to integrate occasional crystal meth use into their lives with minimal impact, others are using to a degree that can have problematic consequences for health as well as social and economic wellbeing.</p>", "<p>This paper explores how reported instances of problematic crystal meth use among gay men and HIV positive men are affecting the work of general practitioners (GPs) in Australia. Although there has been growing concern in gay communities about rates of crystal meth use [##REF##16211470##12##], we believe this to be the first analysis of the experiences of Australian GPs who work with gay men and HIV positive men who use crystal meth.</p>" ]
[ "<title>Methods</title>", "<p>The data reported in this paper are drawn from the Primary Health Care Project on HIV and Depression, an interdisciplinary and multi-method study exploring the complex relationships between sexuality, HIV and depression. Written informed consent was obtained from all participants. Ethics approval was granted by the National Research and Evaluation Ethics Committee of the Royal Australian College of General Practitioners (Approval No: RACGP NREEC 05/35) and the Human Research Ethics Committees of The University of New South Wales and The University of Adelaide.</p>", "<p>The study methodology has been described elsewhere [##REF##18704222##13##,##UREF##4##14##], but there are several aspects that are important to report in relation to this paper. First, this paper is focused on data gathered in the first of three stages of the project. This stage comprised semi-structured interviews with s100 HIV prescriber GPs, eg. primary care physicians trained and accredited to prescribe HIV medication. Sixteen interviews were conducted with GPs (14 male, 2 female) who were based in three Australian cities: two state capitals, one large (Sydney) and one small (Adelaide); and one rural-coastal city. The seven practices in which the GPs were based each treated a high caseload of the gay or homosexually active men and HIV positive men that lived in those areas.</p>", "<p>The second important aspect of the method is that this paper is focused on the experiences reported by the eleven GPs who were based in Sydney, each of whom raised the issue of problematic patient crystal meth use during the interviews. A semi-structured interview format was employed in the interviews to allow for consistency as well as flexibility in remaining responsive to the range of issues and ideas that can emerge in an open-ended conversation. Following the aims of the study, GPs were asked to focus on their experiences as they related to the treatment of gay men and HIV positive men. However, there was minimal guidance in terms of what the GPs may or may not find most topical or challenging about those experiences. A thematic analysis of deidentified transcripts was applied using NVivo 7 [##UREF##5##15##] and following the principles of open and axial coding [##UREF##6##16##]. Reliability was established through a process of independent coding and discussion amongst the research team. This process identified key themes on: how gender and sexuality differentially shaped the experience of depression in gay men in comparison with heterosexual men [##REF##18704222##13##]; how a long term and trusting relationship between doctor and patient increased the likelihood of a timely diagnosis of depression in HIV positive men [##UREF##7##17##]; and how the growing range of clinical and contextual challenges for GPs working in HIV affected their capacity to manage depressive illness in both gay men and in HIV positive men.</p>", "<p>The issue of patient crystal meth use was only raised in the eleven interviews conducted with Sydney-based GPs. These eleven GPs represent more than one third of all of the s100 HIV prescriber GPs working in the inner suburbs of Sydney [##UREF##8##18##]. The rest of this paper will explore five key themes in the reported experiences of these eleven GPs, focusing on their observations of problematic crystal meth use in gay men and in HIV positive men in Sydney. It is important to note that GPs often spoke about these two patient populations interchangeably. Where a distinction was clear this has been reported as such, however, the more common conflation or substitution of their identifying features is discussed in the final section of the paper.</p>" ]
[ "<title>Results</title>", "<title>Increasing problem</title>", "<p>The first and most common theme in the reported experiences of GPs is that crystal meth use has dramatically increased in recent years among the populations of gay men and HIV positive men they treat in Sydney:</p>", "<p>I mean recreational drugs have always been around, but not to the extent where people are not complying with their medication so much and getting depressed, and getting physically and mentally ill with things like crystal meth ... I mean I've been away and then this last year this seems to be an ongoing problem that people are ... trying to deal with. [GP3]</p>", "<p>An important characteristic of this theme is that not only is crystal meth use growing, but that problematic levels of crystal meth use are being observed in gay men and positive men who do not have a history of problematic levels of drug use:</p>", "<p>I'm really surprised at the level of crystal use out there.... And the folk who I would least expect to actually access it, because it's so freely available, and part of the party scene now, who are now accessing and who are now using. And have been actually now shall we say 'hooked in', and are now becoming regular users. And I've been quite surprised at who's actually taking this up. Now these are folk that I would assume would have some degree of control over their actions... I'm talking about men in their thirties and forties who have been around, who know the scene, but who are now actively involved in crystal use ... I'm, it really worries me the extent of addiction that's actually happening, about people I would least expect. [GP7]</p>", "<title>Associations with depression</title>", "<p>A second theme focuses on the observed associations between problematic levels of crystal meth use and depression in both gay men and in HIV positive men:</p>", "<p>In terms of depression at the moment, one of our major problems in this environment at the moment would be the crystal 'epidemic', or whatever you want to call it. And that can make a lot of people very depressed! [GP4]</p>", "<p>But I would say, amphetamine use is pretty high and linked with this depression, particularly linked with depression. [GP10]</p>", "<p>However, this association is not seen as simple or straightforward; rather, the associations between depression and crystal meth use are represented as quite complex and difficult to tease apart:</p>", "<p>I mean most boys using large amounts of crystal are depressed. And how you address that in the context of perhaps ongoing crystal use and work out what's depression as such or what's a drug effect, or why someone's using crystal, I think that's a major problem we're dealing with at the moment. [GP4]</p>", "<p>Well, why are people [having] drug and alcohol issues in the first place? Is it because there is a pre existing depression and this is actually part of trying to mask that? It's drawing a long bow to say that will, going [down] that path, cause a depression. Is there a pre existing depression here anyway? [GP7]</p>", "<title>Treatment challenges</title>", "<p>A third theme describes the various ways in which crystal meth use creates treatment challenges for GPs, including in the effective clinical management of depression:</p>", "<p>If someone's injecting a lot of amphetamines, it's very hard to diagnose depression. Also medication's probably not going to be particularly useful. [GP6]</p>", "<p>Post party depression. It's very common, and then a few days later they're fine. When people are withdrawing from substances, I've seen people get diagnosed, put on major, major tranquillisers and antidepressants. And I don't, I'm not sure if they really needed it. [GP9]</p>", "<p>There's a lot of substance abuse that negates antidepressant therapy, things like that. I mean, [antidepressant drug] isn't going to do much good if people are doing crystal meth every weekend, do you know what I mean? ... And the patients don't necessarily tell you that they have these problems, or that they are using these things. It's very common to see. Gay men come in here and say, \"I'm depressed and I really want to do something about it. I've heard about antidepressant medication. I want to go on it.\" And they don't tell you, even if you ask them specifically, that they are using stimulants recreationally, until some time later. Yeah, that's a big obstacle. [GP5]</p>", "<p>Treatment challenges are further complicated when considering the clinical management of HIV alongside co-morbid depression and drug use:</p>", "<p>However, with the advent of our HIV population, with the use of, particularly the use of ice, particularly the use of crystal... I have actually seen stable people who I thought were quite well controlled on their, either their cognitive behavioural therapy or their antidepressant medication, talk to me about suicidal intent coming down off their crystal. And that's creating some problems for me at the moment. [GP7]</p>", "<p>Positive men and depression? No, it's this whole sort of soup of depression, self esteem, drug use. Trying to tease them out and treat. So I think you really need to make sure you're treating all of them. [GP11]</p>", "<title>Health services and health care</title>", "<p>A fourth theme focuses on the capacity of health services to provide effective care for gay men and HIV positive men who are experiencing problematic levels of crystal meth use, particularly in terms of their mental health care needs:</p>", "<p>There needs to really urgently, urgently address the drug issue. People who become acutely depressed or psychotic or both, under the influence of drugs. And rehabilitation for it. Because generally they're just sent out of hospital. They ring you up and go, \"They've just been discharged. Filled them up with tranquillisers and discharged them on Monday morning\". And there's no follow up, there's nothing ... And then the person doesn't come back, like I can't get hold of them. So I don't, I think there has to be a better system in place so that these people don't cause social harm by going berserk in the community as well ... And to get someone into a private psychiatrist interested in drug and alcohol can take months. And that, even costlier are those private rehab places. [GP9]</p>", "<p>Related to health service capacity are the attitudes of health practitioners, including the observed reluctance of some referral specialists to accept clients who use crystal meth:</p>", "<p>And I won't name names, but I send to, a lot of these people, to a certain psychiatrist. And he said, \"Don't send me any more!\" [laughs] Because he's fed up with them. And I would be too, I guess. [GP9]</p>", "<p>GPs commonly stressed the importance of providing non-judgmental support to men who were using crystal meth, whether or not they felt it was a problematic behaviour. This aspect of the theme implies that effective health care requires the absence of both social prejudice and criminal implications:</p>", "<p>Because most people would come to us for a sexual health reason in the beginning and like the atmosphere or like the doctors, or whatever. Most of them liked the non judgemental ... And being in the city, they liked being able to say that they were feeling a bit tired but could it have been the five trips and crystal they had on the weekend. And we didn't ring the police and fall off our chairs. So they'd come back with an earache. [GP8]</p>", "<title>Workforce issues</title>", "<p>The final theme describes some key issues facing the GP workforce members who provide care to these populations of gay men and HIV positive men in inner city Sydney. This includes GPs feeling unprepared to support the number of men seeking treatment for crystal meth addiction and associated co-morbidities:</p>", "<p>And I'm seeing people I'd least expect saying to me, \"I can't stand this. I've just got to get off this. How do I do it? [GP7]</p>", "<p>There's a problem with crystal meth usage in Sydney, all right? And dealing with addiction and substance abuse, I feel less comfortable with. I don't feel I have the expertise to deal with that, so I tend to send them that way for that. [GP11]</p>", "<p>An important aspect of this theme is the potential for burnout in those health care workers – particularly GPs who are HIV medication prescribers – who are responding to problematic levels of crystal meth use on top of a growing range of other contextual stressors:</p>", "<p>I think it's nasty at the moment, and I think it's nastier for the health worker too. Especially with crystal meth ... people are nasty, they're horrible and aggressive. And you become it too, I think. Because that's the only way you can, because you've got to make very strong borders, boundaries for them to not cross. And people become more unreliable. They're narkier, they have nasty withdrawals. And I think it's a big issue. And I think it's also a very big issue with a lot of, like people working in HIV where there may be... A lot of people are saying there's no evidence, but there's a lot of anecdotal talk ... And that a lot of people are going to burn out. And as you know there are fewer and fewer doctors wanting to do it because they've been told that it's really hard to do HIV. It's not that hard, it just takes a lot of effort and will. And I think similarly with the mental health services. And you've got HIV and you've got depression, it will wear people out. And then you've got drugs that make people nasty and horrible, and weird and inconsolable. Then, it can wear a lot of people out. [GP9]</p>" ]
[ "<title>Discussion</title>", "<p>There are a number of limitations to this study which must be acknowledged. Firstly, the number of GP interviews conducted for the study is small. However, in keeping with the principles of qualitative epistemology, examining a small amount of qualitative data is a legitimate research tool, particularly if the objective of the analysis is – as in this case – to understand particular and contextual experiences rather than to make a claim to representativeness and generalisability [##UREF##5##15##,##UREF##6##16##]. In addition, although only sixteen interviews were conducted for this stage of the study, this sample can be seen to represent a large proportion of the general practice workforce currently providing care to gay men and HIV positive men in the three cities included in the study. For example, the eleven Sydney-based GPs interviewed represent more than one third of the total number of s100 HIV prescriber GPs working in the inner suburbs of Sydney. Secondly, although the issue of crystal meth use was not raised as an urgent problem by the GPs based in the other two sites – one a smaller Australian city, the other a rural-coastal city – this does not mean that crystal meth use or drug and alcohol use more broadly is not a problem at all in those locations. On the contrary, study data not reported here suggests that drug and alcohol use is a significant issue for many of the gay men and HIV positive men living in those cities.</p>", "<p>Another important limitation to consider is that, in this study, the GPs often spoke about the two populations of gay men and HIV positive men as almost interchangeable. Sometimes a section of transcript that began with an observation on gay men would slip into a commentary on HIV – and vice versa – with little, if any, acknowledgement on the part of the GP that this was occurring. This may be because almost all of the HIV positive men these GPs treat are also gay, and that a very high proportion of the gay men they treat are also HIV positive. Each of these GPs is based in a practice that has adopted these populations as the focal point of their work, and which has over many years developed a reputation for expertise and understanding in the health and social issues that face those groups of men. The physical location of inner city Sydney is also important here, as all four practices are embedded within social and spatial networks associated with both gay and HIV positive 'community'. The fact that these GPs had little sense of the differences between these two groups – at least in terms of how they talked about issues such as crystal meth use – suggests that forcing an arbitrary distinction between them may not be useful, if only in this context.</p>", "<p>Nonetheless there are some key implications that can be identified from the data reported in this paper. The experiences of these GPs suggests that health practitioners may benefit from broadening their understandings of how to anticipate and respond to problematic levels of crystal meth use in their patients. This is likely to increase the capacity of GPs and others health practitioners – particularly those working in gay men's health and HIV health promotion – to intervene at an early stage in order to mitigate the impact of crystal meth use on co-morbid mental illness and other health issues [##UREF##1##7##]. These GPs articulated a complex, multi-factorial model of the relationships between drug use, depression, sexuality and HIV in their patients. Thus, it is important for health practitioners to address each of these aspects in turn, rather than prioritising one as more urgent than another. Of course, there are some exceptions, such as the fact that the diagnosis and treatment of depression must usually wait until problematic levels of crystal meth use have been addressed. Equally, if drug use is affecting compliance with HIV medications then it is critical that drug use be firstly addressed in order to prevent the development of drug resistance. A 'stepped care' approach is likely to be most appropriate, particularly for managing co-morbid crystal meth use and depression [##UREF##9##19##]. Guidelines for the management of general practice patients with crystal meth use problems need to be further developed to address specific issues associated with gay men and HIV positive men [##UREF##10##20##].</p>", "<p>Another implication relates to an understanding of the role that drug use plays in the lives of many gay men. Following Worth and Rawstorne, it is important to appreciate 'these phenomena as social relationships that are part of wider community norms and practices' [##REF##16211470##12##]. GPs and other health practitioners must build trust with their gay male patients to ensure that patients are willing to disclose the full nature of their drug use and other potentially stigmatised practices. Evidence drawn from the quantitative arm of this study suggests that this group of GPs has a heightened awareness for detecting depression in gay men and HIV positive men [##UREF##7##17##], demonstrated in a high level of concordance between GP assessments and patient screening for depression [##UREF##11##21##]. In a similar sense, there is a clear need for education programs to train health practitioners – including referral specialists – to better appreciate the issues pertaining to crystal meth use in gay men and in HIV positive men. In addition to training, however, health services need to receive higher levels of resourcing and support to assist the health workforce to cope with the often difficult behaviour of patients experiencing problematic levels of crystal meth use. To avoid an ever increasing burden being placed on primary care and community services, there needs to be an increased resourcing of acute care and referral services to improve their availability, accessibility and expertise in crystal meth issues.</p>" ]
[ "<title>Conclusion</title>", "<p>This is the first study of its kind to report on the experiences of GPs who work with gay men and HIV positive men in Sydney in relation to crystal meth use and associated depression. Five key themes were identified. Commencing from the belief that crystal meth use is an increasing problem in a growing number of these men, these GPs also observed a strong if complex association between crystal meth use and depression. Patient crystal meth use is most often experienced by GPs as a series of challenges in the clinical management of depression, further complicated for those men who are HIV positive. These GPs also expressed serious concerns regarding the capacity of existing health services to cope with crystal meth use, including a perceived stigma among some health practitioners, and a consequent requirement for GPs to provide open-minded, non-judgemental care. Finally, particular issues facing the health workforce were identified including a sense of GPs being unprepared to cope with problematic levels of crystal meth use in their patients as well as a more general lack of capacity (and possible unwillingness) in the HIV and mental health workforce due to a growing range of contextual stressors. Despite some study limitations, key implications can be identified. Further investigation is required in order to consider whether these findings are reproducible in other general practice settings. Other issues that would be particularly useful to explore further include the relationship between depression, drug use and HIV medication regimens, as well as the characteristics and challenges facing the HIV general practice workforce in Australia.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>This paper describes the experiences of Australian general practitioners (GPs) in managing problematic crystal methamphetamine (crystal meth) use among two groups of male patients: gay men and HIV positive men.</p>", "<title>Methods</title>", "<p>Semi-structured qualitative interviews with GPs with HIV medication prescribing rights were conducted in Sydney, Adelaide and a rural-coastal town in New South Wales between August and October 2006. Participants were recruited from practices with high caseloads of gay and HIV positive men.</p>", "<title>Results</title>", "<p>Sixteen GPs were recruited from seven practices to take part in interviews. Participants included 14 male GPs and two female GPs, and the number of years each had been working in HIV medicine ranged from two to 24. Eleven of the GPs who were based in Sydney raised the issue of problematic crystal meth use in these two patient populations. Five key themes were identified: an increasing problem; associations with depression; treatment challenges; health services and health care; workforce issues.</p>", "<title>Conclusion</title>", "<p>Despite study limitations, key implications can be identified. Health practitioners may benefit from broadening their understandings of how to anticipate and respond to problematic levels of crystal meth use in their patients. Early intervention can mitigate the impact of crystal meth use on co-morbid mental illness and other health issues. Management of the complex relationships between drug use, depression, sexuality and HIV can be addressed following a 'stepped care' approach. General practice guidelines for the management of crystal meth use problems should address specific issues associated with gay men and HIV positive men. GPs and other health practitioners must appreciate drug use as a social practice in order to build trust with gay men to encourage full disclosure of drug use. Education programs should train health practitioners in these issues, and increased resourcing provided to support the often difficult task of caring for people who use crystal meth. Greater resourcing of acute care and referral services can shift the burden away from primary care and community services. Further investigation should consider whether these findings are reproducible in other general practice settings, the relationship between depression, drug use and HIV medication, and challenges facing the HIV general practice workforce in Australia.</p>" ]
[ "<title>Abbreviations</title>", "<p>GP: general practitioner; Crystal meth: Crystal methamphetamine</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>DCS: contributed to the planning and reporting of the work described in the article. CEN: contributed to the conduct and reporting of the work described in the article. LM: contributed to the conduct and reporting of the work described in the article. SCK: contributed to the planning and reporting of the work described in the article. MRK: contributed to the planning and reporting of the work described in the article. All authors read and approved the final manuscript.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from all participants to publish their data in deidentified form, including in peer reviewed journals. Deidentified written consent forms are available for review by the Editor-in-Chief of this journal.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2296/9/45/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>The study was funded by the Australian Government National Health and Medical Research Council General Practice Clinical Research Program. The authors' work is independent of the funders.</p>" ]
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[{"surname": ["Degenhardt", "Topp"], "given-names": ["L", "L"], "article-title": ["Crystal methamphetamine use among polydrug users in Sydney\u2019s dance party subculture: characteristics, use patterns and associated harms"], "source": ["Int J Drug Policy"], "year": ["2003"], "volume": ["14"], "fpage": ["17"], "lpage": ["24"], "pub-id": ["10.1016/S0955-3959(02)00200-1"]}, {"surname": ["Leonard", "Dowsett", "Slavin", "Mitchell", "Pitts"], "given-names": ["W", "G", "S", "A", "M"], "source": ["Crystal clear: The social determinants of gay men's use of crystal methamphetamine in Victoria"], "year": ["2008"], "publisher-name": ["Melbourne , Australian Research Centre in Sex, Health & Society, La Trobe University"]}, {"collab": ["NCHSR and NCHECR"], "source": ["Sydney Gay Community Periodic Survey"], "year": ["2007"], "publisher-name": ["Sydney , National Centre in HIV Social Research and National Centre in HIV Epidemiology and Clinical Research, The University of New South Wales"]}, {"collab": ["NCHECR"], "source": ["HIV/AIDS, viral hepatitis and sexually transmissible infections in Australia: Annual Surveillance Report 2007"], "year": ["2007"], "publisher-name": ["Sydney , National Centre in HIV Epidemiology and Clinical Research, The University of New South Wales, Sydney; Australian Institute of Health and Welfare, Canberra"]}, {"surname": ["Newman", "Mao", "Kippax", "Kidd", "Saltman", "Digiusto", "K\u00f6rner", "Rawstorne", "Donohue", "McMurchie", "Ellis", "Rogers", "Booth", "Andrews", "Pell", "Watson", "Westacott"], "given-names": ["C", "L", "S", "M", "D", "E", "H", "P", "W", "M", "D", "G", "A", "G", "C", "J", "R"], "source": ["Primary Health Care Project on HIV and Depression [poster] : October; Melbourne.\n\t\t\t\t\t"], "year": ["2006"]}, {"surname": ["Bazeley"], "given-names": ["P"], "source": ["Qualitative data analysis with NVivo"], "year": ["2007"], "edition": ["2nd"], "publisher-name": ["London , Sage"]}, {"surname": ["Strauss", "Corbin"], "given-names": ["A", "J"], "source": ["Basics of qualitative research: Techniques and procedures for developing grounded theory"], "year": ["1998"], "edition": ["2nd"], "publisher-name": ["London , Sage Publications"]}, {"surname": ["K\u00f6rner", "Newman", "Mao", "Kippax", "Kidd", "Saltman"], "given-names": ["H", "C", "L", "S", "M", "D"], "collab": ["and the HIV and Depression team"], "source": ["\u2018It's really a myriad of different signals, not just the textbook\u2019: The challenges of diagnosing and managing the depression of HIV-positive gay men in general practice [poster]: July; Marseille, France.\n\t\t\t\t\t"], "year": ["2007"]}, {"collab": ["ASHM"], "source": ["ASHM Directory: HIV, hepatitis and related services 2007-2008"], "year": ["2007"], "publisher-name": ["Sydney , Australian Society for HIV Medicine Inc."]}, {"surname": ["Baker", "Dawe"], "given-names": ["A", "S"], "article-title": ["Amphetamine use and co-occurring psychological problems: Review of the literature and implications for treatment"], "source": ["Aust Psychol"], "year": ["2005"], "volume": ["40"], "fpage": ["87"], "lpage": ["94"]}, {"surname": ["Jenner", "Baker", "Whyte", "Carr"], "given-names": ["L", "A", "I", "V"], "source": ["Management of patients with psychostimulant use problems - guidelines for general practitioners"], "year": ["2004"], "publisher-name": ["Canberra , Australian Government Department of Health and Ageing"]}, {"surname": ["Mao", "Kippax", "Newman", "Andrews", "Rogers", "Saltman", "Kidd"], "given-names": ["L", "SC", "CE", "G", "G", "DC", "M"], "article-title": ["Rates of depression among men attending high HIV caseload general practices in Australia"], "source": ["Mental Health in Family Medicine"]}]
{ "acronym": [], "definition": [] }
21
CC BY
no
2022-01-12 14:47:25
BMC Fam Pract. 2008 Aug 15; 9:45
oa_package/1d/95/PMC2529282.tar.gz
PMC2529283
18713466
[ "<title>Background</title>", "<p>In vertebrates, the cysteine- and glycine-rich proteins (CRPs) encoded by the <italic>Csrp </italic>genes are evolutionarily conserved proteins that define a subset of zinc-binding LIM domain proteins. As structural hallmarks, these proteins exhibit two LIM domains with a characteristic spacing, adjacent glycine-rich repeats, and a potential nuclear localization signal [##REF##12539241##1##]. Originally, this family of LIM domain proteins included three members (CRP1, CRP2, CRP3/MLP) that were independently isolated in the course of different experimental strategies [##REF##2115670##2##, ####REF##8361751##3##, ##REF##7954791##4####7954791##4##]. Subsequently, based on structural and sequence similarities, the thymus LIM protein (TLP) was grouped into this subclass of LIM domain proteins [##REF##11713292##5##]. The four CRPs possess significant differences in their temporal and spatial patterns of expression raising interesting questions regarding the physiological and biological significance of the CRP multigene family [##REF##9341203##6##]. For example, it is uncertain if these proteins perform unique functions or substitute for each other within a living organism. The cell types and organs that express the different CRPs suggest several hypothetical functions for this group of LIM domain proteins, including possible roles in organization and stabilization of the contractile myofibrillar/cytoskeletal apparatus [##REF##9341203##6##, ####REF##9039266##7##, ##REF##10751147##8####10751147##8##], maintenance of cellular functions [##REF##8361751##3##], differentiation [##REF##11713292##5##], transcriptional regulation [##REF##12530967##9##], and in the establishment of fibrogenic responses [##REF##11672422##10##]. In addition to this potential functional versatility, there is growing evidence supporting the notion that the two LIM domains of CRPs serve as protein interfaces mediating specific protein-protein interactions thereby arranging two or more protein constituents into nuclear transcription or cytoskeletal complexes [##REF##9722554##11##,##REF##10231520##12##]. In this regard, the CRP3 protein (also termed MLP for muscle LIM protein) is best characterized. It is a positive regulator of myogenic differentiation that was first identified in a screen for genes that become transcriptionally upregulated as a result of skeletal muscle denervation [##REF##7954791##4##]. In accordance, the overexpression of MLP in C2 myoblasts potentiates myogenic differentiation [##REF##7954791##4##] and the absence of the <italic>Csrp3/Mlp </italic>gene product causes a phenotype of dilated cardiomyopathy underscoring the hypothesis that CRP3/MLP is an essential regulator of cardiac muscle development [##REF##9039266##7##]. In line with this hypothesis, the morphological and clinical picture of dilated cardiomyopathy in humans is associated with altered <italic>Csrp3/Mlp </italic>expression [##REF##10851202##13##] and <italic>Csrp3/Mlp </italic>mutations were found in families suffering from dilated as well as from hypertrophic cardiomyopathy [##REF##12642359##14##,##REF##12507422##15##].</p>", "<p>Together, CRP1 and CRP2 were shown to be potent smooth muscle differentiation cofactors triggering the conversion of pluripotent 10T1/2 fibroblasts into smooth muscle cells when overexpressed together with serum response factor (SRF) and GATA proteins [##REF##12530967##9##]. Compatible with this presumed function is the finding that CRP2 is present at highest levels in arterial samples [##REF##8626582##16##,##REF##9341203##6##]. Moreover, a recent report demonstrated that CRP2 can effectively switch on smooth muscle gene activity in adult cardiac myocytes [##REF##17185421##17##] suggesting that CRP2 has essential functions in controlling smooth muscle gene activity.</p>", "<p>Furthermore, during embryogenesis and in adult tissue, <italic>Csrp2 </italic>gene expression is also prominently associated with mesenchyme and epithelia [##REF##9815145##18##,##REF##11748300##19##]. Interestingly, compared to other CRP family members, CRP2 expression begins early in gestation and has a distinct pattern of tissue distribution during cardiovascular development. [##REF##9815145##18##]. CRP2 is expressed transiently in early embryonic cardiomyocytes similar to smooth muscle cell markers like α-smooth muscle actin, calponin, and SM22α [##REF##9815145##18##] but its expression is downregulated in adult cardiomyocytes. Additionally, it was demonstrated that the expression of CRP2 is downregulated with cellular dedifferentiation induced by oncogenic transformation, injury, or wound healing [##REF##8361751##3##,##REF##8626582##16##,##REF##11672422##10##].</p>", "<p>Recently, it was demonstrated that the loss of CRP2 did not result in any apparent gross vascular defects or altered expression of smooth muscle cell markers [##REF##16269651##20##]. Moreover, vascular development, morphology, cell proliferation, endothelial regeneration and the expression of several characteristic smooth muscle specific genes were similar between WT and <italic>Csrp2 </italic>nulls. However, the loss of CRP2 is correlated with increased neointima formation in response to vascular injury. Furthermore, vascular smooth muscle cells isolated from mice lacking CRP2 migrated more rapidly in response to PDGF-BB with an increased activation of the Rho GTPase Rac1 suggesting that <italic>Csrp2 </italic>and its protein product CRP2 are functionally linked to cell migration [##REF##16269651##20##].</p>", "<p>We here report about the generation and characterization of a similar <italic>Csrp2 </italic>null mouse model. We demonstrate that these deficient mice are viable and fertile, exhibiting a mild cardiac phenotype in which the cardiomyocytes display a slight increase in their thickness, indicating moderate hypertrophy at the cellular level. In line with these findings, the peculiarity of heart architecture reflected by the typical arrangement of intercalated disc-associated proteins (i.e. β-catenin, N-RAP, connexin-43) was altered suggesting that CRP2 is involved in the organization of the cytoskeleton in cardiac muscle cells.</p>" ]
[ "<title>Methods</title>", "<title>Isolation of murine <italic>Csrp2 </italic>sequences</title>", "<p>By using a cDNA specific for rat <italic>Csrp2 </italic>as a probe, we isolated different genomic <italic>Csrp2 </italic>clones from a mouse genomic 129SvJ library. The overall organization of the <italic>Csrp2 </italic>gene was in agreement with previous reports [##REF##9553112##35##]. The sequence of a ~17.3 kbp fragment of one clone (clone 5o) containing exon 3 through 6 of <italic>Csrp2 </italic>was sequenced and deposited [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"AY533303\">AY533303</ext-link>].</p>", "<title>Chromosomal Assignment of murine <italic>Csrp2 </italic>using the T31 radiation hybrid mapping panel</title>", "<p>DNAs from one hundred cell lines of the T31 mouse radiation hybrid (RH) panel (Research Genetics, Invitrogen, Paisley, UK) were typed using two independent sets of A23 hamster and 129 mouse DNA controls. The individual genomic PCRs were performed in reactions (50 μl) containing 50 ng of DNA, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl<sub>2</sub>, 400 μM of each dNTP, and 2 Units <italic>Taq </italic>polymerase (Roche), respectively. Primers used were: 5'-d(GAGACCGACATCTTAGGACAG)-3' and 5'-d(GATTGTGATGAGCTGCAGGCG)-3'. PCR conditions were: initial denaturation at 95°C for 5 min, 35 cycles of amplification (94°C for 1 min, 50°C for 1 min, 72°C for 3 min), final extension at 72°C for 10 min, and cooling to 4°C. The PCR products were separated on 1.8% agarose in 1× TBE and visualized by ethidium bromide staining. Data from this whole-genome radiation hybrid mapping were electronically submitted, analyzed, and deposited [MGI: 1202907] at the Jackson Laboratory, Bar Harbor, MA [##UREF##0##36##].</p>", "<title>Gene targeting</title>", "<p>The targeting construct was created by a two-step cloning strategy (see also Additional file ##SUPPL##4##5##). In a first step, the 3.4 kbp <italic>Bgl</italic>II fragment containing nt 4984 to nt 8393 [Genbank: <ext-link ext-link-type=\"gen\" xlink:href=\"AY533303\">AY533303</ext-link>] was subcloned and a blunted 1.7-kb <italic>Xho</italic>I/<italic>Sal</italic>I fragment containing a neomycin resistance cassette was cloned into the <italic>Stu</italic>I-site at nt 7292. Subsequently, the enlarged fragment was cloned back into the original 17.3 kbp genomic clone. All cloning boundaries were verified by sequencing. The resulting targeting vector was digested with <italic>Xho</italic>I cutting at position 1506 and in vector pBS-SKII. The 1.5 kbp <italic>Xho</italic>I fragment was removed by gel electrophoresis and later applied in Southern blot analysis as 5' external probe, and the remaining targeting vector was transfected into embryonic stem (ES) cells (129 SvJ) by electroporation. ES cells that had incorporated the transgene were subsequently maintained on mitotically inactive mouse embryonic fibroblast feeder layers and selected in the presence of 400 μg G418/ml. A total of 142 neomycin-resistant ES clones were picked, and their genomic DNA was isolated, digested with <italic>Bam</italic>HI, separated on 1.0% agarose gels, and transferred to Hybond-N membranes (Amersham Pharmacia, Braunschweig, Germany). For the identification of homologous recombinants, Southern blots were performed using the <sup>32</sup>P-labeled ~1.5-kbp external 5' fragment as a probe. The ES cell clones that showed correct targeting were injected into C57BL/6J blastocysts. Subsequently, the composites were transferred into pseudopregnant foster mice and resulting chimeras were mated. The congenic strain was produced by repeated backcrosses into the C57BL/6J strain and embryos and sperms from N10 generation were cryoconserved [EM: 01784] by the European Mouse Mutant Archive [##UREF##1##37##].</p>", "<title>Genotyping</title>", "<p>Genomic DNA isolated from tail biopsies of the offspring were genotyped by PCR using primer combinations 5'-d(CAGCAGTAGAGCTCCGAAGCTCC)-3' (ex4for) and 5'-d(CTACCTTCCCAGCTCCAATGATC)-3' (ex4rev), or primer combination 5'-d(CTGCTCTTTACTGAAGGCTCTTT)-3' (neofor) and ex4rev resulting in fragments of 218 (wild-type) or 422 bp (k.o.), respectively.</p>", "<title>Quantitative analysis of <italic>Csrp2 </italic>transcripts</title>", "<p>Gene expression of <italic>Csrp2 </italic>was monitored by real-time PCR as described in detail elsewhere [##REF##12633614##31##]. To correct for differences in quantity between RNA samples, data of amounts of <italic>Csrp2 </italic>transcripts were normalized to those of β-actin.</p>", "<title>Isolation and immunostaining of cardiomyocytes</title>", "<p>Primary cultures of cardiomyocytes were prepared, maintained and stained as described previously [##REF##15226401##38##]. The mouse monoclonal antibodies sarcomeric α-actinin (clone EA53) and DAPI to stain the nuclei were obtained from Sigma; Alexa633-conjugated phalloidin to visualise F-actin was purchased from Invitrogen. Cy3-conjugated anti mouse and Cy2-conjugated anti rabbit antibodies were from Jacksom Immunochemicals (via Stratech Scientific, Newmarket, UK). Confocal micrographs of stained cardiomyocytes were taken in a Zeiss LSM 510 confocal microscope equipped with argon, helium-neon and blue diode lasers, using a 25×/0.8 oil immersion and a 63×/1.4 oil immersion lens, respectively.</p>", "<title>Histological analysis</title>", "<p>Tissue sections from various organs were fixed with 4% paraformaldehyde in phosphate-buffered saline for 24 hours and analyzed according to standard procedures. For the analysis of murine hearts, 4 μm thick sections were cut along the frontal axis and stained with hematoxylin/eosin and Sirius red. Longitudinal cut areas of the left vetricular wall below the aortic valve were analysed for cardiomyocyte diameters using an internal microscopic size standard (mouse erythrocyte, 7 μm) and counting 20 high-power fields (HPF) of three (<italic>Csrp</italic>2<sup>-/-</sup>) or four (WT) animals. In all experiments, homozygous mutant mice were compared to wild-type siblings. Cryosections (10 μm thickness) were prepared from equivalent ventricular regions of age-matched wild-type, <italic>Csrp2</italic><sup>-/-</sup>, and <italic>MLP</italic><sup>-/- </sup>mice and were immunostained for N-RAP [##REF##11352937##24##,##REF##9295142##39##], connexin-43 (Chemicon Int., Temecula, CA), β-catenin (Sigma-Aldrich, Taufkirchen, Germany), and sarcomeric α-actinin [##REF##10658210##40##] as described previously [##REF##11352937##24##].</p>", "<title>SDS-PAGE, immunoblotting, and generation of CRP expression plasmids</title>", "<p>Whole-cell extracts from transfected COS-7 cells or kidney lysates were prepared following standard procedures. Equal amounts of proteins (30 μg) were resolved in NuPAGE™ Bis-Tris gels (Novex, Invitrogen, Karlsruhe, Germany) and electro-blotted onto a Protran membrane (Schleicher &amp; Schuell). Proteins were electroblotted onto nitrocellulose membranes (Schleicher &amp; Schuell, Dassel, Germany) and unspecific binding sites were blocked in TBST [10 mM Tris/HCl; 150 mM NaCl; 0.1% (v/v) Tween 20; pH 7.6] containing 5% (w/v) nonfat milkpowder. Primary antibodies employed were directed against the myc-epitope (M5546, Sigma), β-actin (A5441, Sigma) and CRP2 [##REF##16169118##41##]. They were diluted in 2.5% (w/v) nonfat milkpowder in TBST and visualized using horseradish peroxidase-conjugated anti-mouse- or anti-rabbit-IgG (Santa Cruz) and the Supersignal chemiluminescent substrate (Pierce, Bonn, Germany). Expression vectors for murine CRP1, CRP2 and CRP3 were prepared in vector pCMV-Myc (Clontech, Heidelberg, Germany). Therefore heart mRNA was reversed transcript and specific cDNA for murine <italic>Csrp1</italic>, <italic>Csrp2</italic>, and <italic>Csrp3 </italic>was generated using primers Csrp1-1 TCT CCC TGG ACA GAG CAG AAT G, Csrp1-2 CTC ACT CTG AGT GAA CCA AGG C, Csrp-2-1 CTC CCT CCT CCC ACT CGG AAT G, Csrp2-2 TTA CTG GTT CAC ACC ATT ACT GAG C, Csrp3-1 TTG GCC CAG AGT CTT CAC CAT G, and Csrp3-2 AGC AGG CAG CTT CAC TCC TTC, respectively. The cDNAs were cloned into pGEM-T-Easy vector (Promega, Madison, WI), sequenced and subcloned into the <italic>Eco</italic>RI site of expression vector pCMV-Myc. Transfection was done using the FuGene transfection reagent (Roche, Mannheim, Germany).</p>", "<title>Electron microscopic studies</title>", "<p>Tissue pieces from equivalent regions of the left ventricle taken from 8–10 month old male mice (3 animals each, 4–5 slices per animal) were fixed and prepared for electron microscopy as described before [##REF##11352937##24##]. The ultrathin sections were stained with uranyl acetate, air-dried and examined with a Philips transmission electron microscope TEM 400.</p>", "<title>Echocardiographic measurements</title>", "<p>Five 8-month-old male animals each (wildtype, <italic>Csrp2</italic><sup>-/-</sup>) were anaesthetized with a combination of ketamine (100 mg/kg) and xylamine (5 mg/kg) to perform echocardiographic examination using a Sonos 5500 from Philips Medical Systems equipped with a 12 MHz transducer. The thicknesses of the anterior and posterior walls of left ventricle of respective animals were measured in the 2-D directed M-mode.</p>" ]
[ "<title>Results</title>", "<title>Chromosomal localization of the murine <italic>Csrp2 </italic>gene</title>", "<p>By use of the T31 mouse/hamster radiation hybrid (RH) panel [##REF##9414320##21##,##REF##12529315##22##] containing a set of 100 different DNAs from somatic cell hybrids, we localized the murine <italic>Csrp2 </italic>gene to Chromosome 10. In this analysis with the highest anchor LOD of 23.2 was assigned to D10Mit150 with a best-fit location between markers D10Mit203 proximal and D10Mit150 central, confirming the gene position that was recently launched by the Mouse Genome Sequencing Consortium (NT_09500). Noteworthy, this region is syntenic to human chromosomal region 12q21.1 (Fig. ##FIG##0##1##), essentially the region to which the human orthologue was previously assigned [##REF##9286703##23##].</p>", "<title>Targeted disruption of the <italic>Csrp2 </italic>gene</title>", "<p>For generation of the targeting vector we isolated and sequenced a ~17.3 kbp genomic clone of the murine <italic>Csrp2 </italic>gene [Genbank:<ext-link ext-link-type=\"gen\" xlink:href=\"AY533303\">AY533303</ext-link>]. The targeting construct was generated by insertion of a neomycin resistance cassette into the <italic>Stu</italic>I restriction site of exon 4 and contained 1386 bp upstream and 14413 bp downstream sequences (for more details see Method section). After transfection of ES cells with the targeting construct, we tested 142 transformants by Southern blot using an external hybridization probe. Sixteen of these ES clones were found to have incorporated the targeted vector by homologous recombination. Subsequently, ES clones carrying the disrupted <italic>Csrp2 </italic>gene were injected into C57BL/6J blastocysts, and transferred into the uteri of pseudopregnant recipients. The mouse chimeras were identified by the inspection of the proportion of coat agouti skin color ranging from complete white to near 80% black. Crosses between chimeras and C57BL/6J mice revealed that the ES cell genome was transmitted through the germline, as indicated by the agouti skin color of the offspring. F1 hybrids tested to be heterozygous for the disrupted gene were backcrossed (up to N10) into the C57BL/6J strain background. To generate <italic>Csrp2 </italic>null mice, we interbred heterozygous animals and genotyped litters after weaning at 4 weeks of age using a genotyping PCR strategy (Fig. ##FIG##1##2A##). Furthermore, we performed Southern blot hybridization using an external probe to demonstrate that the targeting construct was correctly inserted into the <italic>Csrp2 </italic>locus (Fig. ##FIG##1##2B##). To demonstrate the absence of specific <italic>Csrp2 </italic>transcripts, we performed Northern blot analysis (Fig. ##FIG##1##2C##) and quantitative PCR (Additional file ##SUPPL##0##1##) revealing that the level of <italic>Csrp2 </italic>mRNA was half that of the wild type in heterozygous <italic>Csrp2</italic><sup>+/- </sup>null mouse. However, an aberrant RNA species that appeared at very low level was expressed in gene-disrupted mice (cf. Fig. ##FIG##1##2C##). Sequence analysis of a cDNA generated by reverse transcription of the respective mRNA species revealed that this aberrant message was generated by artificial splicing of exon 3 to the downstream neomycin/exon4 boundary (Additional file ##SUPPL##0##1C##). This RNA does not produce any protein product at any size as tested by Western blot analysis (Fig. ##FIG##1##2D##).</p>", "<title>CRP2 deficient mice are viable and fertile</title>", "<p>Mice homologous for the targeted deletion had no gross phenotypic abnormalities, and development and reproductive function appeared normal. DNA analysis of 489 progeny (225 females, 264 males) derived from <italic>Csrp2</italic><sup>+/- </sup>intercrosses revealed that <italic>Csrp2</italic><sup>-/- </sup>mice were born in the predicted 1:2:1 Mendelian distribution (128 wild type, 243 heterozygotes, and 118 nulls). Mating of homozygous males with homozygous females produced viable offspring of normal litter size at normal frequency indicating that CRP2-deficient mice were fertile and pregnancies were carried out to full term. To detect possible structural defects induced by the absence of <italic>Csrp2</italic>, we comparatively examined formalin-fixed tissue sections of adult wt and <italic>Csrp2</italic><sup>-/- </sup>mice. We found that sections taken from kidney, skeletal muscle, heart, liver, lung, brain, thymus, stomach, or intestine were indistinguishable from those of control mice (Additional file ##SUPPL##1##2##, and not shown).</p>", "<title>Detailed analysis of heart architecture</title>", "<p>Previous reports have demonstrated that the absence of another CRP family member (i.e. CRP3/MLP) reproduces the morphological and clinical picture of dilated cardiomyopathy and heart failure in humans [##REF##9039266##7##]. Furthermore, independent studies have shown that <italic>Csrp2 </italic>expression is detectable in both vascular and venous smooth muscle cells and in cardiomyocytes throughout embryogenesis suggesting an important role for <italic>Csrp2 </italic>in the developing heart and cardiovascular system [##REF##9815145##18##,##REF##11748300##19##]. Therefore, we decided to direct our attention to potential alterations of the heart. Compared to <italic>Csrp3/Mlp</italic><sup>-/-</sup>, the <italic>Csrp2 </italic>nulls had no apparent degeneration or enlargements, and the weight and size of hearts taken from <italic>Csrp2</italic><sup>-/- </sup>mice were indistinguishable from control littermates. However, a more detailed morphometric analysis of myocardial sections revealed that the thickness/diameter of (longitudinally cut) cardiomyocytes was significantly higher in the <italic>Csrp2</italic>-disrupted mice (wt: 15.5 ± 0.8; 15.8 ± 1.0; 15.8 ± 1.1; 15.2 ± 1.1 <italic>versus </italic>ko: 17.4 ± 1.1; 17.5 ± 1.3; 17.0 ± 0.9 μm) (Fig. ##FIG##2##3##, Table ##TAB##0##1##).</p>", "<p>Based on the knowledge that the Z discs of <italic>Csrp3/MLP</italic><sup>-/- </sup>mice show misalignment and the fact that CRP2 has affinity for α-actinin [##REF##12507422##15##,##REF##9341203##6##], we next envisaged to comparatively analyse the Z-disc defining the lateral boundaries of the sarcomere. To do so, we first tested if CRP2 is expressed in adult cultured cardiomyocytes (Additional file ##SUPPL##2##3##). This analysis revealed that, compared to the regular arrangement of α-actinin and F-actin, CRP2 showed a more irregular staining pattern in cultured cardiomyocytes. In the <italic>in vivo </italic>situation, CRP2 was most prominently localized at the intercalated disc (Fig. ##FIG##3##4##). However, in contrast to <italic>Mlp</italic><sup>-/- </sup>cardiomyocytes in which the myofibrils are somewhat disorganized, the localization pattern of α-actinin, an integral component of the Z-disc is indistinguishable in wild-type and <italic>Csrp2</italic><sup>-/- </sup>cardiomyocytes (Fig. ##FIG##4##5A##). Furthermore, the intercalated discs were more convoluted in <italic>Csrp2</italic><sup>-/- </sup>mouse hearts than in hearts of wild type littermates (Fig. ##FIG##4##5B##) but less pronounced than in the <italic>Csrp3/Mlp </italic>null mice (not shown). We next addressed the question whether a lack of CRP2 expression would lead to a change of the molecular composition of the intercalated disc in addition to its altered ultrastructure, as previously described for the <italic>Csrp3/Mlp </italic>knock out mice [##REF##9039266##7##,##REF##11352937##24##]. Although less pronounced than in the <italic>Csrp3/Mlp </italic>knock out mice, we found that <italic>Csrp2 </italic>knock outs show also increased signal for the adherens junction protein β-catenin at the intercalated disc, while there is less signal for connexin-43, the component of the gap junctions (Fig. ##FIG##5##6A##). Likewise, the slight increase of cardiomyocyte thickness in <italic>Csrp2</italic><sup>-/- </sup>mice were detectable confirming the diameter measurements described above. To test if the increased signals determined for N-RAP and β-catenin are due to a higher expression of these proteins or an altered distribution, we performed Western blot analysis (Fig. ##FIG##5##6B##). We found that the expression of both proteins was unmodified compared to normal control mice, while we confirmed the increase in expression of both proteins in <italic>Csrp3</italic><sup>-/-</sup>/<italic>Mlp</italic><sup>-/- </sup>deficient mice.</p>", "<title>Expression of <italic>Csrp1 </italic>and <italic>Csrp3 </italic>in CRP2 deficient mice</title>", "<p>To investigate whether expression of <italic>Csrp1 </italic>and <italic>Csrp3 </italic>was affected by the absence of CRP2, we isolated RNAs from different organs and compared the transcript levels of respective genes in wild-type and homozygous mutant mice. Total RNA was isolated from different tissues and the relative amounts of <italic>Csrp1 </italic>and <italic>Csrp3/Mlp </italic>mRNAs were determined by Northern blot analysis. We found that the transcriptional activity of <italic>Csrp1 </italic>and <italic>Csrp3 </italic>genes in <italic>Csrp2</italic><sup>-/- </sup>mice was indistinguishable from controls (Additional file ##SUPPL##3##4##).</p>" ]
[ "<title>Discussion</title>", "<p>CRP2 belongs to the CRP family of LIM domain proteins that are evolutionarily conserved. The sequence of mouse CRP2 displays 99.5%, 97.9%, and 97.4% amino acid identity to human, quail, and chicken CRP2, respectively. Therefore, it is supposed that this LIM domain protein has a critically, evolutionarily conserved role for the development or maintenance of vital processes within organisms. Consistent with this hypothesis is the observation that <italic>Csrp2 </italic>expression is detectable in a number of cell types during embryogenesis, including mesenchyme, vascular smooth muscle cells, and epithelial derivatives [##REF##11748300##19##]. It was also demonstrated that <italic>Csrp2 </italic>is expressed in the cardiovascular system at early time points during mouse development indicating an important role in vascular smooth muscle differentiation [##REF##9815145##18##]. Based on the knowledge that CRP2 and other CRPs can bind to zyxin, α-actinin, and α-actin [##REF##9314536##25##,##REF##9341203##6##,##REF##14741346##26##], it is tempting to speculate that these proteins are bridging molecules that are important for the organization or stabilization of cytoskeletal structures. This is also supported by structural data showing that the two tandemly arranged LIM domains are completely independent folded units that are linked by a highly flexible spacer [##REF##9722554##11##,##REF##10231520##12##].</p>", "<p>Moreover, <italic>Csrp2 </italic>was originally identified from normal quail embryo fibroblasts that were screened for genes suppressed in the course of oncogenic transformation [##REF##8361751##3##]. Subsequently, it was demonstrated that the transcriptional suppression of <italic>Csrp2 </italic>is generally linked to the transformed state of cells [##REF##7499425##27##] suggesting that CRP2 might have tumor-suppressor activity. In this context it is remarkable that the reintroduction of CRP2 into human breast and colorectal cancer cell lines was sufficient to significantly decrease colony formation [##REF##15313885##28##]. However, all these findings demonstrate that indeed CRP2 must have specific functions both in development and control of cancer cell growth. Considering the postulated roles and the observed expression pattern of CRP2, the finding that mice lacking a functional <italic>Csrp2 </italic>gene exhibit a quite mild phenotype was unexpected but is in agreement with a recent report characterizing a similar <italic>Csrp2 </italic>gene disruption in mice [##REF##16269651##20##]. No gross morphological or physiological abnormalities were detected, suggesting that <italic>Csrp2 </italic>is dispensable for normal development. This finding is somewhat surprising, since the ablation of the CRP3/MLP or TLP genes is associated with striking morphological and functional alterations [##REF##9039266##7##,##REF##11713292##5##]. Possibly, there exists a functional redundancy that is known from other myogenic factors [##REF##1330322##29##,##REF##1423602##30##]. This would give rise to \"cryptic robustness\" to cells and organs expressing different CRPs. Conversely, a loss of function mutation or an experimental inactivation of a <italic>Csrp </italic>gene would predominantly affect cells or organs exclusively expressing this family member. There are some good arguments supporting this \"quantitative model of CRP function\". First, different independent studies have shown that CRP3/MLP is exclusively expressed in heart at high level and to a lower extent in skeletal muscle [##REF##7954791##4##,##REF##9815145##18##,##REF##10751147##8##,##REF##12633614##31##]. The abundance of <italic>Csrp3/Mlp </italic>transcripts in these organs is several times higher than those of <italic>Csrp1 </italic>and <italic>Csrp2 </italic>(Additional file ##SUPPL##3##4##). Therefore, it is reasonable that the CRP quantities expressed by these genes cannot compensate for CRP3/MLP. Consistent with our hypothesis, it is not surprising that <italic>Csrp3/Mlp </italic>nulls have a strong cardiac phenotype [##REF##9039266##7##] and <italic>Csrp3/Mlp </italic>mutations are associated with heart failure in humans [##REF##12642359##14##,##REF##12507422##15##]. Secondly, TLP is highly expressed in the thymus and accordingly mice deficient for TLP have alterations of normal thymus function [##REF##11713292##5##]. Third, our data presented in this study indicate that the loss of CRP2 has only a mild cardiac phenotype. Compared to the <italic>Csrp3/Mlp </italic>knock out model [##REF##9039266##7##], the cardiac alterations are much less pronounced. The localisation of CRP2 (Fig. ##FIG##3##4##) and the alterations in tissue distribution of N-RAP and β-catenin found in animals lacking CRP2 (Fig. ##FIG##5##6##) point to a functionality of CRP2 in the organisation of the intercalated disc. In this regard, CRP2 might cooperate with other CRPs. It is tempting to speculate that during heart development <italic>Csrp3/Mlp </italic>or <italic>Csrp1 </italic>may be in the position to compensate for the loss of CRP2. This principle may also be true for the development of other organs. <italic>Csrp2 </italic>is broadly expressed in mouse embryos and adults [##REF##9815145##18##,##REF##11748300##19##,##REF##12633614##31##]. Likewise, <italic>Csrp1 </italic>is widely expressed in mouse tissues [##REF##9815145##18##,##REF##10090149##32##] and expression coincides with that of <italic>Csrp2 </italic>(Additional file ##SUPPL##4##5##). Noteworthy, the <italic>Csrp1 </italic>and <italic>Csrp2 </italic>genes are expressed in early vertebrate embryos and are spatially regulated in mouse [##REF##11748300##19##,##REF##10090149##32##]. In this regard, the recent finding that both, CRP1 and CRP2, are able to convert pluripotent 10T1/2 fibroblasts into smooth muscle cells [##REF##12530967##9##] demonstrates that these CRPs share some functions. Together, these observations are in agreement with the notion that individual CRPs share redundant functions and may explain the mild phenotype of the <italic>Csrp2</italic><sup>-/- </sup>mouse and the surprising observation that <italic>Csrp2 </italic>thought to be involved in key biological processes could be removed without affecting the organism.</p>", "<p>A recent report has shown that CRP2 physically associates with other cardiovascular lineage regulators, such as serum response factor (SRF) and GATA proteins, thereby synergistically activating the transcription of smooth muscle cell target genes (i.e. smooth muscle myosin heavy-chain, calponin, smooth muscle α-actin, caldesmon, SM22α) with contractile abilities [##REF##17185421##17##]. Therefore, it is possible that the lack of CRP2 cause alterations in expression of respective genes resulting in cardiomyocytes displaying a slight increase in thickness. CRP2 and its close homologue CRP1 are co-expressed during early cardiovascular development and both CRPs share similar functionality in facilitating transcriptional activity of the SRF-GATA-complex, especially of promoters encoding the SMC target genes [##REF##12530967##9##]. Therefore, it is reasonable that CRP1 in the <italic>Csrp2 </italic>nulls can substitute for CRP2 resulting in the observed mild phenotype. Noteworthy, this functional substitution is not overall complete. In preliminary echocardiography using five animals each we found indications that the disruption of the <italic>Csrp2 </italic>gene is associated with a lower left ventricular wall thickness and fractional shortening (Additional file ##SUPPL##5##6##) indicating reduced heart function in respective nulls.</p>", "<p>However, the lack of an \"obvious <italic>Csrp2</italic><sup>-/- </sup>phenotype\" in an animal kept under laboratory conditions constitutes no compelling argument against a unique and indispensable role of a gene in the normal physiology and the specialized <italic>in vivo </italic>function of CRP2 may become apparent only after appropriate challenges to the mice. In this context, it might be significant that apart from the shared binding affinity of CRP1, CRP2 and CRP3/MLP to zyxin and α-actinin, we and others have isolated proteins specifically interacting with individual CRPs [##REF##9234731##33##,##REF##10751147##8##,##REF##10924333##34##,##REF##11672422##10##]. The ability of CRPs to discriminate between different target proteins may the basis for subtle differences in functionality. Such an overlap of functional redundancy with protein specific activities was also demonstrated in the myogenic basic helix-loop-helix (HLH) genes [##REF##1330322##29##,##REF##1423602##30##]. Comparable to the <italic>Csrp2</italic><sup>-/- </sup>nulls, the inactivation of <italic>MyoD </italic>leads to up-regulation of the myogenic HLH gene <italic>Myf-5 </italic>and results in normal development [##REF##1330322##29##]. It is possible that during early development and differentiation of different myogenic lineages, myogenic factors and also CRPs act cooperatively, but exert distinct function at later stages, when the fine-tuning of cellular programs happens.</p>", "<p>The demonstration that the <italic>Csrp2 </italic>gene is silenced during cellular transformation and CRP2 protein induces growth-inhibitory effects when overexpressed in cancer cells points to a critical role in the control of normal cell growth. Future studies will clarify whether mice or cells lacking CRP2 will be more susceptible to tumor promoters or other physiological stress factors. Moreover, the phenotype of mice lacking several members of the CRP family will be highly informative as it directly addresses the question of mutual functional substitution and redundancy.</p>" ]
[ "<title>Conclusion</title>", "<p>We conclude that the LIM domain protein CRP2 is involved in normal cardiomyocyte development. The loss of CRP2 influences the fine architecture of the intercalated disc morphology.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The cysteine and glycine rich protein 2 (CRP2) encoded by the <italic>Csrp2 </italic>gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 <italic>in vivo</italic>, we disrupted the <italic>Csrp2 </italic>gene in mice and analysed the resulting phenotype.</p>", "<title>Results</title>", "<p>A ~17.3 kbp fragment of the murine <italic>Csrp2 </italic>gene containing exon 3 through 6 was isolated. Using this construct we confirmed the recently determined chromosomal localization (Chromosome 10, best fit location between markers D10Mit203 proximal and D10Mit150 central). A gene disruption cassette was cloned into exon 4 and a mouse strain lacking functional <italic>Csrp2 </italic>was generated. Mice lacking CRP2 are viable and fertile and have no obvious deficits in reproduction and survival. However, detailed histological and electron microscopic studies reveal that CRP2-deficient mice have subtle alterations in their cardiac ultrastructure. In these mice, the cardiomyocytes display a slight increase in their thickness, indicating moderate hypertrophy at the cellular level. Although the expression of several intercalated disc-associated proteins such as β-catenin, N-RAP and connexin-43 were not affected in these mice, the distribution of respective proteins was changed within heart tissue.</p>", "<title>Conclusion</title>", "<p>We conclude that the lack of CRP2 is associated with alterations in cardiomyocyte thickness and hypertrophy.</p>" ]
[ "<title>Authors' contributions</title>", "<p>JFS was responsible for the maintenance of the <italic>Csrp2 </italic>null strain including backcrossing to N10, genotyping, Northern- and Western blot experimentation, and helped in sequencing the ~17.3 kbp genomic fragment. MM has performed the mouse manipulation necessary for generation the <italic>Csrp2 </italic>null strain. EE has performed the experimentation for heart architecture analysis and the isolation/staining of murine cardiomyocytes. SW and DS provided their technical skills in all experiments. KG performed the quantitative real time PCR experiments. RB performed the histological analysis of mouse tissues and calculated cardiomyocyte diameters. RW cloned the disruption construct, sequenced part of the <italic>Csrp2 </italic>gene, performed the experiments necessary for chromosomal assignments, and drafted the manuscript.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>We thank Lucy Rowe (TJL Fine Mapping Laboratory/The Jackson Laboratory Mapping Panels, Scientific Services Department, The Jackson Laboratory, ME) for assistance in analysis of the mouse mapping data, Friederike Cuello for the generous donation of freshly isolated cardiomyocytes, Günter Hollweg for expert help in electron microscopic analysis, Jutta Kirfel for production of histological sections, and Felix Gramley for expert help in echocardiographic measurements. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) to RW.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Assignment and map location of murine <italic>Csrp2 </italic>on Chromosome 10</bold>. We typed the 100 cell hybrid DNAs (1–100) of the mouse T31 whole-genome-radiation hybrid panel and two sets of independent A23 hamster (H) and 129 mouse (M) controls by PCR to determine the chromosomal map location of <italic>Csrp2</italic>. The mapping results of this analysis were deposited under accession no. MGI: 1202907 at the Jackson Laboratory and the murine <italic>Csrp2 </italic>gene localized to chromosome 10. In the figure, the entire T31 RH Chr 10 framework map is depicted on the left of the figure with the overall length calculated from the framework data [##UREF##2##42##]. The centromere is depicted by a black circle at the top of the map. The enlarged segment of distal Chromosome 10 is shown with respective framework markers listed to the left of the chromosome bar and a selection of mapped genes to the right. The distances between loci are calculated based on only the listed data sets, and unscored radiation hybrid cell line data are inferred where the data on either side of the missing score are in agreement. Blocks of human synteny are indicated to the right of the RH map, based on information from the NCBI's locus link [##UREF##3##43##]. Note all locus names should be in italics, but are shown in plain text for readability.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Targeted disruption of the mouse <italic>Csrp2 </italic>gene</bold>. <bold>(A) </bold>A neomycin resistance cassette (neo) was incorporated into <italic>Csrp2 </italic>(exon 4) as outlined in the Method section. The different <italic>Csrp2 </italic>alleles (WT <italic>vs </italic>k.o.) from progeny of mating heterozygotes were discriminated by PCR analysis of tail biopsy DNAs. The positions of the oligonucleotide primers used for amplification of the wild-type (218 bp) and mutant (422 bp) PCR fragments are indicated. In the genotyping experiment shown, the different DNAs were genotyped as homozygous null (-/-), heterozygous (+/-) or wild-type (+/+). <bold>(B) </bold>Southern hybridisation of littermate offspring from heterozygous intercrosses genotyped as heterozygote (WT), wild-type (WT), or homozygote null (<italic>Csrp2</italic><sup>-/-</sup>). The DNA was digested with <italic>Bam</italic>HI, fragments were separated in a 1% agarose gel and transferred to a nylon membrane. The blot was hybridized with an external 1.5 kbp <italic>Xho</italic>I probe (see Methods), resulting in fragments of ~7.3 (knock out) or ~12.6 kb (wild-type) in size. <bold>(C) </bold>Northern blot of total kidney RNA isolated from wild-type (+/+), heterozygote (+/-), and homozygote (-/-) <italic>Csrp2 </italic>null mice. The RNAs were hybridized with a <italic>Csrp2 </italic>specific cDNA probe. The autoradiograph showed the typical 1.2. kb <italic>Csrp2 </italic>signal in <italic>Csrp</italic>2<sup>+/+</sup>, a weaker band with <italic>Csrp2</italic><sup>+/-</sup>, and a faint band with the <italic>Csrp2</italic><sup>-/- </sup>mice. To verify the integrity of RNAs, the blot was subsequently hybridized with a <italic>GAPDH</italic>-specific cDNA probe. <bold>(D) </bold>Western blot of kidney homogenates extracted from wild-type (WT) and mutant mice (<italic>Csrp2</italic><sup>-/-</sup>). As a positive control and to demonstrate the specificity of the CRP2 specific antibody, cell extracts taken from COS-7 cells that were transfected with myc-epitope tagged version of murine CRP1, CRP2 and CRP3 were taken. The expression of these proteins was demonstrated by subsequent probing with a myc-epitope specific antibody. In the <italic>Csrp2 </italic>nulls, no CRP2 band at any size was detected.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Alterations in cardiomyocyte thickness and intercalated disc morphology in <italic>Csrp2</italic><sup>-/- </sup>mice</bold>. Morphometric analysis of longitudinal cut areas of the left ventricular wall below the aortic valve from wild-type <bold>(A) </bold>and <italic>Csrp2</italic><sup>-/- </sup><bold>(B) </bold>siblings were analysed for cardiomyocyte diameters using a microscope with an internal size scale. For details refer Material and Method section and Table 1. The space bar in each figure part represents 100 μM.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>CRP2 is preferentially associated with the intercalated disc</bold>. Confocal micrographs of immunostained sections from wild type mice show that a strong signal for CRP can be detected at the intercalated disc (green signal in overlay in top two rows). Preimmune serum only picks up extracellular matrix in control sections (green signal in bottom two rows). The sections were counterstained for the Z-disc protein α-actinin (red signal in overlays) and with DAPI to visualise the nuclei (blue signal in overlays). The space bar represents 10 μM.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Structure of the Z discs from wild-type, <italic>Csrp</italic>2<sup>-/-</sup>, and <italic>Csrp3/Mlp</italic><sup>-/- </sup>mice</bold>. <bold>(A) </bold>The structures of Z discs from wild-type, <italic>Csrp</italic>2<sup>-/-</sup>, and <italic>Csrp3/Mlp</italic><sup>-/- </sup>were displayed by immunofluorescence microscopy using an antibody directed against sarcomeric α-actinin. The space bar represents 10 μM.<bold>(B) </bold>Heart sections of wild-type mice (WT) and <italic>Csrp2 </italic>nulls (<italic>Csrp2</italic><sup>-/-</sup>) were infiltrated with an expoxy resin and examined in a Philips TEM 400 transmission microscope. The intercalated discs are each marked by arrowheads. Note the moderate and pronounced convolution of the membrane at the intercalated disc of <italic>Csrp2 </italic>null compared to wild type mice. (Original magnification × 9.000). <bold>(C) </bold>Heart sections of wild-type mice (WT) and <italic>Csrp2 </italic>nulls (<italic>Csrp2</italic><sup>-/-</sup>) at higher magnification (× 18.000). For electron microscopic analysis three hearts taken from each genotype were analysed. The most representative images are shown in <bold>(B) </bold>and <bold>(C)</bold>.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Markers of heart dysfunction</bold>. <bold>(A) </bold><italic>Csrp2 </italic>nulls show less pronounced alterations in the distribution of intercalated disc-associated proteins than observed in <italic>Csrp3/Mlp </italic>disrupted mice. Micrographs of longitudinal frozen sections of ventricular tissues from wild-type (panel <italic>A, D, G</italic>), <italic>Csrp3/Mlp</italic><sup>-/- </sup>(panel <italic>B, E, H</italic>) and <italic>Csrp2</italic><sup>-/- </sup>(panel <italic>C, F, I</italic>) mice, stained with antibodies against β-catenin (panel <italic>A-C</italic>), connexin-43 (panel <italic>D-F</italic>) and N-RAP (panel <italic>G-I</italic>) antibodies. While β-catenin and N-RAP expression are significantly upregulated in <italic>Csrp3/Mlp </italic>deficient mice (panel B, H) and to a lesser extent in <italic>Csrp2</italic><sup>-/- </sup>mice (panel C, I), connexin-43 expression is reduced in <italic>Csrp3/Mlp</italic><sup>-/- </sup>as well as <italic>Csrp2</italic><sup>-/- </sup>mice (panel F). The space bar represents 10 μM.<bold>(B) </bold>Western blot analysis of N-RAP and β-catenin. Heart extracts from normal (WT), <italic>Csrp2</italic>- and MLP-deficient mice were probed with antibodies specific for N-RAP and β-catenin. Equal loading was demonstrated in Ponceau Red stain.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Morphometric analysis of myocardial sections</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">No. of HPF</td><td align=\"center\" colspan=\"7\">thickness/diameter (μm) of cardiomyocytes</td></tr><tr><td/><td colspan=\"7\"><hr/></td></tr><tr><td/><td align=\"center\">WT</td><td align=\"center\">WT</td><td align=\"center\">WT</td><td align=\"center\">WT</td><td align=\"center\">Csrp2<sup>-/-</sup></td><td align=\"center\">Csrp2<sup>-/-</sup></td><td align=\"center\">Csrp2<sup>-/-</sup></td></tr></thead><tbody><tr><td align=\"center\">1</td><td align=\"center\">14.6</td><td align=\"center\">16.7</td><td align=\"center\">15.9</td><td align=\"center\">15.9</td><td align=\"center\">16.7</td><td align=\"center\">16.3</td><td align=\"center\">17.1</td></tr><tr><td align=\"center\">2</td><td align=\"center\">16.7</td><td align=\"center\">16.3</td><td align=\"center\">16.3</td><td align=\"center\">14.2</td><td align=\"center\">20</td><td align=\"center\">19.4</td><td align=\"center\">17.5</td></tr><tr><td align=\"center\">3</td><td align=\"center\">14.9</td><td align=\"center\">13.7</td><td align=\"center\">17.1</td><td align=\"center\">14.2</td><td align=\"center\">17.9</td><td align=\"center\">20</td><td align=\"center\">16.3</td></tr><tr><td align=\"center\">4</td><td align=\"center\">15.2</td><td align=\"center\">15.9</td><td align=\"center\">14.2</td><td align=\"center\">14.6</td><td align=\"center\">19.4</td><td align=\"center\">17.9</td><td align=\"center\">16.3</td></tr><tr><td align=\"center\">5</td><td align=\"center\">14</td><td align=\"center\">14.2</td><td align=\"center\">14.2</td><td align=\"center\">14.9</td><td align=\"center\">15.6</td><td align=\"center\">15.6</td><td align=\"center\">15.9</td></tr><tr><td align=\"center\">6</td><td align=\"center\">16.7</td><td align=\"center\">15.6</td><td align=\"center\">15.9</td><td align=\"center\">13.5</td><td align=\"center\">15.9</td><td align=\"center\">16.7</td><td align=\"center\">18.4</td></tr><tr><td align=\"center\">7</td><td align=\"center\">16.7</td><td align=\"center\">15.2</td><td align=\"center\">15.2</td><td align=\"center\">13.7</td><td align=\"center\">19.4</td><td align=\"center\">17.5</td><td align=\"center\">17.1</td></tr><tr><td align=\"center\">8</td><td align=\"center\">15.2</td><td align=\"center\">15.9</td><td align=\"center\">14.2</td><td align=\"center\">14.2</td><td align=\"center\">17.1</td><td align=\"center\">17.5</td><td align=\"center\">18.9</td></tr><tr><td align=\"center\">9</td><td align=\"center\">14.9</td><td align=\"center\">16.7</td><td align=\"center\">17.5</td><td align=\"center\">17.5</td><td align=\"center\">18.9</td><td align=\"center\">16.7</td><td align=\"center\">16.7</td></tr><tr><td align=\"center\">10</td><td align=\"center\">14.2</td><td align=\"center\">17.5</td><td align=\"center\">17.5</td><td align=\"center\">16.7</td><td align=\"center\">17.1</td><td align=\"center\">19.4</td><td align=\"center\">17.9</td></tr><tr><td align=\"center\">11</td><td align=\"center\">15.9</td><td align=\"center\">16.7</td><td align=\"center\">15.9</td><td align=\"center\">15.9</td><td align=\"center\">17.5</td><td align=\"center\">20</td><td align=\"center\">17.9</td></tr><tr><td align=\"center\">12</td><td align=\"center\">15.6</td><td align=\"center\">15.9</td><td align=\"center\">15.6</td><td align=\"center\">15.6</td><td align=\"center\">17.9</td><td align=\"center\">17.9</td><td align=\"center\">17.5</td></tr><tr><td align=\"center\">13</td><td align=\"center\">14.9</td><td align=\"center\">16.3</td><td align=\"center\">15.2</td><td align=\"center\">14.9</td><td align=\"center\">15.6</td><td align=\"center\">17.9</td><td align=\"center\">17.1</td></tr><tr><td align=\"center\">14</td><td align=\"center\">14.9</td><td align=\"center\">17.5</td><td align=\"center\">15.9</td><td align=\"center\">15.2</td><td align=\"center\">15.9</td><td align=\"center\">15.6</td><td align=\"center\">17.9</td></tr><tr><td align=\"center\">15</td><td align=\"center\">16.7</td><td align=\"center\">16.7</td><td align=\"center\">14.9</td><td align=\"center\">14.2</td><td align=\"center\">16.7</td><td align=\"center\">16.3</td><td align=\"center\">17.1</td></tr><tr><td align=\"center\">16</td><td align=\"center\">14.9</td><td align=\"center\">14.9</td><td align=\"center\">15.9</td><td align=\"center\">14.6</td><td align=\"center\">18.9</td><td align=\"center\">17.1</td><td align=\"center\">16.7</td></tr><tr><td align=\"center\">17</td><td align=\"center\">16.3</td><td align=\"center\">15.6</td><td align=\"center\">15.6</td><td align=\"center\">14.6</td><td align=\"center\">17.9</td><td align=\"center\">17.9</td><td align=\"center\">15.6</td></tr><tr><td align=\"center\">18</td><td align=\"center\">15.9</td><td align=\"center\">14.9</td><td align=\"center\">14.2</td><td align=\"center\">17.1</td><td align=\"center\">15.9</td><td align=\"center\">16.7</td><td align=\"center\">15.2</td></tr><tr><td align=\"center\">19</td><td align=\"center\">15.2</td><td align=\"center\">15.2</td><td align=\"center\">17.1</td><td align=\"center\">16.7</td><td align=\"center\">17.1</td><td align=\"center\">16.7</td><td align=\"center\">16.3</td></tr><tr><td align=\"center\">20</td><td align=\"center\">15.6</td><td align=\"center\">14.2</td><td align=\"center\">17.5</td><td align=\"center\">15.9</td><td align=\"center\">15.9</td><td align=\"center\">16.3</td><td align=\"center\">15.9</td></tr><tr><td align=\"center\">Mean</td><td align=\"center\">15.5</td><td align=\"center\">15.8</td><td align=\"center\">15.8</td><td align=\"center\">15.2</td><td align=\"center\">17.4</td><td align=\"center\">17.5</td><td align=\"center\">17.0</td></tr><tr><td align=\"center\">SD</td><td align=\"center\">0.8</td><td align=\"center\">1.0</td><td align=\"center\">1.1</td><td align=\"center\">1.1</td><td align=\"center\">1.1</td><td align=\"center\">1.3</td><td align=\"center\">0.9</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional File 1</title><p><bold>Quantitative real-time RT-PCR</bold>. <bold>(A) </bold>Kidney RNAs from <italic>Csrp2</italic><sup>+/+</sup>, <italic>Csrp2</italic><sup>+/-</sup>, and <italic>Csrp2</italic><sup>-/- </sup>littermates were reverse-transcribed and analyzed for <italic>Csrp2 </italic>expression using a LightCycler protocol (<italic>left panel</italic>). Data acquired were normalized to β-actin and relative intensities were compared to <italic>Csrp2</italic>-expression in <italic>CSRP2</italic><sup>+/+ </sup>mice (set to 100). The relative expression of <italic>Csrp2 </italic>obtained by real time PCR in normal and in <italic>Csrp2 </italic>nulls was confirmed by Northern blot (<italic>right panel</italic>). <bold>(B) </bold>The amplicon from <italic>Csrp2</italic><sup>-/- </sup>mice was sequenced showing that the aberrant mRNA results from an artificial splice event between exon3 and the downstream neo/exon4 boundary inserting 25 bps.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional File 2</title><p><bold>Tissue morphology</bold>. Tissue slices of adult <italic>Csrp2</italic><sup>-/- </sup><bold>(A-D) </bold>and wild type control mice <bold>(A'-D') </bold>taken from renal cortex <bold>(A, A') </bold>and pelvis <bold>(B, B')</bold>, skeletal muscle <bold>(C, C')</bold>, and liver <bold>(D, D') </bold>were Hematoxylin-Eosin-stained and analysed by light microscopy. The space bar in each figure represents 100 μM.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional File 3</title><p><bold>CRP2 expression in cultured adult murine cardiomyocytes</bold>. <bold>(A) </bold>Cultured murine cardiomyocytes were permeabilized and stained with an antibody specific for CRP2 or a preimmuneserum (<italic>inlet</italic>). The cells were washed and incubated with a second antibody that was coupled with alkaline phosphatase. After extensive washing the cells were then incubated with the fast red substrate (DAKO, Hamburg, Germany) and pictures were taken in a standard light microscope. <bold>(B-D) </bold>Cardiomyocytes were simultaneously stained for CRP2 <bold>(B)</bold>, α-actinin <bold>(C) </bold>and F-actin <bold>(D) </bold>and analysed by confocal microscopy. The space bar represents 10 μM.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S4\"><caption><title>Additional File 4</title><p><bold>Analysis of <italic>Csrp </italic>expression in <italic>Csrp2 </italic>deficient mice</bold>. Northern blot analysis from RNAs isolated from different organs of wild-type (+/+) and <italic>Csrp2</italic><sup>-/- </sup>mice were analysed for expression of <italic>Csrp1</italic>, <italic>Csrp2</italic>, and <italic>Csrp3/Mlp</italic>. The ethidium bromide-stained gel is shown to demonstrate equal loading of RNA samples.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S5\"><caption><title>Additional File 5</title><p><bold>Organisation and disruption of the murine <italic>Csrp2 </italic>gene</bold>. <bold>(A) </bold>The <italic>Csrp2 </italic>gene contains one non-coding (E1) and 5 coding exons (E2-E6) that are marked by white or black boxes. For cloning of the disruption construct a 17.3 kbp fragment of the <italic>Csrp2 </italic>gene spanning E1 to E6 was isolated and a <italic>neo </italic>cassette was inserted into the <italic>Stu</italic>I site of exon 4. For details see Materials and Method section. <bold>(B) </bold>The localisation of the external hybridisation probe used for verification of successful insertion by Southern blot is depicted as a solid red line. This probe detects a ~12.6 kb <italic>Bam</italic>HI fragment in wild type (<italic>Csrp2</italic>) and a ~7.3 kb <italic>Bam</italic>HI fragment in <italic>Csrp2 </italic>nulls (Mut <italic>Csrp2</italic>). Animals heterozygous for the disruption allele show both fragments in Southern blot analysis (cf. Fig. ##FIG##1##2B##).</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S6\"><caption><title>Additional File 6</title><p>Echocardiography in wildtype and <italic>Csrp2 </italic>nulls.</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><p>Abbreviation used: HPF, high-power field.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1471-213X-8-80-1\"/>", "<graphic xlink:href=\"1471-213X-8-80-2\"/>", "<graphic xlink:href=\"1471-213X-8-80-3\"/>", "<graphic xlink:href=\"1471-213X-8-80-4\"/>", "<graphic xlink:href=\"1471-213X-8-80-5\"/>", "<graphic xlink:href=\"1471-213X-8-80-6\"/>" ]
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[{"article-title": ["The Jackson Laboratory"]}, {"article-title": ["The European Mouse Mutant Archive"]}, {"article-title": ["The Jackson Laboratory"]}, {"article-title": ["The National Center for Biotechnology Information"]}]
{ "acronym": [], "definition": [] }
43
CC BY
no
2022-01-12 14:47:25
BMC Dev Biol. 2008 Aug 19; 8:80
oa_package/b1/3a/PMC2529283.tar.gz
PMC2529284
18706114
[ "<title>Introduction</title>", "<p>Premature cardiovascular disease is the leading cause of death in patients with end stage renal disease (ESRD) treated with dialysis. The relationship between cardiovascular (CV) risk factors and CV events is less clear in ESRD than in the general population, with paradoxical relationships between both cholesterol and blood pressure and CV risk[##REF##9690224##1##,##REF##10070915##2##]. The presence of increased arterial stiffness is a predictor of CV morbidity and mortality in ESRD[##REF##11566918##3##,##REF##10318666##4##]. Conventionally, this has been assessed by measurement of pulse wave velocity (PWV). More recently, cardiovascular magnetic resonance (CMR) has been used to identify tissue abnormalities in the heart of ESRD patients[##REF##16508657##5##]. This imaging modality also permits contemporaneous visualisation of large arteries and direct measurement of aortic function, providing an integrated assessment of both ventricular and vascular function in one examination. Aortic distensibilty (AD), the relative change in aortic size throughout the cardiac cycle relative to blood pressure, is readily assessed with CMR. Given the strong relationship between vascular stiffness and outcome in ESRD, CMR measures of vascular function may identify patients at increased CV risk. We have demonstrated in a previous pilot study using CMR that aortic stiffness is increased in patients with ESRD[##REF##17136027##6##]. More recently aortic stiffness has been demonstrated with CMR to be increased in patients with even mild chronic kidney disease (CKD), equivalent to findings in patients with heart failure with preserved systolic function[##UREF##0##7##].</p>", "<p>To date however, the relationship between aortic distensibilty (AD), as measured with CMR and long term outcome has not been assessed. Thus, we assessed the relationship between CMR measures of vascular function and conventional CV risk factors in ESRD patients and studied the long term prognostic implications of CMR measures of vascular function.</p>" ]
[ "<title>Methods</title>", "<title>Subjects</title>", "<p>Studies were performed on 144 patients with chronic kidney disease (CKD) stage 5[##REF##11904577##8##]. All patients were assessed as part of a CV screening program for renal transplantation and therefore were either on dialysis therapy or expected to start dialysis within six months. All patients gave written, informed consent and the study was approved by the local ethics committee.</p>", "<title>CMR technique</title>", "<p>CMR was performed using a 1.5 Tesla MR scanner (Sonata, Siemens, Erlangen, Germany). In haemodialysis patients imaging was performed on the post-dialysis day whereas peritoneal dialysis patients were studied at their \"dry weight\". Aortic volume was acquired from cine CMR images in the transverse plane of the ascending aorta, obtained at the level of the main pulmonary artery, using a steady-state free precession (true FISP) sequence, TR = 3.2 ms, TE = 1.6 ms, FA = 60°, FoV 276 × 340 mm, pixel dimensions 2.3 × 1.3 mm, slice thickness = 7 mm (Figure ##FIG##0##1##). The approximately 10 second breath-hold CMR resulted in images with a temporal resolution of 22.5 ms. During AD measurement brachial blood pressure was measured using an oscillometric device (Schiller Magscreen, Schiller AG, Baar, Switzerland).</p>", "<p>Left ventricular (LV) mass and function were measured using a true FISP sequence to acquire cine images in long axis planes (vertical long axis, horizontal long axis, LV outflow tract) followed by sequential short axis LV cine loops (8 mm slice thickness, 2 mm gap between slices) from the atrio-ventricular ring to the apex. LV mass index (LVMI) was calculated by normalising LV mass to the body surface area. Overall, scan time was approximately 30 minutes.</p>", "<p>All CMR analyses were performed using conventional analysis software (Argus, Siemens, Erlangen, Germany). Cross sectional aortic contours were defined by manual planimetry throughout the cardiac cycle, with resulting aortic volume defined as the measured cross sectional area multiplied by the slice thickness. This calculation was performed automatically by the analysis program.</p>", "<title>Measurement of aortic distensibilty and volumetric arterial strain</title>", "<p>Aortic distensibilty (AD) was calculated from change in aortic volume and simultaneous brachial blood pressure using the formula:</p>", "<p></p>", "<p>where (Aortic volume)<sub>max </sub>and (Aortic volume)<sub>min </sub>are the maximal and minimal calculated aortic volumes during the cardiac cycle. Additionally, aortic volumetric arterial strain (VAS), which is non-pressure dependent, was calculated from the formula:</p>", "<p></p>", "<p>As a ratio of change in aortic volume, VAS does not have any units.</p>", "<title>Clinical data</title>", "<p>All patients underwent conventional cardiovascular risk factor assessment including history, clinical examination, ECG as well as routine haematology, biochemical, and lipid profile. A history of ischaemic heart disease (IHD) was defined as previous clinically documented myocardial infarction/angina pectoris or previous coronary revascularisation procedure. Peripheral vascular disease was defined by clinically documented intermittent claudication with prior consultation by a vascular surgeon or the need for a previous peripheral vessel revascularisation procedure. Blood was drawn at the time of scanning for lipids and C – reactive protein, with other haematological and biochemical parameters (haemoglobin, dialysis adequacy, calcium-phosphate product) assessed from the mean of readings taken on the three consecutive months preceding the scan.</p>", "<title>Follow up</title>", "<p>Follow up data was collected from date of the CMR scan using electronic patient records. Death and cardiovascular events (myocardial infarction, cerebrovascular event, coronary revascularisation and amputation for peripheral vascular disease) were collected as end points.</p>", "<title>Statistical methods</title>", "<p>Correlations between cardiac dimensions, measures of arterial function and continuous clinical variables were assessed with Pearson and Spearman co-efficients. Differences between groups were tested by student's t-test and Mann-Whitney-U test. Measures of vascular function were compared between patients who died or had a vascular event during the follow up period with these measures divided into tertiles or quartiles and subjected to an unadjusted survival analysis by the Kaplan-Meier method. Statistical significance was determined by the log-rank test. Cox survival analysis was performed to assess the influence of multiple variables on outcome. Variables identified as possibly influential on outcome by univariate analysis were then entered into a forward stepwise regression model. As exponential values logarithmic transformation was used for AD and VAS values to simplify interpretation of values in the regression analyses. Analyses were performed using SPSS 13.0 software package (SPSS Inc., Chicago, IL., USA).</p>" ]
[ "<title>Results</title>", "<title>Subjects</title>", "<p>Patient demographics at the time of scan are shown in Table ##TAB##0##1##. 144 patients had aortic VAS measurement and 122 patients had measurement of AD available for analysis. 22 patients did not have blood pressure readings to calculate AD (either due to bilateral arteriovenous fistulae used as vascular access for dialysis making recording impossible, or due to blood pressure being recorded non-synchronously with the AD trace).</p>", "<title>Clinical correlates of aortic distensibilty and volumetric arterial strain</title>", "<p>All scans were analysed by a single observer (P.B.M). No significant differences in patient demographics or vascular function were exhibited between patients with advanced renal failure not yet on dialysis therapy (34 patients; 24.6%) and those established on dialysis (110 patients) and therefore patients were combined as a single group. AD and VAS demonstrated a negative correlation with age (AD R = -0.44, p &lt; 0.001, VAS R = -0.44, p &lt; 0.001).</p>", "<p>There were no significant correlations between haemoglobin, dialysis adequacy (urea reduction ratio in haemodialysis or creatinine clearance in peritoneal dialysis), time on dialysis, lipid parameters, C-reactive protein, calcium, phosphate or calcium phosphate product and AD or VAS. There were no significant differences in AD or VAS between genders. AD and VAS were reduced in patients with diabetes mellitus (diabetics – median AD 1.8 × 10<sup>-3 </sup>vs. non-diabetics 2.8 × 10<sup>-3 </sup>mmHg<sup>-1</sup>, p = 0.001; VAS 0.11 vs. 0.15, p = 0.001), patients with a history of ischaemic heart disease (median AD 1.8 × 10<sup>-3 </sup>vs. 2.6 × 10<sup>-3 </sup>mmHg<sup>-1</sup>, p = 0.028; VAS 0.11 vs. 0.15, p = 0.009) or peripheral vascular disease (median aortic distensibilty 1.2 × 10<sup>-3 </sup>vs. 1.8 × 10<sup>-3 </sup>mmHg<sup>-1</sup>, p = 0.005; VAS 0.14 vs. 0.07, p = 0.015). No significant differences in AD or VAS were demonstrated between patients treated with statins or requiring antihypertensive therapy. There were no significant differences between AD between dialysis modalities. 58 patients (40.3%) had a functioning arteriovenous fistula or graft at the time of scanning. There were no significant differences in AD or VAS between patients with an arteriovenous fistula or graft and those without.</p>", "<p>Weak but statistically significant negative correlations were demonstrated between AD and LVMI (R = -0.21, p = 0.021) and end systolic volume (R = -0.18, p = 0.048) but no other LV dimension. Aortic VAS correlated with markers of cardiac function – left ventricular ejection fraction (R = 0.23, p = 0.006) and stroke volume (0.19, p = 0.024).</p>", "<title>Relationship between CMR measures of vascular function on survival and combined mortality and cardiovascular events</title>", "<p>Follow up data were available for all 144 patients. Overall survival data was analysed for the whole cohort including analyses of the relationship between VAS and outcome. For analyses of the relationship between AD and outcome, only the 120 patients who only results of AD available were studied. The median follow up period was 719 days (interquartile range 375 days). There were 20 deaths, giving an overall mean death rate of 76.2 per 1000 patient years. Patients who died had significantly lower AD than those who where alive at the end of follow up. There were no significant differences in VAS between survivors and patients who died. Patients who died had significantly higher systolic and pulse pressure than survivors (Figures ##FIG##1##2##, ##FIG##2##3##), but there were no significant differences in diastolic blood pressure (Table ##TAB##1##2##).</p>", "<p>During the follow up period there were an additional 12 non-fatal CV events (five patients had a myocardial infarction; four patients underwent coronary revascularisation, two patients undergoing amputation for peripheral vascular disease and one patient had a cerebrovascular event). These patients had significantly lower AD and VAS than those who remained event free (median AD in patients with CV events 1.5 × 10<sup>-3 </sup>vs. 2.8 × 10<sup>-3 </sup>mmHg<sup>-1</sup>, p = 0.006; VAS 0.12 vs. 0.14, p = 0.012; Figure ##FIG##3##4##). There were no significant difference in blood pressure between patients who remained event free and patients who had a non fatal CV event.</p>", "<p>Combining death with non fatal CV events as a combined CV end point, patients who reached this combined end point had significantly lower AD (median AD 1.7 × 10<sup>-3 </sup>vs. 2.7 × 10<sup>-3 </sup>mmHg<sup>-1</sup>, p &lt; 0.001), VAS (0.12 vs. 0.14, p = 0.006) and higher systolic blood pressure (mean 152.9 vs. 136.5 mmHg, p = 0.001) and pulse pressure (mean 67.0 vs. 54.5 mmHg, p = 0.001) than those who did not. There were no significant differences in diastolic blood pressure (mean 85.9 vs. 82.0 mmHg, p = 0.154) between those who reached the combined end point and those who did not.</p>", "<p>In a Cox forward stepwise regression model assessing patient survival; diabetes, systolic blood pressure and AD were significant independent predictors of patient survival (Table ##TAB##2##3##). In a similar analysis assessing the combined CV end point of death or a vascular event; diabetes, AD and aortic VAS were significant predictors of events (Table ##TAB##3##4##). Due to their close interdependence, blood pressure variables and AD were entered individually into the each model to determine their influence. Similarly either AD or VAS was entered individually to the model but not together.</p>" ]
[ "<title>Discussion</title>", "<p>This is the first study to examine the relationship between AD assessed with CMR and outcome in ESRD. Patients with ESRD have previously been demonstrated to have increased aortic stiffness compared to controls, represented by reduced AD and VAS with associated vessel dilatation[##REF##17136027##6##]. In keeping with the relationship between arteriosclerosis and advancing age, there was a significant relationship between reduced AD and VAS and age. AD and VAS were both lower in patients at highest CV risk, i.e. those with diabetes, ischaemic heart disease and/or peripheral vascular disease. No other factors such as drug therapy, adequacy of dialysis, duration of renal replacement therapy, dialysis modality or any laboratory parameter appeared to be significantly associated with AD or VAS.</p>", "<p>One major potential limitation of our study is that calculation of AD is based on cross-sectional volume of the vessel wall and pulse pressure. For practical reasons, direct aortic blood pressure measurement has been substituted by non-invasive indirect brachial blood pressure. Brachial blood pressure is not entirely representative of central haemodynamics and our methods do not permit assessment of the phenomenon of central to peripheral systolic and pulse pressure amplification, as can be measured with tonometry. It is difficult to dissociate any clinical effect associated with changes in AD from that due directly to pulse pressure, which is a well described predictor of outcome in ESRD. Nonetheless, as it is otherwise impossible to assess aortic stiffness without documentation of pressure within the vessel lumen, the approach used in this study has been used widely[##UREF##0##7##,##REF##12450602##9##, ####REF##15611948##10##, ##REF##14740462##11####14740462##11##]. For this reason, aortic VAS, the fractional increase in aortic volume during the cardiac cycle was also used. This does not depend on blood pressure variables and in this study displayed similar relationships with clinical variables and outcome. However, there is limited literature to support its use. Other studies have demonstrated that PWV can be measured with CMR. There are advantages to using measuring PWV compared to AD as it is load-independent, negating the necessity for blood pressure measurements. However, at the time of initiation of this study, there were few studies in any patient group other than healthy volunteers[##REF##15611948##10##,##REF##11583892##12##], and unfortunately CMR derived PWV was not measured in this study cohort.</p>", "<p>The relationship between these markers of aortic function and LV dimensions show a weak but significant relationship between increased aortic stiffness and increasing LVMI and end systolic volume, suggesting that reduced AD may increase LV wall tension and hence cardiac hypertrophy. Aortic VAS, which is independent of blood pressure, correlated with markers of systolic function, namely ejection function and stroke volume, suggesting that in the failing heart where systolic blood pressure is lower, increased arterial stiffness remains deleterious to ventricular performance.</p>", "<p>A variety of studies have addressed the role of AD as a marker of either CV risk or relating AD to cardiac performance in other groups at high risk of CV disease. Data relating CMR measures of arterial function to long term outcome are scarce. In otherwise healthy individuals, obese subjects have been shown by CMR to have increased aortic cross sectional area and decreased aortic elasticity[##REF##12521634##13##,##REF##18275595##14##]. AD is reduced in patients with heart failure and correlates with exercise capacity[##REF##12450602##9##]. One study in hypertensive patients has demonstrated that AD increases following treatment with nicardipine or alacepril but not trichlormethiazide, independent of changes in pulse pressure[##REF##10516870##15##]. More recently, in patients with non diabetic CKD, AD has been shown to correlate with GFR demonstrating that CMR can quantify the impact of reduced kidney function on vascular function in early CKD[##UREF##0##7##]. Additionally, endothelial function can be studied using CMR to by assessing cross sectional flow mediated dilation of the brachial artery[##REF##15542292##16##].</p>", "<p>Independent predictors of mortality during the follow up period were diabetes, AD and systolic blood pressure. Due to their close interdependence, systolic blood pressure and aortic distensibilty could not be independently assessed. When a combined end point of death, non-fatal myocardial infarction, cardiac revascularisation, amputation for peripheral vascular disease and cerebrovascular event was used, only diabetes and AD or VAS were independent predictors of events. Surprisingly, age was not a predictor of outcome, suggesting that since age and AD were closely correlated, vascular, rather than temporal, aging is a more important determinant of survival. In keeping with other studies, time on renal replacement therapy was significantly longer in those patients who died[##REF##10318666##4##], but this was not independently associated with outcome. Therefore in this patient group, AD was a predictor of mortality and/or vascular events independent of age and dialysis vintage.</p>", "<p>Haemodynamic factors which promote arterial remodelling present in ESRD, although not specific to kidney disease, include age and blood pressure. Factors specific to ESRD include arteriovenous shunts and chronic volume overload[##REF##8840292##17##]. Increased blood flow through a shunt promotes arterial remodelling although may not lead to detectible changes in measures of vascular function[##REF##12324910##18##]. We could find no difference in AD or VAS in patients with an arteriovenous shunt and those without. As novel marker of increased CV risk, no data exist on the natural history of progression of AD, or whether it can be improved by therapeutic intervention. Our results suggest that as there was no significant difference between those CKD 5 patients on dialysis compared to pre-dialysis patients, the major derangement in vascular function occurs earlier during the progression of CKD. One assumes that reduced AD is a consequence of arterial calcification[##REF##12937218##19##], although we cannot confirm this as CMR is unable to display calcific lesions due to their absence of water content.</p>", "<p>A large number of studies have been performed to assess the determinants of arterial stiffness in ESRD, and its relation to long term survival, using either incremental elastic modulus, PWV or augmentation index as markers of arterial stiffness. In keeping with our results, factors associated with increased arterial stiffness include age, diabetes, and systolic blood pressure (or pulse pressure) [##REF##10067793##20##, ####REF##10844634##21##, ##REF##14669860##22####14669860##22##]. Other factors associated with arterial stiffness include serum calcium or the presence of inflammation[##REF##14669860##22##]. Surprisingly, these factors were not predictors of vascular function in our study. Arterial stiffness has been repeatedly demonstrated to be an independent predictor of all cause and cardiovascular mortality in haemodialysis patients[##REF##11566918##3##,##REF##10318666##4##,##REF##11897754##23##].</p>", "<p>Aside from the issue of use of brachial blood pressure to calculate central haemodynamics, our study has some other limitations. Although direct comparison between CMR and calcification is not possible, CMR has emerged as a technique for imaging atheroma. A direct comparison between vascular function and dark blood imaging looking at aortic wall thickness and atheroma would be of interest but unfortunately was not performed. Measurement of AD is still a relatively novel technique and its widespread reproducibility is yet to be established. In house blinded analysis has demonstrated an inter-observer variability of 4.6%, although this needs to be reproduced in other centres, particularly as there are potential sources of error in both data acquisition and analysis as well as in blood pressure measurements. As we have previously used CMR with late gadolinium enhancement (LGE) to analyse myocardial tissue composition in ESRD[##REF##16508657##5##], it would have been of interest to compare myocardial tissue abnormalities with vascular function. However, as a link between gadolinium and nephrogenic systemic fibrosis has emerged, this comparison is impossible[##REF##17704357##24##]. It is unfortunate that it was not possible to acquire brachial blood pressure on every patient due to some patients having multiple vascular procedures on both upper limbs. Finally, CMR technique is relatively expensive and time consuming to analyse. With future development of automated analysis software, faster analysis of AD will be possible.</p>" ]
[ "<title>Conclusion</title>", "<p>This study has demonstrated that vascular function can be measured using CMR in ESRD. Increased aortic stiffness, indicated by reduced AD and reduced VAS are associated with risk factors for CV disease. Both AD and VAS were independent predictors of combined vascular events and mortality. AD predicted all cause mortality. Therefore, CMR offers a novel non-invasive tool to assess vascular function in patients ESRD. AD and VAS are potential targets for therapeutic intervention to reduce cardiovascular risk in ESRD.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Increased arterial stiffness is associated with mortality in patients with chronic kidney disease. Cardiovascular magnetic resonance (CMR) permits assessment of the central arteries to measure aortic function.</p>", "<title>Methods</title>", "<p>We studied the relationship between central haemodynamics and outcome using CMR in 144 chronic kidney disease patients with estimated glomerular filtration rate &lt;15 ml/min (110 on dialysis). Aortic distensibilty and volumetric arterial strain were calculated from cross sectional aortic volume and pulse pressure measured during the scan.</p>", "<title>Results</title>", "<p>Median follow up after the scan was 24 months. There were no significant differences in aortic distensibilty or aortic volumetric arterial strain between pre-dialysis and dialysis patients. Aortic distensibilty and volumetric arterial strain negatively correlated with age. Aortic distensibilty and volumetric arterial strain were lower in diabetics, patients with ischaemic heart disease and peripheral vascular disease. During follow up there were 20 deaths. Patients who died had lower aortic distensibilty than survivors. In a survival analysis, diabetes, systolic blood pressure and aortic distensibilty were independent predictors of mortality. There were 12 non-fatal cardiovascular events during follow up. Analysing the combined end point of death or a vascular event, diabetes, aortic distensibilty and volumetric arterial strain were predictors of events.</p>", "<title>Conclusion</title>", "<p>Deranged vascular function measured with CMR correlates with cardiovascular risk factors and predicts outcome. CMR measures of vascular function are potential targets for interventions to reduce cardiovascular risk.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>Guarantor of integrity of entire study PBM, study concepts/study design PBM, AD, KGB, TS, JEF, HJD, AGJ data collection and analysis PBM, AD, KGB, RKP, RAPW, TS, JE, AGJ, statistical analysis PBM, RKP, AGJ, manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors.</p>" ]
[ "<title>Acknowledgements</title>", "<p>Patrick Mark was funded by a British Heart Foundation Junior Fellowship. The Glasgow Cardiac Magnetic Resonance Unit is supported by a British Heart Foundation grant. The study was additionally supported by Darlinda's Charity for Renal Research and the Chief Scientist's Office, Scotland.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Representative images demonstrating sagittal view (left) used to plan transverse plane aortic images (right) with schematic tracing of ascending aorta (AA).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Kaplan-Meier survival curves for all cause mortality with patients stratified by systolic blood pressure tertile</bold>.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Kaplan-Meier survival curves for all cause mortality with patients stratified by aortic distensibilty tertile.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>Kaplan-Meier survival curves for survival to either CV end point or death with patients stratified by aortic VAS quartile.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Background demographics of patients studied</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\">Number</td><td align=\"left\">144</td><td/></tr><tr><td align=\"left\">Age</td><td align=\"left\">51.5</td><td align=\"left\">(11.2)</td></tr><tr><td align=\"left\">Male (%)</td><td align=\"left\">90</td><td align=\"left\">(62.5)</td></tr><tr><td align=\"left\">Height (m)</td><td align=\"left\">169.5</td><td align=\"left\">(9.8)</td></tr><tr><td align=\"left\">Weight (kg)</td><td align=\"left\">75.9</td><td align=\"left\">(16.1)</td></tr><tr><td align=\"left\">On dialysis (%)</td><td align=\"left\">110</td><td align=\"left\">(76.4)</td></tr><tr><td align=\"left\"> Haemodialysis</td><td align=\"left\">61</td><td align=\"left\">(42.4)</td></tr><tr><td align=\"left\"> Peritoneal dialysis</td><td align=\"left\">49</td><td align=\"left\">(34.0)</td></tr><tr><td align=\"left\">RRT time (months)</td><td align=\"left\">6.0</td><td align=\"left\">(42.0)</td></tr><tr><td align=\"left\">Past history of IHD (%)</td><td align=\"left\">24</td><td align=\"left\">(16.7)</td></tr><tr><td align=\"left\">Diabetes (%)</td><td align=\"left\">46</td><td align=\"left\">(31.9)</td></tr><tr><td align=\"left\">Smoker (%)</td><td/><td/></tr><tr><td align=\"left\"> Never</td><td align=\"left\">83</td><td align=\"left\">(57.6)</td></tr><tr><td align=\"left\"> Current</td><td align=\"left\">38</td><td align=\"left\">(26.4)</td></tr><tr><td align=\"left\"> Ex</td><td align=\"left\">23</td><td align=\"left\">(16.0)</td></tr><tr><td align=\"left\">SBP (mmHg)</td><td align=\"left\">140.2</td><td align=\"left\">(24.2)</td></tr><tr><td align=\"left\">DBP (mmHg)</td><td align=\"left\">82.9</td><td align=\"left\">(12.8)</td></tr><tr><td align=\"left\">Cross sectional aortic volume (mL)</td><td align=\"left\">5.0</td><td align=\"left\">(2.0)</td></tr><tr><td align=\"left\">Aortic distensibilty (x 10<sup>-3 </sup>mmHg<sup>-1</sup>)</td><td align=\"left\">2.4</td><td align=\"left\">(2.0)</td></tr><tr><td align=\"left\">Aortic volumetric arterial strain</td><td align=\"left\">0.13</td><td align=\"left\">(0.09)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Demographic data for patients who were alive and dead at the end of the follow up period</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\"><bold>Alive</bold></td><td align=\"center\" colspan=\"2\"><bold>Dead</bold></td><td align=\"center\"><bold>p value</bold></td></tr></thead><tbody><tr><td align=\"left\">Number</td><td align=\"center\">124</td><td align=\"center\">(86.1)</td><td align=\"center\">20</td><td align=\"center\">(13.9)</td><td/></tr><tr><td align=\"left\">Age</td><td align=\"center\">51.5</td><td align=\"center\">(11.3)</td><td align=\"center\">51.9</td><td align=\"center\">(11.1)</td><td align=\"center\">0.876</td></tr><tr><td align=\"left\">Male (%)</td><td align=\"center\">80</td><td align=\"center\">(64.5)</td><td align=\"center\">10</td><td align=\"center\">(50.0)</td><td align=\"center\">0.213</td></tr><tr><td align=\"left\">On dialysis (%)</td><td align=\"center\">92</td><td align=\"center\">(74.2)</td><td align=\"center\">18</td><td align=\"center\">(90.0)</td><td align=\"center\">0.122</td></tr><tr><td align=\"left\">RRT time (months)</td><td align=\"center\">6.0</td><td align=\"center\">(26.0)</td><td align=\"center\">36.0</td><td align=\"center\">(107.0)</td><td align=\"center\">0.023</td></tr><tr><td align=\"left\">Past history of IHD (%)</td><td align=\"center\">19</td><td align=\"center\">(15.3)</td><td align=\"center\">5</td><td align=\"center\">(25.0)</td><td align=\"center\">0.281</td></tr><tr><td align=\"left\">Diabetes (%)</td><td align=\"center\">34</td><td align=\"center\">(27.4)</td><td align=\"center\">12</td><td align=\"center\">(60.0)</td><td align=\"center\">0.004</td></tr><tr><td align=\"left\">Smoker (%)</td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Never</td><td align=\"center\">73</td><td align=\"center\">(58.9)</td><td align=\"center\">10</td><td align=\"center\">(50.0)</td><td/></tr><tr><td align=\"left\"> Current</td><td align=\"center\">30</td><td align=\"center\">(24.2)</td><td align=\"center\">8</td><td align=\"center\">(40.0)</td><td align=\"center\">0.304</td></tr><tr><td align=\"left\"> Ex</td><td align=\"center\">21</td><td align=\"center\">(16.9)</td><td align=\"center\">2</td><td align=\"center\">(10.0)</td><td/></tr><tr><td align=\"left\">SBP (mmHg)</td><td align=\"center\">137.3</td><td align=\"center\">(23.6)</td><td align=\"center\">156.3</td><td align=\"center\">(21.4)</td><td align=\"center\">0.001</td></tr><tr><td align=\"left\">DBP (mmHg)</td><td align=\"center\">82.1</td><td align=\"center\">(12.9)</td><td align=\"center\">87.4</td><td align=\"center\">(11.7)</td><td align=\"center\">0.092</td></tr><tr><td align=\"left\">PP (mmHg)</td><td align=\"center\">55.20</td><td align=\"center\">(17.1)</td><td align=\"center\">69.0</td><td align=\"center\">(17.1)</td><td align=\"center\">0.001</td></tr><tr><td align=\"left\">Cross sectional aortic volume (mL)</td><td align=\"center\">5.0</td><td align=\"center\">(1.9)</td><td align=\"center\">5.1</td><td align=\"center\">(2.3)</td><td align=\"center\">0.634</td></tr><tr><td align=\"left\">Aortic distensibilty (x 10<sup>-3 </sup>mmHg<sup>-1</sup>)</td><td align=\"center\">2.4</td><td align=\"center\">(2.0)</td><td align=\"center\">2.1</td><td align=\"center\">(2.1)</td><td align=\"center\">0.009</td></tr><tr><td align=\"left\">Aortic volumetric arterial strain</td><td align=\"center\">0.12</td><td align=\"center\">(0.09)</td><td align=\"center\">0.15</td><td align=\"center\">(0.10)</td><td align=\"center\">0.176</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Patient survival</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"3\"><bold>Univariate analysis</bold></td><td align=\"center\" colspan=\"3\"><bold>Multivariate analysis</bold></td></tr><tr><td align=\"left\"><bold>Variable</bold></td><td align=\"center\"><bold>HR</bold></td><td align=\"center\"><bold>(95.0% CI)</bold></td><td align=\"center\"><bold>p</bold></td><td align=\"center\"><bold>HR</bold></td><td align=\"center\"><bold>(95.0% CI)</bold></td><td align=\"center\"><bold>p</bold></td></tr></thead><tbody><tr><td align=\"left\">Diabetes</td><td align=\"center\">3.031</td><td align=\"center\">(1.036, 8.866)</td><td align=\"center\">0.043</td><td align=\"center\">4.214</td><td align=\"center\">(1.631, 10.886)</td><td align=\"center\">0.003</td></tr><tr><td align=\"left\">SBP (mmHg)</td><td align=\"center\">1.015</td><td align=\"center\">(0.992, 1.038)</td><td align=\"center\">0.197</td><td align=\"center\">1.022</td><td align=\"center\">(1.000, 1.044)</td><td align=\"center\">0.049</td></tr><tr><td align=\"left\">Log aortic distensibilty</td><td align=\"center\">0.072</td><td align=\"center\">(0.008, 0.694)</td><td align=\"center\">0.023</td><td align=\"center\">0.135</td><td align=\"center\">(0.019, 0.948)</td><td align=\"center\">0.044</td></tr><tr><td align=\"left\">Log aortic VAS</td><td align=\"center\">0.107</td><td align=\"center\">(0.007, 1.732)</td><td align=\"center\">0.116</td><td/><td/><td/></tr><tr><td align=\"left\">Duration RRT (days)</td><td align=\"center\">1.000</td><td align=\"center\">(1.000, 1.000)</td><td align=\"center\">0.421</td><td/><td/><td/></tr><tr><td align=\"left\">Age (years)</td><td align=\"center\">1.015</td><td align=\"center\">(0.969, 1.063)</td><td align=\"center\">0.526</td><td/><td/><td/></tr><tr><td align=\"left\">Gender (ref male)</td><td align=\"center\">0.833</td><td align=\"center\">(0.312, 2.226)</td><td align=\"center\">0.716</td><td/><td/><td/></tr><tr><td align=\"left\">Haemoglobin (g/dL)</td><td align=\"center\">0.910</td><td align=\"center\">(0.670, 1.236)</td><td align=\"center\">0.547</td><td/><td/><td/></tr><tr><td align=\"left\">Albumin (g/L)</td><td align=\"center\">0.942</td><td align=\"center\">(0.839, 1.058)</td><td align=\"center\">0.311</td><td/><td/><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Combined CV event and death survival</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"3\"><bold>Univariate analysis</bold></td><td align=\"center\" colspan=\"3\"><bold>Multivariate analysis</bold></td></tr><tr><td align=\"left\"><bold>Variable</bold></td><td align=\"center\"><bold>HR</bold></td><td align=\"center\"><bold>(95.0% CI)</bold></td><td align=\"center\"><bold>p</bold></td><td align=\"center\"><bold>HR</bold></td><td align=\"center\"><bold>(95.0% CI)</bold></td><td align=\"center\"><bold>p</bold></td></tr></thead><tbody><tr><td align=\"left\">Diabetes</td><td align=\"center\">2.989</td><td align=\"center\">(1.276 7.004)</td><td align=\"center\">0.012</td><td align=\"center\">3.575</td><td align=\"center\">(1.669 7.655)</td><td align=\"center\">0.001</td></tr><tr><td align=\"left\">Log aortic distensibilty</td><td align=\"center\">0.052</td><td align=\"center\">(0.009 0.319)</td><td align=\"center\">0.001</td><td align=\"center\">0.066</td><td align=\"center\">(0.013 0.347)</td><td align=\"center\">0.001</td></tr><tr><td align=\"left\">Log aortic VAS</td><td align=\"center\">0.021</td><td align=\"center\">(0.002 0.206)</td><td align=\"center\">0.001</td><td align=\"center\">0.026</td><td align=\"center\">(0.004 0.175)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\">Duration RRT (days)</td><td align=\"center\">1.000</td><td align=\"center\">(1.000 1.000)</td><td align=\"center\">0.560</td><td/><td/><td/></tr><tr><td align=\"left\">SBP (mmHg)</td><td align=\"center\">1.014</td><td align=\"center\">(0.995 1.032)</td><td align=\"center\">0.142</td><td/><td/><td/></tr><tr><td align=\"left\">Age (years)</td><td align=\"center\">1.023</td><td align=\"center\">(0.984 1.062)</td><td align=\"center\">0.248</td><td/><td/><td/></tr><tr><td align=\"left\">Gender (ref male)</td><td align=\"center\">0.827</td><td align=\"center\">(0.368 1.860)</td><td align=\"center\">0.646</td><td/><td/><td/></tr><tr><td align=\"left\">Haemoglobin (g/dL)</td><td align=\"center\">0.958</td><td align=\"center\">(0.748 1.225)</td><td align=\"center\">0.731</td><td/><td/><td/></tr><tr><td align=\"left\">Albumin (g/L)</td><td align=\"center\">0.984</td><td align=\"center\">(0.896 1.080)</td><td align=\"center\">0.728</td><td/><td/><td/></tr></tbody></table></table-wrap>" ]
[ "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M1\" name=\"1532-429X-10-39-i1\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mtext>Aortic distensibilty</mml:mtext><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mo stretchy=\"false\">[</mml:mo><mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>Aortic volume</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>max</mml:mi><mml:mo>⁡</mml:mo></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>Aortic volume</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>min</mml:mi><mml:mo>⁡</mml:mo></mml:mrow></mml:msub><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">[</mml:mo><mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>Aortic volume</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>min</mml:mi><mml:mo>⁡</mml:mo></mml:mrow></mml:msub><mml:mo>∗</mml:mo><mml:mtext>pulse pressure</mml:mtext><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow></mml:mfrac></mml:mrow></mml:semantics></mml:math></disp-formula>", "<disp-formula><mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" id=\"M2\" name=\"1532-429X-10-39-i2\" overflow=\"scroll\"><mml:semantics><mml:mrow><mml:mtext>VAS</mml:mtext><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mo stretchy=\"false\">[</mml:mo><mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>Aortic volume</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>max</mml:mi><mml:mo>⁡</mml:mo></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>Aortic volume</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>min</mml:mi><mml:mo>⁡</mml:mo></mml:mrow></mml:msub><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">[</mml:mo><mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>Aortic volume</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>min</mml:mi><mml:mo>⁡</mml:mo></mml:mrow></mml:msub><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow></mml:mfrac></mml:mrow></mml:semantics></mml:math></disp-formula>" ]
[]
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[ "<table-wrap-foot><p>Results are show as mean with standard deviation in parenthesis or number with percentage in parenthesis as appropriate, except for RRT time and measures of vascular function which are displayed as median and inter quartile range.</p></table-wrap-foot>", "<table-wrap-foot><p>Results are show as mean with standard deviation in parenthesis or number with percentage in parenthesis as appropriate except for RRT time and measures of vascular function which are displayed as median and inter quartile range. Tests of significance are t-test and Chi-squared between groups, except for measures of vascular function where Mann-Whitney-U was used.</p></table-wrap-foot>", "<table-wrap-foot><p>Cox survival analysis for patient survival. Because of their close correlation SBP aortic distensibilty and aortic VAS were entered separately into the model. Results for other variables are shown with systolic blood pressure in the model</p></table-wrap-foot>", "<table-wrap-foot><p>Cox survival analysis for patient survival to either death or cardiovascular event. Because of their close correlation SBP aortic distensibilty and aortic VAS were entered separately into the model. Results for other variables are shown with systolic blood pressure in the model.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1532-429X-10-39-1\"/>", "<graphic xlink:href=\"1532-429X-10-39-2\"/>", "<graphic xlink:href=\"1532-429X-10-39-3\"/>", "<graphic xlink:href=\"1532-429X-10-39-4\"/>" ]
[]
[{"surname": ["Edwards", "Ferro", "Townend", "Steeds"], "given-names": ["NC", "CJ", "JN", "RP"], "article-title": ["Aortic Compliance and Arterial-Ventricular Coupling in Early Chronic Kidney Disease: a Pattern Resembling Heart Failure with Preserved Ejection Fraction"], "source": ["Heart"], "year": ["2008"], "lpage": [" in press"]}]
{ "acronym": [], "definition": [] }
24
CC BY
no
2022-01-12 14:47:25
J Cardiovasc Magn Reson. 2008 Aug 18; 10(1):39
oa_package/2a/9e/PMC2529284.tar.gz
PMC2529285
18652663
[ "<title>Background</title>", "<p>Cervical carcinoma is the second most common malignancy affecting women [##REF##8784885##1##]. Most cases are squamous cell carcinomas (SCCs), which develop from precursor squamous intraepithelial lesions (SILs). At present it is not possible to discriminate progressive from non progressive SIL, leading to over-treatment of large numbers of women, with attendant physical and psychological morbidity.</p>", "<p>Persistent infection by high risk human papillomavirus (HR-HPV) represents the most significant risk factor in development of cervical carcinoma [##REF##2171125##2##,##REF##11673826##3##], with HPV16 being the virus type most commonly seen in SCC. In cervical malignancy HPV is usually integrated into the host genome. Integration is characterised by retention of the viral oncogenes E6 and E7, and by disruption or loss of expression of the viral transcriptional repressor E2, leading to deregulated production of E6 and E7 [##REF##15479788##4##].</p>", "<p>Various PCR based assays have been devised to measure HPV copy number and physical state in cell lines and clinical samples [##REF##16672403##5##, ####REF##17109343##6##, ##REF##17568661##7##, ##REF##15297527##8##, ##REF##17060938##9##, ##REF##12951209##10##, ##REF##14625048##11##, ##REF##16681753##12##, ##REF##11880410##13####11880410##13##], with a view to deriving clinically or biologically useful information. Viral load is often determined from levels of genes present in episomes and integrants (i.e. E6 or E7), while physical state is inferred from the E2/E6 (or E2/E7) ratio. The latter approach is based on the notion that episome only infections would harbour an equivalent copy number of E2 and E6, giving an E2/E6 ratio of 1, while integrated virus with loss of E2 would give an E2/E6 ratio of 0, and a mixed episomal and integrant infections would give ratios between 0 and 1.</p>", "<p>Both TaqMan and SYBR green strategies have been developed for HPV gene PCR, with the latter approach offering advantages of simplicity and low cost. Existing methods adjust for sample cellularity, generally determining HPV gene copy numbers per unit mass of genomic DNA (gDNA), per copy of an independent host calibrator gene, or with reference to a cell line [##REF##16672403##5##, ####REF##17109343##6##, ##REF##17568661##7##, ##REF##15297527##8####15297527##8##,##REF##14625048##11##, ####REF##16681753##12##, ##REF##11880410##13##, ##REF##16386784##14####16386784##14##]. These approaches are potentially liable to error, for example through inaccuracies in DNA concentration measurement, or from errors in handling and pipetting template DNAs for the construction of separate calibration curves. Small inaccuracies in quantification or dispensing of template DNA can translate to large over- or under-estimates of viral content, on account of the large dynamic range and sensitivity of real time PCR.</p>", "<p>Despite these concerns, and the potential use of quantitative PCR (qPCR) data in clinical decision-making, remarkably few previous studies have undertaken detailed technical evaluation of the performance of PCR methods for assessing HPV load and physical state. The existing reports have typically been carried out using DNA mixtures or cell lines, but not both, and have only rarely been performed to a good standard [##REF##12951209##10##,##REF##14625048##11##]. Where quantitative data is available it generally suggests substantial inaccuracy. One study showed a ten fold difference in the absolute values for HPV quantification between two laboratories (attributable to differences in the calibration standards used) [##REF##12951209##10##], while another determined a viral load in the cervical SCC cell line SiHa of 37 copies per cell [##REF##11880410##13##], which is far in excess of the approximately three copies actually present [##REF##3029430##15##,##REF##2990217##16##].</p>", "<p>As the literature contains no adequate evaluation of the errors inherent in SYBR green qPCR analysis of HPV gene copy number, the present study sought to rectify this omission. We undertook detailed assessment of a modified SYBR green based qPCR strategy for absolute quantification of the copy number of HPV16 E2 and E6 genes, compared to a host diploid genome. We produced a single clone, NA6, containing the amplicons HPV16 E2, HPV16 E6 and host gene hydroxymethylbilane synthase (HMBS; located at 11q23.3) in a 1:1:1 ratio, for use in generating external calibration curves. In principle, this system may offer advantages of simplicity and comparability. Firstly, all external calibration curves are generated from one template dilution series, rather than several series required with independent calibration constructs (for example, three would be required for measurement of E2, E6 and HMBS), thereby reducing the amount of manual handling needed. Secondly, the amount of host gDNA in each test sample can be quantified directly with reference to the HMBS standard curve, rather than relying on an estimation of gDNA concentration by spectrophotometry. Thirdly, a standardised calibration construct such as NA6 may reduce inter-laboratory variation in HPV gene quantification.</p>", "<p>We have undertaken rigorous evaluation of the performance of HPV16 SYBR green qPCR and present data documenting the potential sources of error, even in the 'best case scenario' of cell line analysis. We first used mixtures of HPV16 DNA and host gDNA, then cervical keratinocyte cell lines in which the HPV16 copy number and physical state were determined by Southern blot and densitometry, and finally HPV16-positive SCC tissue samples. We demonstrate the error range that should be anticipated when quantifying HR-HPV gene copy number by SYBR green qPCR. We also show that E2/E6 ratios are of limited use in assessing HR-HPV physical state, being of value only for identifying the subset of integrant-containing cells in which the E2/E6 ratio is near zero.</p>" ]
[ "<title>Methods</title>", "<title>Cell lines</title>", "<p>The HPV16 positive cervical SCC cell lines SiHa, and CaSki and the HPV negative cervical SCC line C33A (all at high passage) were obtained from the American Type Culture Collection (ATCC). We also used early and late passages from one long-term culture series of the W12 cell line (W12 Series1; W12.Ser1). W12 was established from a cervical low grade SIL and in long term culture recapitulates cervical neoplastic progression genetically and phenotypically [##REF##16505361##17##, ####REF##14973079##18##, ##REF##12460913##19##, ##REF##2173795##20####2173795##20##]. All cell lines were propagated in monolayer culture, as described previously [##REF##14973079##18##,##REF##17516585##21##] or by the ATCC.</p>", "<title>Genomic DNA extraction</title>", "<p>Genomic DNA (gDNA) was prepared from pelleted cells by overnight digestion with proteinase K, brief phenol chloroform extraction, ethanol precipitation, and removal of contaminating RNA by RNAse A. The purified gDNA was quantified by spectrophotometry.</p>", "<title>Cloning of E2, E6 and HMBS amplicons into NA6</title>", "<p>E2, E6 and HMBS amplicons were cloned separately, following PCR amplification, into pcr<sup>®</sup>2.1 vector using the original TA cloning system (Invitrogen). Briefly, fragments of the E2 hinge region (81 bp), and E6 gene (80 bp) were amplified from the pSP64-HPV16 construct (which contains the 7.9 kb full length HPV16 genome [##REF##2173795##20##]), while a section of HMBS (118 bp) was amplified from peripheral blood lymphocyte gDNA. PCR was carried out on an Applied Biosystems 9700 using the AmpliTaq Gold system (Perkin Elmer, UK) and comprised 1 × Taq Buffer, 1.5 mM MgCl2, 200 μM dNTPs, 300 μM Primer Pairs, and 2 U of Taq polymerase. PCR primers and cycling conditions are listed in Table ##TAB##0##1##.</p>", "<p>In order to construct a single clone containing each amplicon in a 1:1:1 ratio, the following cloning strategy was adopted. The E2 and E6 clones were linearised with XbaI, and the products ligated with T4 DNA ligase. Using the ligated product as template, PCR with E2 forward and E6 reverse primers generated a single E2-E6 product for subsequent TA cloning. Following selection, a single E2-E6 clone and a separate HMBS clone were digested with HindIII, and similarly ligated together. PCR with E2 forward and HMBS reverse primers generated a single product that following TA cloning, selection and sequencing, was shown to contain a single copy of the E2, E6 and HMBS amplicons (Figure ##FIG##0##1##). The final clone, named NA6, is 4,382 bp and has a molecular weight of 2.892 × 10<sup>6 </sup>Da.</p>", "<title>Preparation of NA6 calibration curves</title>", "<p>Stock NA6 was prepared at a concentration of 100 μg/ml. Ten fold serial dilutions were undertaken to produce a titration series representing, 1 ng/μl, 100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, 10 fg/μl and 1 fg/μl of NA6. From this, calibration curves for E2, E6 and HMBS were produced in quadruplicate. For each of the four qPCR runs, each dilution point was assayed in triplicate. qPCR was undertaken using 2 μl of template DNA in a 25 μl PCR. For the most concentrated point within the dilution series (1 ng/μl), 2 ng of NA6 equates to 4.176 × 10<sup>8 </sup>copies of each amplicon. For the most dilute point, 2 fg equates to 4.176 × 10<sup>2 </sup>copies. Additional file ##SUPPL##0##1##, 'NA6 Standard Curves' worksheet, presents the raw data used in calibration curve construction.</p>", "<title>SYBR Green qPCR</title>", "<p>All qPCR was carried out using an Opticon I thermal cycler (MJ Research) with SYBR Green JumpStart qPCR Kits (Sigma, UK). Reactions comprised 1× SYBR Green mix, 500 nM primer pairs, and 2 μl of template. In initial optimisation experiments we investigated the effects of primer concentration on crossing point (Cp) determination. We found that a primer concentration of 500 nM was optimal, and that a reduction to 300 nM resulted in PCR artefacts at low template concentrations. It was found that a template volume of 2 μl could be dispensed more accurately and reproducibly than 1 μl volumes (data not shown).</p>", "<p>The following cycling conditions were employed: initial denaturation 94°C 2 min, followed by 40 cycles of 94°C 20 seconds, 58°C 20 seconds, 72°C 20 seconds, 76°C 15 seconds, plate read. A final extension of 72°C 10 minutes, and melting curve of 65°C to 90°C, 1°C/second transition were incorporated. Opticon raw data was exported to Microsoft Excel for analysis.</p>", "<title>Data analysis</title>", "<p>Absolute quantification strategies require that the fluorescence threshold used to derive the calibration curve is also applied to the sample data. Hence it may be anticipated that little effect would be experienced by varying the fluorescence threshold (Ft), provided that the Ft passes through the centre of the log transformed reaction curve data. However, we found that gross changes in absolute quantification were seen for relatively small changes in Ft following theoretical evaluations (data not shown). To avoid issues related to placement of Ft values, an automated derivation of optimum Ft assignment was implemented. Fluorescence thresholds were calculated for each of the four calibration curves according to the cycle before second derivative maxima method, and then averaged during the process of producing the calibration curve. The E2, E6 and HMBS Ft values were then applied to all sample data in order to calculate crossing points.</p>", "<p>Calibration curves for E2, E6 and HMBS were constructed by plotting crossing points (Cp) versus the log of template copy number. For copy number determination in test samples, the fluorescence threshold (Ft), primer efficiency (E) and numbers of molecules at fluorescence threshold (Nt) were taken to be constants and were determined as the means of the four standard curve replicates for each amplicon. Hence, for an unknown sample, number of copies is given by equation 1.</p>", "<p></p>", "<p>Calibration curve primer efficiencies were determined by equation 2, and numbers of molecules at threshold by equation 3.</p>", "<p></p>", "<p></p>", "<p>The coefficient of variation between data obtained in replicate calibration curves represented the ratios of standard deviations over the mean, multiplied by 100%.</p>", "<p>Gene copy numbers in test samples were obtained by comparing the Cp value to those in the relevant external calibration curve. A Microsoft Excel template was prepared to calculate viral loads, and E2/E6 ratios (see Additional file ##SUPPL##0##1##). For all samples, viral load per diploid genome was determined by dividing E2 and E6 copy numbers by half the HMBS copy number. In addition, for the cell lines, the viral load per cell was derived using the known ploidy status.</p>", "<title>Southern hybridisation</title>", "<p>In order to validate the qPCR method, comparisons were made with HPV16 copy numbers determined in cervical keratinocyte cell lines by Southern blotting. 5 μg of gDNA was digested at 37°C overnight with either BamH1, PstI or HindIII, and electrophoresed on a 0.8% agarose gel. Agarose gels were depurinated in 0.25 M HCl, denatured in 0.5 M NaOH/1.5 M NaCl and neutralised in 0.5 M tris HCl/3 M NaCl before transfer to Hybond N membrane. UV cross-linked membranes were prehybridised in 20 ml of hybridisation buffer for 4 hrs at 65°C prior to introduction of <sup>32</sup>P-dCTP labelled probe. Probes were generated from the previously described E2 and E6 amplicons (81 and 80 bp respectively) cloned into pcr2.1 TA cloning vector, or from full length HPV16 (7.9 kbp) excised from pSP64-HPV16 using BamH1, and labelled using RediPrime labelling kit. Hybridisation was for 16 hr, followed by stringency washing of membranes in 2 × SSC/0.1% SDS and 0.1 × SSC/0.1% SDS twice for 15 minutes, and then autoradiography.</p>", "<title>Clinical samples</title>", "<p>Cervical squamous cell carcinoma samples were kindly provided by Dr Geetashree Mukherjee from the archives of the Kidwai Memorial Institute of Oncology, Bangalore, India. All tissue samples were obtained with informed consent, anonymised, and used with approval from the Kidwai Local Research Ethics Committee (reference: PER/CAB-I/D-I/13/01).</p>", "<p>gDNA was prepared from 14 snap frozen HPV16-positive cervical SCC samples, as described elsewhere [##REF##17516585##21##]. The mean total yields were insufficient for Southern blotting to be performed to provide comparative data (data not shown). Additional file ##SUPPL##0##1##, 'Assessment of Clinical Samples' worksheet, presents the raw data from the qPCR analysis of the clinical sample viral load and E2/E6 ratios.</p>" ]
[ "<title>Results</title>", "<title>Validation of NA6 standard curves using mixtures of virus and host DNA</title>", "<p>In generating the NA6 construct and preparing the calibration curves (see Materials and Methods), PCR primers were chosen to give amplicons of similar length (Table ##TAB##0##1##). The E2 primers, amplifying bp 3,361–3,442 of the HPV16 genome (GenBank accession <ext-link ext-link-type=\"gen\" xlink:href=\"AF125673\">AF125673</ext-link>), were located in the part of the E2 open reading frame (ORF) that is most often deleted on HPV16 integration [##REF##16672403##5##]. The E6 primers were located in the E6 ORF (bp 94–174), while the HMBS primers spanned an exon-intron border. After cloning, the 1:1:1 relationship of the E2, E6 and HMBS amplicons in NA6 were confirmed by sequence analysis (Figure ##FIG##0##1##).</p>", "<p>The SYBR green qPCR method used was developed following numerous optimisation experiments to determine ideal primer concentration, template gDNA concentration and cycling parameters (see Materials and Methods). We analysed melting curves of reaction products to verify specificity of primer binding and thereby circumvent any issues of non-specific SYBR green fluorescence. Following four replicate qPCR runs, seven point external calibration curves were generated for E2, E6 and HMBS. Figure ##FIG##1##2A – C## shows representative qPCR data used to generate standard curves for E2 (Fig ##FIG##1##2A##; from replicate 1 of 4), E6 (Fig ##FIG##1##2B##; from replicate 2 of 4), and HMBS (Fig ##FIG##1##2C##; from replicate 3 of 4). Quantification of each gene was linear over six logs, with no evidence to suggest competition between the PCR targets in the NA6 construct.</p>", "<p>Tight correlations were observed between the values obtained for each amplicon in the replicate qPCR runs, as illustrated for the E2 amplicon in Figure ##FIG##1##2D## (R<sup>2 </sup>= 0.996). For each amplicon, the mean Cp for each titration point 1 ng/μl to 1 fg/μl was plotted to generate the final calibration curves, which are shown in Figure ##FIG##1##2E##, together with their respective line equations and correlation values.</p>", "<p>Despite the apparent concordance between replicate runs, when we calculated viral gene copy number using calibration curves generated from individual qPCR runs, we observed inter-assay differences in the copy number values generated. Each of the four replicate calibration curves (Replicate 1 to Replicate 4) for E2, E6 and HMBS amplicons, as well as the final mean calibration curve derived from the quadruplicate runs, were used to calculate gene copy number according to a series of theoretical crossing points (Cp; the cycle number where the fluorescence threshold was crossed), ranging from a Cp of 10 to 30 (Table ##TAB##1##2##). The inter-run coefficients of variation in quantification of gene copy number at each crossing point ranged from approximately 4% to 31% (see penultimate row of each section of Table ##TAB##1##2##), which is in agreement with previously published findings [##REF##16672403##5##,##REF##12951209##10##,##REF##14625048##11##]. There was no apparent trend according to crossing point, with as much variation at low Cp values as at high Cp values (Table ##TAB##1##2##), indicating that, in contrast to other methods [##REF##12951209##10##,##REF##14625048##11##], template concentration did not affect inter-assay variation. Nevertheless, this data indicates that there is unavoidable run to run variation in HPV qPCR, reflecting for example, the consequences of repeated manual handling in replicate runs. We therefore suggest that the generation of a reliable standard curve for HPV gene quantification requires averaging of replicate calibration runs, rather than depending on a single run.</p>", "<p>We next assessed the ability of our absolute qPCR strategy accurately to quantify viral E2 and E6 copy number at a broad range of concentrations within a background of human DNA. The raw data from this work is presented in Additional file ##SUPPL##0##1## 'Accuracy Test' worksheet. Test DNA stock was generated by mixing 50 ng of human gDNA with 10 pg of the plasmid pSP64-HPV16 (which contains full-length HPV16 [##REF##2173795##20##]) per microliter. The mixture was then used to produce a three point serial dilution (Table ##TAB##2##3##). The starting population theoretically represented 760,000 E2 and E6 amplicons, and 28,571 HMBS amplicons per 2 μl of NA6 template (the volume of template loaded in each PCR reaction), which equated to a load of 53 HPV16 copies per diploid genome.</p>", "<p>DNA concentrations of each titration point were determined by spectrophotometry, measuring each sample three times. The mean values so determined were 51 ng/μl, 11 ng/μl and 3 ng/μl (Table ##TAB##2##3##) and these were used to calculate the anticipated amounts of the amplicons of E2, E6 and HMBS in the 2 μl of template assayed, for comparison with the values determined by qPCR.</p>", "<p>Table ##TAB##2##3## shows the mean data for each test sample (obtained from three replicate qPCR runs), compared with the predicted amounts of amplicon. Moderate variation (17.5–39.7% for E2 and 5.2–18.9% for E6) was seen at each concentration point; with greatest overall error at the lowest test sample concentration (3 ng/μl of gDNA; representing about 1,600 diploid cells per 2 μl of sample), where the limits of accuracy of qPCR at low template concentrations had presumably been exceeded.</p>", "<p>When we determined viral gene copy number per diploid genome, we obtained figures of approximately 25 E6 copies per diploid genome in the 51 ng/μl sample; 46 E6 copies per diploid genome in the 11 ng/μl sample; and 78 E6 copies per diploid genome in the 3 ng/μl sample, compared to the anticipated 53 copies per diploid genome (Table ##TAB##2##3##). These figures represent errors of 53%, 13%, and 47% respectively. The error is not surprising given the run to run variation we observed in the qPCR optimisation reactions, plus the fact that the experiments we performed required serial dilution of test samples and measurement of DNA concentrations by spectrophotometry, of which represent potential sources of error. This error in absolute quantification of HPV gene copy number by qPCR should be anticipated in future studies.</p>", "<title>Estimation of viral load and physical status in HPV16 infected cell lines</title>", "<p>We next assessed the performance of NA6-based absolute SYBR green qPCR in cervical keratinocyte cell lines in which the copy number and physical state of HPV16 were determined using Southern blotting and densitometry. Raw data from this assessment is presented in Additional file ##SUPPL##0##1## 'Assessment of Cell Lines' worksheet.</p>", "<p>We undertook Southern hybridisation of BamH1 and HindIII digested gDNA from the cell lines C33A, W12.Ser1p16, W12.Ser1p57, CaSki, and SiHa (Figure ##FIG##2##3##, panels A-C). We used three different probes: full length HPV16 (7.9 Kbp), and the previously cloned E2 and E6 amplicons (81 and 80 bp respectively). For each probe, target sequence load was determined by autoradiographic comparison with copy number controls generated from full length HPV16, excised from the pSP64-HPV16 construct, and used at a range of 500 to 1 copy per 5 μg of host gDNA (right-hand lanes in Figure ##FIG##2##3A##). Viral loads were determined per cell, taking ploidy into account. The data was compared with viral loads determined by qPCR assessment of HPV16 E2 or E6 amplicon copies per diploid genome (Table ##TAB##3##4##). The HPV negative cervical SCC cell line C33A showed no signal by Southern blot and the viral gene copy number values determined by qPCR were also zero.</p>", "<p>Using full length HPV16 as probe, W12.Ser1p16 (known to be diploid [##REF##14973079##18##]) was found to contain approximately 100 HPV16 episomes per cell, with no detectable integrants. qPCR indicated 143 E6 copies per diploid genome and 148 E2 copies per diploid genome. This represented a viral load of 143 copies per cell. The mean E2/E6 ratio determined from the absolute qPCR data was 1.04, as would be predicted in cells containing episomes only, although the range in triplicate qPCR runs was 0.76 to 1.32.</p>", "<p>By Southern blotting using full length HPV16, W12.Ser1p57 (known to be near tetraploid [##REF##14973079##18##]) was episome free and only a faint signal indicative of a single viral integrant was seen after 72 hrs exposure (Figure ##FIG##2##3A##). There were no detectable signals using the E2 or E6 amplicons as probe, even after 8 days of exposure, reflecting the predictably limited sensitivity of Southern blotting using 80 or 81 bp probes. By qPCR we observed a relatively wide range of copy number values in W12.Ser1p57, consistent with increased error at low sample concentrations (see Table ##TAB##2##3##). The mean copy number values determined from the absolute quantification data were 0.2 E2 copies and 0.3 E6 copies per diploid genome, indicating a viral load of approximately one copy per tetraploid cell (after rounding up). The mean E2/E6 ratio determined from the qPCR data was 0.72, with a range of 0.57 to 3.82. The presence of amplifiable E2 in W12.Ser1p57 is not surprising, as we previously determined retention of the hinge region in the HPV16 integrant in these cells [##REF##14973079##18##].</p>", "<p>SiHa cervical SCC cells (which are near triploid [##REF##7887426##22##]) were shown by Southern blotting using full length HPV16 to be episome free and to contain approximately 3 integrants per cell, in keeping with previous reports [##REF##2990217##16##,##REF##10423141##23##]. Southern hybridisation using the E6 probe also suggested a viral load of approximately 3 copies per cell, whereas the E2 probe did not detect a signal. By qPCR SiHa was calculated to contain 3.4 copies of E6 per diploid genome, indicating a viral load of 5 copies per triploid cell (after rounding down). By qPCR, SiHa was also calculated to contain 0.7 copies of E2 per diploid genome, indicating error at the limits of qPCR performance, as the E2 hinge region is not retained in SiHa [##REF##3029430##15##]. The mean E2/E6 ratio for SiHa was 0.20, with a range of 0.11 to 0.24.</p>", "<p>CaSki cervical SCC cells (which are near tetraploid [##REF##7887426##22##]) were shown by Southern blotting using full length HPV16 probe to be episome free and to contain more than 1,000 integrated copies per cell. By qPCR, CaSki was determined to carry approximately 2,272 E2 copies, and 869 E6 copies per diploid genome, the latter equating to a viral load of 1,738 E6 copies per tetraploid cell. The mean E2/E6 ratio for CaSki was 2.61, with a range of 1.37 to 3.46. Southern hybridisation using the E2 and E6 probes confirmed the presence of greater copy numbers of E2 than E6 in CaSki (Figure ##FIG##2##3##; compare panels B and C), and 1-dimensional analysis of the autoradiogram gave an E2/E6 ratio of approximately 2.</p>", "<p>The data from the detailed cell line analysis confirms that errors in SYBR green qPCR-based HPV16 gene quantification should be allowed for, especially at low copy number. Nevertheless, the viral loads determined from mean E6 copy numbers were reasonably close to the values shown by Southern blotting and can be considered to provide a useable indication of actual loads. The E2/E6 ratios also showed considerable variation, which again was greatest at low copy number. Moreover, when taking mean ratio values, our data shows that while integrated HPV16 in the absence of episomes may produce a low E2/E6 ratio, the presence of a high E2/E6 ratio, even one greater than 1.0, does not exclude the presence of integrated HPV16 only.</p>", "<title>Estimation of viral load and physical state of HPV16 in cervical clinical samples</title>", "<p>The SYBR green qPCR method was also used to assess copy number of HPV16 genes in frozen tissue samples from fourteen HPV16-positive cervical SCCs (Table ##TAB##4##5##). Viral loads were determined as E6 copy number per diploid genome, and could broadly be classified into three levels; low (up to 50 viral copies per diploid genome); medium (approximately 300 to 700 viral copies per diploid genome); and high (above 2000 viral copies per diploid genome, seen in one sample only).</p>", "<p>E2/E6 ratios were also determined for the SCCs. In three samples (n55, G9, G31), E2/E6 ratios were 0.06 or less, indicative of fully integrated HPV16 only (Table ##TAB##4##5##, '*' marked samples). In a fourth sample (G1) the ratio was 0.21, while in ten other samples it ranged from 0.38–1.55 (Table ##TAB##4##5##, right hand column). Such values have previously been considered to indicate the presence of episomes, with or without coexistence of integrants [##REF##16672403##5##,##REF##14625048##11##,##REF##11880406##24##]. However, in view of our findings with cell lines, we concluded that these values may also represent integrated HPV16 only and that, consequently, no information regarding HPV16 physical state could be drawn with confidence from the E2/E6 values for these eleven samples.</p>" ]
[ "<title>Discussion</title>", "<p>HPV load and physical status have been claimed to be useful parameters for clinical evaluation of cervical squamous cell neoplasia [##REF##17568661##7##,##REF##16483642##25##, ####REF##17607770##26##, ##REF##14984955##27##, ##REF##15386375##28##, ##REF##16214397##29####16214397##29##]. Various PCR methodologies are described for estimation of HPV load, type and physical state [##REF##15297527##8##,##REF##12951209##10##,##REF##16145162##30##, ####REF##12576600##31##, ##REF##12843067##32##, ##UREF##0##33##, ##REF##12873165##34##, ##REF##17229868##35####17229868##35##]. Most of these techniques rely on the use of cloned full length HPV16 in order to produce a calibration curve for absolute quantification, with viral loads generally presented in terms of copies per unit mass of gDNA. However, thorough critical testing of these methods, using approaches similar to those in the present study, has been reported only rarely, and not at all for SYBR green based strategies.</p>", "<p>Quantification of E2 and E6 relative to gDNA mass is subject to sources of potential error, including the need to quantify gDNA by spectrophotometry. Indeed it appears to have generated an error in the assessment of viral load in SiHa, which was determined in one study to be 172,991 copies of E6 in 50 ng of gDNA [##REF##11880410##13##]. 50 ng of gDNA is equivalent to 4,630 triploid cells, giving a viral load in SiHa of 37 copies per cell, a substantial overestimate of the copy number demonstrated by Southern blotting. Such error may be reduced by using a calibration curve generated from a single clone, such as NA6, containing E2, E6 and HMBS amplicons in a 1:1:1 ratio. The NA6 standard curve enables the number of diploid genomes in a sample to be determined from the HMBS copy number. Although we have not performed direct comparisons, this approach should offer some advantages over alternative standardisation methods, as it negates the need to determine each sample concentration accurately, or to have knowledge of ploidy, and provides a reference point for comparison of unrelated specimens and PCR runs.</p>", "<p>We undertook rigorous testing of the performance of SYBR green absolute qPCR in determining HR-HPV gene copy number. In experiments using virus host DNA mixtures we observed substantial run to run variation in PCR data and identified potential sources of error in copy number determination. Firstly, we observed that using an individual calibration curve produced variation in gene quantification of up to 31%, emphasizing the need to derive a standard curve from multiple replicate qPCR runs. Four such runs were used in our study.</p>", "<p>Secondly, when we investigated the ability of SYBR green qPCR to derive gene copy numbers from test gDNA in which known amounts of HPV16 DNA had been added, we found moderate variation in quantification of individual E2 and E6 amplicons. This is consistent with the inherent potential for error in the procedures that we undertook in the relevant experiments, including estimating DNA concentration by spectrophotometry and serially diluting test samples. Estimates of viral load that were internally normalised to HMBS copy number showed error rates of 13–53%, a limitation that should be kept in mind when performing qPCR work.</p>", "<p>In contrast to other studies in the literature, we also critically evaluated the performance of our SYBR green qPCR method in quantifying HPV16 gene copy number in a range of cervical keratinocyte cell lines. We observed reasonable agreement between viral loads determined by qPCR and Southern blotting, with some overestimation of low viral copy number by qPCR. The SYBR green method that we used determined an HPV16 copy number in SiHa of 5 copies per cell, which is an order of magnitude more accurate than a study that used gDNA levels quantified by spectrophotometry [##REF##11880410##13##].</p>", "<p>Our cell line data therefore suggests that our estimates of viral load (ranging from 2 to 2,000 copies per diploid genome) in a set of fourteen HPV16 positive cervical SCC clinical samples are likely to be broadly accurate, albeit subject to the errors that we demonstrated using HPV16/gDNA mixtures. Taken together, our detailed evaluation supports the use of SYBR green HPV16 qPCR in studies attempting to correlate viral copy number with clinical outcome using tissue specimens, at least where the degree of sampling of abnormal tissue is known.</p>", "<p>We observed a wide range of E2/E6 ratios in cell lines and clinical samples. In integrant only SiHa cells the E2/E6 ratio was substantially greater than 0, at 0.2 (0.11–0.24), while in integrant only CaSki cells it was 2.61 (1.37–3.46), as a result of multiple integrants with greater representation of E2 than E6. These values (and their ranges) are attributable to the errors implicit in HPV gene quantification by qPCR, as well as to the retention of E2 sequences in some integrants. The latter may be full-length E2, as in CaSki, or alternatively fragments of E2 retaining the hinge region that is amplified by the most frequently used gene quantification primers (including those in the present study). While the hinge is the region of E2 that is most commonly deleted in HPV16 integration, it may also be retained; as is the case in the integrant in W12.Ser1p57 [##REF##14973079##18##]. It should be noted that an excess of E2 copy number over E6 copy number is likely to be rare in cervical SCCs and only encountered in cells with high level genomic instability, such as CaSki. On the other hand, W12.Ser1p57 appears to be more representative of cells in vivo, with a low copy number of integrated HPV16. In W12.Ser1p57 we observed a mean E2/E6 ratio of 0.72, with a range of 0.57 to 3.82.</p>", "<p>The E2/E6 ratios in episome-containing W12Ser1p16 cells ranged from 0.76–1.32 in triplicate assays. Nagao et al observed E2/E6 ratios of 0.61–1.13 in cervical carcinomas that were confirmed to contain HPV16 episomes only [##REF##11880406##24##]. This group also identified cases with mixed integrant and episome infection in which the E2/E6 ratio was 0.41–0.55, consistent with their findings from experiments mixing plasmids containing full length HPV16 DNA and HPV6 E6 DNA [##REF##11880406##24##]. Our current data suggests that E2/E6 ratios in this range could also reflect integrated HPV16 only and the presence of episomes cannot be assumed in such cases.</p>", "<p>In our opinion, only very low E2/E6 ratios are likely to be good indicators of the integrant only state. We propose an E2/E6 ratio of 0.10 or less, as values greater than this are unlikely to discriminate reliably between integrant only samples and samples where episomes are present. A previous study showed that E2/E6 ratios under 0.10 were only seen where integrated HPV16 DNA was in 10 fold or greater excess over episomal HPV16 DNA [##REF##16672403##5##]. However even these findings are rather questionable, as the qPCR assay used (in which gene copy numbers were referenced to a fixed mass of gDNA) gave an E2/E6 ratio of 0.25 when integrated and episomal DNA were present in a 1:1 mixture and 0.56 when episomal DNA was in 10 fold excess. Moreover, as with most published reports, no absolute copy number values were reported in the study [##REF##16672403##5##].</p>", "<p>E2/E6 ratios less than 0.10 are likely to be encountered rarely. Indeed, in our study we observed such values in only three of 14 HPV16 positive cervical SCCs. In the other cases, we consider that no reliable conclusions regarding viral state could be drawn from the E2/E6 ratio, indicating its limited usefulness when applied to clinical samples and uncharacterized cell lines. Based on these findings, we suggest that data from previous studies using the E2/E6 ratio to examine the physical state of HPV16 in clinical samples may not be accurate, and, in particular may have overestimated the frequency of mixed integrant and episomal infections in cervical neoplasia [##REF##16672403##5##,##REF##11880410##13##].</p>" ]
[]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16 gene copy numbers. We assessed a modified method, in which external calibration curves were generated from a single construct containing HPV16 E2, HPV16 E6 and the host gene hydroxymethylbilane synthase in a 1:1:1 ratio.</p>", "<title>Results</title>", "<p>When testing dilutions of mixed HPV/host DNA in replicate runs, we observed errors in quantifying E2 and E6 amplicons of 5–40%, with greatest error at the lowest DNA template concentration (3 ng/μl). Errors in determining viral copy numbers per diploid genome were 13–53%. Nevertheless, in cervical keratinocyte cell lines we observed reasonable agreement between viral loads determined by qPCR and Southern blotting. The mean E2/E6 ratio in episome-only cells was 1.04, but with a range of 0.76–1.32. In three integrant-only lines the mean E2/E6 ratios were 0.20, 0.72 and 2.61 (values confirmed by gene-specific Southern blotting). When E2/E6 ratios in fourteen HPV16-positive cervical carcinomas were analysed, conclusions regarding viral physical state could only be made in three cases, where the E2/E6 ratio was ≤ 0.06.</p>", "<title>Conclusion</title>", "<p>Run-to-run variation in SYBR green qPCR produces unavoidable inaccuracies that should be allowed for when quantifying HPV gene copy number. While E6 copy numbers can be considered to provide a useable indication of viral loads, the E2/E6 ratio is of limited value. Previous studies may have overestimated the frequency of mixed episomal/integrant HPV infections.</p>" ]
[ "<title>Authors' contributions</title>", "<p>IR devised the experiments, prepared the NA6 calibrator clone, and carried out accuracy tests and assessment of cell lines by qPCR. GN undertook assessment of clinical samples by qPCR and NF prepared calibration curves. W12 cell line establishment, propagation and gDNA preparations were made by MS and MRP. MTH prepared other cell line gDNA and contributed to the development of the qPCR method. AT provided statistical input and advised on qPCR mathematics. IR and NC wrote the manuscript. This work was funded by MRC and CRUK programme grants held by NC.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>The authors thank Scott Allen Rauch for permission to employ and distribute XlXtrFun and Andrew Pope for permission to employ and distribute IntersectComplex.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Sequence of NA6 clone insert demonstrates that E2, E6 and HMBS amplicons are present in equimolar amounts</bold>. The M13 forward primer of pcr2.1 TA vector is shown at 195 bp in bold, underlined. E2, E6 and HMBS amplicons are shown in bold italics, with primer binding sites underlined. The M13 reverse primer is at 850 bp, bold and underlined.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Generation of mean NA6 calibration curve for E2, E6 amd HMBS quantification</bold>. Panels A – C show representative data from the four replicate PCR runs using serially diluted NA6 as template (2 ng – 2 fg), from which the mean calibration curve for viral quantification was generated. The different curves correspond to different starting amounts of template. The fluorescence thresholds are indicated by the lines of crosses running horizontally. Panel A shows E2 log transformed data from replicate 1 qPCR run, panel B shows HMBS log transformed data from replicate 3 qPCR run, panel C shows E6 log transformed data from replicate 2 qPCR run. Panel D demonstrates the tight correlation between the data points generated for the E2 amplicon at each template concentration in the four replicate runs. Similar findings were made for the E6 and HMBS amplicons. Panel E shows the mean calibration curves for E2, E6 and HMBS used for viral gene copy number quantification, together with the respective line equations.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Southern analysis of HPV16 copy number and physical state in the cervical keratinocyte cell lines used to validate the NA6 qPCR method</bold>. The three panels represent blots probed with full length HPV16 (A), the HPV16 E2 amplicon (B) and the HPV16 E6 amplicon (C). The lanes marked B are BamH1 digests, while the lanes marked H are Hind III digests. The right hand lanes in Panel A show copy number controls for estimates of viral load, ranging from 500 to 1 copies of full length HPV16 per 5 μg of normal peripheral blood lymphocyte gDNA. C33A is HPV16 negative, CaSki contains integrated HPV16 at high copy, SiHa contains integrated HPV16 at low copy, W12.Ser1p16 contains episomal HPV16, and W12.Ser1p57 contains a single HPV16 integrant (arrowed). The inset at the bottom of Panel A shows the faint signal produced by the HPV16 integrant in W12.Ser1p57 following prolonged (72 hr) exposure. For CaSki cells, the signal produced with the E2 probe (Panel B) is considerably greater than that with the E6 probe (Panel C) and additional E2 bands are present (boxed in Panel B), indicative of multiple integration sites.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Primers used to generate NA6 and for qPCR.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Primers</bold></td><td align=\"center\"><bold>Sequence (5' to 3')</bold></td><td align=\"center\"><bold>Product length (bp)</bold></td><td align=\"center\"><bold>Reference</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>E2 F</bold></td><td align=\"left\">AAC GAA GTA TCC TCT CCT GAA ATT ATT AG</td><td align=\"center\">81</td><td align=\"center\">[##REF##11880410##13##]</td></tr><tr><td align=\"left\"><bold>E2 R</bold></td><td align=\"left\">CCA AGG CGA CGG CTT TG</td><td/><td/></tr><tr><td align=\"left\"><bold>E6 F</bold></td><td align=\"left\">GAG AAC TGC AAT GTT TCA GGA CC</td><td align=\"center\">80</td><td align=\"center\">[##REF##11880410##13##]</td></tr><tr><td align=\"left\"><bold>E6 R</bold></td><td align=\"left\">TGT ATA GTT GTT TGC AGC TCT GTG C</td><td/><td/></tr><tr><td align=\"left\"><bold>HMBS F</bold></td><td align=\"left\">GCC TGC AGT TTG AAA TCA GTG</td><td align=\"center\">118</td><td align=\"center\">[##REF##12843067##32##]</td></tr><tr><td align=\"left\"><bold>HMBS R</bold></td><td align=\"left\">CGG GAC GGG CTT TAG CTA</td><td/><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Effect of standard curve selection on E6, E2 and HMBS gene copy number calculations.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>E6</bold></td><td align=\"center\"><bold>Correlation Coefficient (R<sup><bold>2</bold></sup>)</bold></td><td align=\"center\"><bold>Primer Efficiency (E)</bold></td><td align=\"center\"><bold>Number of molecules at threshold (Nt)</bold></td><td align=\"center\" colspan=\"5\"><bold>E6 gene copy numbers over a range of theoretical crossing points</bold></td></tr><tr><td colspan=\"4\"/><td colspan=\"5\"><hr/></td></tr><tr><td/><td/><td/><td/><td align=\"center\"><bold>10</bold></td><td align=\"center\"><bold>15</bold></td><td align=\"center\"><bold>20</bold></td><td align=\"center\"><bold>25</bold></td><td align=\"center\"><bold>30</bold></td></tr></thead><tbody><tr><td align=\"center\"><bold>Final calibration curve for E6</bold></td><td align=\"center\"><bold>0.9963</bold></td><td align=\"center\"><bold>95.01%</bold></td><td align=\"center\"><bold>3.3806E+11</bold></td><td align=\"center\"><bold>4.25E+08</bold></td><td align=\"center\"><bold>1.51E+07</bold></td><td align=\"center\"><bold>5.34E+05</bold></td><td align=\"center\"><bold>1.89E+04</bold></td><td align=\"center\"><bold>6.72E+02</bold></td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"center\"><bold>Replicate 1</bold></td><td align=\"center\">0.9988</td><td align=\"center\">87.79%</td><td align=\"center\">1.43162E+11</td><td align=\"center\">2.62E+08</td><td align=\"center\">1.12E+07</td><td align=\"center\">4.81E+05</td><td align=\"center\">2.06E+04</td><td align=\"center\">8.82E+02</td></tr><tr><td align=\"center\"><bold>Replicate 2</bold></td><td align=\"center\">0.9909</td><td align=\"center\">101.86%</td><td align=\"center\">5.85431E+11</td><td align=\"center\">5.21E+08</td><td align=\"center\">1.56E+07</td><td align=\"center\">4.64E+05</td><td align=\"center\">1.39E+04</td><td align=\"center\">4.13E+02</td></tr><tr><td align=\"center\"><bold>Replicate 3</bold></td><td align=\"center\">0.9982</td><td align=\"center\">96.56%</td><td align=\"center\">3.68007E+11</td><td align=\"center\">4.28E+08</td><td align=\"center\">1.46E+07</td><td align=\"center\">4.97E+05</td><td align=\"center\">1.69E+04</td><td align=\"center\">5.77E+02</td></tr><tr><td align=\"center\"><bold>Replicate 4</bold></td><td align=\"center\">0.9973</td><td align=\"center\">93.84%</td><td align=\"center\">2.55641E+11</td><td align=\"center\">3.41E+08</td><td align=\"center\">1.25E+07</td><td align=\"center\">4.56E+05</td><td align=\"center\">1.67E+04</td><td align=\"center\">6.09E+02</td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td/><td/><td/><td align=\"center\"><bold>Coefficient of variation of replicates</bold></td><td align=\"center\">28.72%</td><td align=\"center\">14.58%</td><td align=\"center\">3.83%</td><td align=\"center\">16.27%</td><td align=\"center\">31.32%</td></tr><tr><td/><td/><td/><td align=\"center\"><bold>Mean copy number of replicates</bold></td><td align=\"center\">3.88E+08</td><td align=\"center\">1.35E+07</td><td align=\"center\">4.74E+05</td><td align=\"center\">1.70E+04</td><td align=\"center\">6.20E+02</td></tr><tr><td/><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"center\"><bold>E2</bold></td><td align=\"center\"><bold>Correlation Coefficient (R<sup><bold>2</bold></sup>)</bold></td><td align=\"center\"><bold>Primer Efficiency (E)</bold></td><td align=\"center\"><bold>Number of molecules at threshold (Nt)</bold></td><td align=\"center\" colspan=\"5\"><bold>E2 gene copy numbers over a range of theoretical crossing points</bold></td></tr><tr><td colspan=\"4\"/><td colspan=\"5\"><hr/></td></tr><tr><td/><td/><td/><td/><td align=\"center\"><bold>10</bold></td><td align=\"center\"><bold>15</bold></td><td align=\"center\"><bold>20</bold></td><td align=\"center\"><bold>25</bold></td><td align=\"center\"><bold>30</bold></td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"center\"><bold>Final calibration curve for E2</bold></td><td align=\"center\"><bold>0.9945</bold></td><td align=\"center\"><bold>90.86%</bold></td><td align=\"center\"><bold>2.42762E+11</bold></td><td align=\"center\"><bold>3.79E+08</bold></td><td align=\"center\"><bold>1.49E+07</bold></td><td align=\"center\"><bold>5.90E+05</bold></td><td align=\"center\"><bold>2.33E+04</bold></td><td align=\"center\"><bold>9.02E+02</bold></td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"center\"><bold>Replicate 1</bold></td><td align=\"center\">0.9887</td><td align=\"center\">88.55%</td><td align=\"center\">1.92376E+11</td><td align=\"center\">3.39E+08</td><td align=\"center\">1.42E+07</td><td align=\"center\">5.96E+05</td><td align=\"center\">2.50E+04</td><td align=\"center\">0.9887</td></tr><tr><td align=\"center\"><bold>Replicate 2</bold></td><td align=\"center\">0.9945</td><td align=\"center\">93.10%</td><td align=\"center\">2.8539E+11</td><td align=\"center\">3.96E+08</td><td align=\"center\">1.48E+07</td><td align=\"center\">5.49E+05</td><td align=\"center\">2.05E+04</td><td align=\"center\">0.9945</td></tr><tr><td align=\"center\"><bold>Replicate 3</bold></td><td align=\"center\">0.9973</td><td align=\"center\">92.70%</td><td align=\"center\">3.11657E+11</td><td align=\"center\">4.41E+08</td><td align=\"center\">1.66E+07</td><td align=\"center\">6.25E+05</td><td align=\"center\">2.35E+04</td><td align=\"center\">0.9973</td></tr><tr><td align=\"center\"><bold>Replicate 4</bold></td><td align=\"center\">0.9976</td><td align=\"center\">89.07%</td><td align=\"center\">1.81623E+11</td><td align=\"center\">3.11E+08</td><td align=\"center\">1.29E+07</td><td align=\"center\">5.33E+05</td><td align=\"center\">2.21E+04</td><td align=\"center\">0.9976</td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td/><td/><td/><td align=\"center\"><bold>Coefficient of variation of replicates</bold></td><td align=\"center\">15.69%</td><td align=\"center\">10.60%</td><td align=\"center\">7.36%</td><td align=\"center\">8.57%</td><td align=\"center\">13.06%</td></tr><tr><td/><td/><td/><td align=\"center\"><bold>Mean copy number of replicates</bold></td><td align=\"center\">3.72E+08</td><td align=\"center\">1.46E+07</td><td align=\"center\">5.76E+05</td><td align=\"center\">2.28E+04</td><td align=\"center\">9.02E+02</td></tr><tr><td/><td/><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"center\"><bold>HMBS</bold></td><td align=\"center\"><bold>Correlation Coefficient (R<sup><bold>2</bold></sup>)</bold></td><td align=\"center\"><bold>Primer Efficiency (E)</bold></td><td align=\"center\"><bold>Number of molecules at threshold (Nt)</bold></td><td align=\"center\" colspan=\"5\"><bold>HMBS gene copy numbers over a range of theoretical crossing points</bold></td></tr><tr><td colspan=\"4\"/><td colspan=\"5\"><hr/></td></tr><tr><td/><td/><td/><td/><td align=\"center\"><bold>10</bold></td><td align=\"center\"><bold>15</bold></td><td align=\"center\"><bold>20</bold></td><td align=\"center\"><bold>25</bold></td><td align=\"center\"><bold>30</bold></td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"center\"><bold>Final calibration curve for HMBS</bold></td><td align=\"center\"><bold>0.9970</bold></td><td align=\"center\"><bold>91.05%</bold></td><td align=\"center\"><bold>2.28115E+11</bold></td><td align=\"center\"><bold>3.52E+08</bold></td><td align=\"center\"><bold>1.38E+07</bold></td><td align=\"center\"><bold>5.44E+05</bold></td><td align=\"center\"><bold>2.14E+04</bold></td><td align=\"center\"><bold>8.40E+02</bold></td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"center\"><bold>Replicate 1</bold></td><td align=\"center\">0.9951</td><td align=\"center\">89.10%</td><td align=\"center\">1.333E+11</td><td align=\"center\">2.28E+08</td><td align=\"center\">9.43E+06</td><td align=\"center\">3.90E+05</td><td align=\"center\">1.61E+04</td><td align=\"center\">6.67E+02</td></tr><tr><td align=\"center\"><bold>Replicate 2</bold></td><td align=\"center\">0.9985</td><td align=\"center\">95.39%</td><td align=\"center\">3.52583E+11</td><td align=\"center\">4.35E+08</td><td align=\"center\">1.53E+07</td><td align=\"center\">5.36E+05</td><td align=\"center\">1.88E+04</td><td align=\"center\">6.60E+02</td></tr><tr><td align=\"center\"><bold>Replicate 3</bold></td><td align=\"center\">0.9990</td><td align=\"center\">90.43%</td><td align=\"center\">2.4809E+11</td><td align=\"center\">3.96E+08</td><td align=\"center\">1.58E+07</td><td align=\"center\">6.31E+05</td><td align=\"center\">2.52E+04</td><td align=\"center\">1.01E+03</td></tr><tr><td align=\"center\"><bold>Replicate 4</bold></td><td align=\"center\">0.9953</td><td align=\"center\">89.26%</td><td align=\"center\">1.78485E+11</td><td align=\"center\">3.03E+08</td><td align=\"center\">1.25E+07</td><td align=\"center\">5.13E+05</td><td align=\"center\">2.11E+04</td><td align=\"center\">8.71E+02</td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td/><td/><td/><td align=\"center\"><bold>Coefficient of variation of replicates</bold></td><td align=\"center\">27.36%</td><td align=\"center\">22.14%</td><td align=\"center\">19.17%</td><td align=\"center\">18.93%</td><td align=\"center\">20.99%</td></tr><tr><td/><td/><td/><td align=\"center\"><bold>Mean copy number of replicates</bold></td><td align=\"center\">3.40E+08</td><td align=\"center\">1.32E+07</td><td align=\"center\">5.18E+05</td><td align=\"center\">2.03E+04</td><td align=\"center\">8.01E+02</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Dynamic range of E2, E6 and HMBS quantification by qPCR.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"center\"><bold>Template DNA per μl</bold></td><td align=\"center\" colspan=\"5\"><bold>Expected quantification in 2 μl</bold></td><td align=\"center\" colspan=\"8\"><bold>Observed quantification with percent change over expected</bold></td></tr><tr><td colspan=\"1\"><hr/></td><td colspan=\"5\"><hr/></td><td colspan=\"8\"><hr/></td></tr><tr><td/><td align=\"center\"><bold>E2</bold></td><td align=\"center\"><bold>E6</bold></td><td align=\"center\"><bold>HMBS</bold></td><td align=\"center\"><bold>E2/E6</bold></td><td align=\"center\"><bold>E6 per diploid genome</bold></td><td align=\"center\" colspan=\"2\"><bold>E2</bold></td><td align=\"center\" colspan=\"2\"><bold>E6</bold></td><td align=\"center\" colspan=\"2\"><bold>HMBS</bold></td><td align=\"center\"><bold>E2/E6</bold></td><td align=\"center\"><bold>E6 per diploid genome</bold></td></tr><tr><td colspan=\"1\"><hr/></td><td colspan=\"1\"><hr/></td><td colspan=\"1\"><hr/></td><td colspan=\"1\"><hr/></td><td colspan=\"1\"><hr/></td><td colspan=\"1\"><hr/></td><td colspan=\"2\"><hr/></td><td colspan=\"2\"><hr/></td><td colspan=\"2\"><hr/></td><td colspan=\"1\"><hr/></td><td colspan=\"1\"><hr/></td></tr><tr><td align=\"center\">51 ng gDNA 10 pg pSP64 HPV16</td><td align=\"center\">760,000</td><td align=\"center\">760,000</td><td align=\"center\">28,571</td><td align=\"center\">1</td><td align=\"center\">53</td><td align=\"center\">893,063</td><td align=\"center\"><bold>+17.5%</bold></td><td align=\"center\">616,115</td><td align=\"center\"><bold>-18.9%</bold></td><td align=\"center\">49,816</td><td align=\"center\"><bold>+74.4%</bold></td><td align=\"center\">1.4</td><td align=\"center\">25</td></tr><tr><td align=\"center\">11 ng gDNA 2.16 pg pSP64 HPV16</td><td align=\"center\">163,922</td><td align=\"center\">163,922</td><td align=\"center\">6,163</td><td align=\"center\">1</td><td align=\"center\">53</td><td align=\"center\">208,404</td><td align=\"center\"><bold>+27.1%</bold></td><td align=\"center\">172,423</td><td align=\"center\"><bold>+5.2%</bold></td><td align=\"center\">7,437</td><td align=\"center\"><bold>+20.7%</bold></td><td align=\"center\">1.2</td><td align=\"center\">46</td></tr><tr><td align=\"center\">3 ng gDNA 60 fg pSP64 HPV16</td><td align=\"center\">44,665</td><td align=\"center\">44,665</td><td align=\"center\">1,679</td><td align=\"center\">1</td><td align=\"center\">53</td><td align=\"center\">26,956</td><td align=\"center\"><bold>-39.7%</bold></td><td align=\"center\">40,597</td><td align=\"center\"><bold>-9.1%</bold></td><td align=\"center\">1,047</td><td align=\"center\"><bold>+37.6%</bold></td><td align=\"center\">0.7</td><td align=\"center\">78</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Comparison of HPV16 viral load and physical state determined by Southern hybridisation with NA6 qPCR analysis of HPV16 E2 and E6 copy numbers in cervical keratinocyte cell lines.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Cell line</bold></td><td align=\"left\"><bold>Ploidy</bold></td><td align=\"center\"><bold>Southern analysis Copy numbers per cell</bold></td><td align=\"center\" colspan=\"4\"><bold>NA6 qPCR analysis</bold></td></tr><tr><td/><td/><td/><td colspan=\"4\"><hr/></td></tr><tr><td/><td/><td/><td align=\"center\"><bold>E2 copies per diploid genome</bold></td><td align=\"center\"><bold>E6 copies per diploid genome</bold></td><td align=\"center\"><bold>Viral load per cell</bold></td><td align=\"center\"><bold>Ratio of E2/E6 (range)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>C33A</bold></td><td align=\"left\">Near triploid</td><td align=\"center\">HPV16 negative</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\"><bold>W12.Ser1p16</bold></td><td align=\"left\">Diploid</td><td align=\"center\">Episomal (approx 100 copies)</td><td align=\"center\">148</td><td align=\"center\">143</td><td align=\"center\">143</td><td align=\"center\">1.04 (0.76–1.32)</td></tr><tr><td align=\"left\"><bold>W12.Ser1p57</bold></td><td align=\"left\">Tetraploid</td><td align=\"center\">Integrated (approx 1 copy)</td><td align=\"center\">0.2</td><td align=\"center\">0.3</td><td align=\"center\">1</td><td align=\"center\">0.72 (0.57–3.82)</td></tr><tr><td align=\"left\"><bold>SiHa</bold></td><td align=\"left\">Triploid</td><td align=\"center\">Integrated (2–3 copies)</td><td align=\"center\">0.7</td><td align=\"center\">3.4</td><td align=\"center\">5</td><td align=\"center\">0.20 (0.11–0.24)</td></tr><tr><td align=\"left\"><bold>CaSki</bold></td><td align=\"left\">Tetraploid</td><td align=\"center\">Integrated (&gt;1000 copies)</td><td align=\"center\">2,272</td><td align=\"center\">869</td><td align=\"center\">1,738</td><td align=\"center\">2.61 (1.37–3.46)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>NA6 qPCR analysis of E2 and E6 copy numbers in cervical SCC samples.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Cervical SCC sample ID</bold></td><td align=\"center\"><bold>E2 copies</bold></td><td align=\"center\"><bold>E6 copies</bold></td><td align=\"center\"><bold>Viral load level</bold></td><td align=\"center\"><bold>Ratio of E2/E6</bold></td></tr></thead><tbody><tr><td align=\"center\"><bold>G30</bold></td><td align=\"center\">2</td><td align=\"center\">1</td><td align=\"center\">Low</td><td align=\"center\">1.28</td></tr><tr><td align=\"center\"><bold>G1</bold></td><td align=\"center\">0</td><td align=\"center\">2</td><td align=\"center\">Low</td><td align=\"center\">0.21</td></tr><tr><td align=\"center\"><bold>G31</bold></td><td align=\"center\">0</td><td align=\"center\">4</td><td align=\"center\">Low</td><td align=\"center\">0.06*</td></tr><tr><td align=\"center\"><bold>n55</bold></td><td align=\"center\">0</td><td align=\"center\">5</td><td align=\"center\">Low</td><td align=\"center\">0.01*</td></tr><tr><td align=\"center\"><bold>G19</bold></td><td align=\"center\">12</td><td align=\"center\">10</td><td align=\"center\">Low</td><td align=\"center\">1.26</td></tr><tr><td align=\"center\"><bold>G12</bold></td><td align=\"center\">35</td><td align=\"center\">35</td><td align=\"center\">Low</td><td align=\"center\">0.99</td></tr><tr><td align=\"center\"><bold>n12</bold></td><td align=\"center\">44</td><td align=\"center\">42</td><td align=\"center\">Low</td><td align=\"center\">1.03</td></tr><tr><td align=\"center\"><bold>n10</bold></td><td align=\"center\">73</td><td align=\"center\">48</td><td align=\"center\">Low</td><td align=\"center\">1.54</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"center\"><bold>G26</bold></td><td align=\"center\">461</td><td align=\"center\">297</td><td align=\"center\">Medium</td><td align=\"center\">1.55</td></tr><tr><td align=\"center\"><bold>G18</bold></td><td align=\"center\">258</td><td align=\"center\">343</td><td align=\"center\">Medium</td><td align=\"center\">0.75</td></tr><tr><td align=\"center\"><bold>G6</bold></td><td align=\"center\">467</td><td align=\"center\">423</td><td align=\"center\">Medium</td><td align=\"center\">1.10</td></tr><tr><td align=\"center\"><bold>G3</bold></td><td align=\"center\">384</td><td align=\"center\">528</td><td align=\"center\">Medium</td><td align=\"center\">0.73</td></tr><tr><td align=\"center\"><bold>G9</bold></td><td align=\"center\">0</td><td align=\"center\">691</td><td align=\"center\">Medium</td><td align=\"center\">0.00*</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"center\"><bold>G11</bold></td><td align=\"center\">954</td><td align=\"center\">2,481</td><td align=\"center\">High</td><td align=\"center\">0.38</td></tr></tbody></table></table-wrap>" ]
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[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Crossing point values used in critical evaluation of HPV16 gene copy number quantification by SYBR green PCR. The Microsoft Excel workbook of Additional file ##SUPPL##0##1## contains four worksheets. <bold>1: NA6 Standard Curves.</bold> The crossing point values used in generation of external calibration curves for absolute quantification of viral E2, viral E6 and host HMBS genes are presented. For each gene, four runs of a seven point NA6 titration series were conducted in triplicate. <bold>2: Accuracy Test.</bold> The crossing point values used to determine viral load and physical state of a three point serial dilution of a mixture of test gDNA and HPV16 plasmid DNA are presented. Three runs were undertaken at each titration point in triplicate. <bold>3: Assessment of Cell Lines.</bold> The crossing point values used to derive viral load and physical state of five cervical carcinoma cell lines are presented. Each cell line was assessed in three separate runs, and each reaction was performed in triplicate. <bold>4: Assessment of Clinical Samples.</bold> The crossing point values used to derive viral load and physical state of 14 squamous cell cervical carcinoma samples are presented. One run was undertaken for each sample, and all reactions were performed in triplicate.</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><p><italic>F </italic>refers to forward primers and <italic>R </italic>to reverse primers.</p></table-wrap-foot>", "<table-wrap-foot><p>In the main part of each section the rows represent the values for the final calibration curve and for the four replicate qPCR runs of the 7 point NA6 titration series, from which the final curve was generated. The right-hand five columns give the gene copy numbers determined from each curve at theoretical crossing points ranging from 10 to 30. In the bottom part of each section the numbers represent the coefficient of variation in mean copy number determined between the four replicate qPCR runs, together with the mean copy number determined from replicates 1 – 4 at each Cp.</p></table-wrap-foot>", "<table-wrap-foot><p>Peripheral blood lymphocyte gDNA was spiked with a known quantity of HPV16 DNA (in the pSP64-HPV16 plasmid). The template DNA was serially diluted, and absolute amounts were measured by spectrophotometry. The template DNA values are given in the first column of the Table. The anticipated gene copy numbers for E2, E6 and HMBS were derived, and are shown in the 'Expected quantification' section. qPCR of the serially diluted samples was performed, and the observed gene copy numbers are reported in the 'Observed quantification' section.</p></table-wrap-foot>", "<table-wrap-foot><p>Values less than 1 were rounded to the nearest whole number when calculating viral load per cell. E2/E6 ratio values were calculated from the qPCR readings for E2 and E6 and rounded to the nearest two decimal places.</p></table-wrap-foot>", "<table-wrap-foot><p>HPV gene copy numbers are per diploid genome. E2/E6 ratio values were calculated from the qPCR readings for E2 and E6 and rounded to the nearest two decimal places. The only samples in which HPV16 physical state could be inferred with confidence are marked by asterisks in the right hand column.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1472-6750-8-57-1\"/>", "<graphic xlink:href=\"1472-6750-8-57-2\"/>", "<graphic xlink:href=\"1472-6750-8-57-3\"/>" ]
[ "<media xlink:href=\"1472-6750-8-57-S1.xls\" mimetype=\"application\" mime-subtype=\"vnd.ms-excel\"><caption><p>Click here for file</p></caption></media>" ]
[{"surname": ["Yoshida", "Sano", "Kanuma", "Owada", "Sakurai", "Fukuda", "Nakajima"], "given-names": ["T", "T", "T", "N", "S", "T", "T"], "article-title": ["Quantitative real-time polymerase chain reaction analysis of the type distribution, viral load, and physical status of human papillomavirus in liquid-based cytology samples from cervical lesions"], "source": ["Int J Gynecol Cancer"], "year": ["2007"]}]
{ "acronym": [], "definition": [] }
35
CC BY
no
2022-01-12 14:47:25
BMC Biotechnol. 2008 Jul 24; 8:57
oa_package/06/eb/PMC2529285.tar.gz
PMC2529286
18680573
[ "<title>Background</title>", "<p>When we are watching a movie, reading a book or playing a computer game we sometimes experience these variants of virtual reality as if they were real. This subjective sensation of presence is referred as \"the feeling of being there\". From an earlier EEG (electroencephalography) study [##REF##16497116##1##] we know that activations in certain brain areas (especially in the prefrontal cortex) are negatively correlated with the subjective feeling of presence in another space (spatial presence). The involvement in a virtual scene can be measured by questionnaires (e.g. MEC-SPQ [##UREF##0##2##]). Moreover, psychophysiological measures (e.g. electro-dermal activity or heart rate variations) are also used to indicate different presence states in a virtual environment (VE) of a person. Mostly, a higher involvement in the virtual reality scenario is accompanied by enhanced responses of the vegetative nervous system such as electrodermal responses and heart rate [##REF##16497116##1##,##REF##15006177##3##, ####REF##17034333##4##, ##REF##15803164##5####15803164##5##].</p>", "<p>In this study we will use \"transcranial direct current stimulation\" to modulate brain activation during the confrontation with a virtual reality scenario. \"Transcranial direct current stimulation\" (tDCS) non-invasively modulates the excitability of a brain region of interest by altering neuronal membrane potentials [##REF##12244081##6##,##REF##10990547##7##]. Anodal tDCS has been found to increase cortical excitability and the potentiation of N-methyl-D-aspartate (NMDA) receptor efficacy, while cathodal tDCS has been found to decrease cortical excitability. Several studies have shown that the effects caused by tDCS last several minutes beyond the period of tDCS application [##REF##12244081##6##, ####REF##10990547##7##, ##REF##11723286##8##, ##REF##18281890##9####18281890##9##]. Until now several studies have shown that tDCS can modulate cognitive and behavioral skills associated with the targeted brain area. For example, anodal tDCS to the left prefrontal cortex was found to increase working memory performance [##REF##15999258##10##] and verbal fluency [##REF##15753425##11##]. Anodal tDCS to the motor cortex contralateral to stroke patients' paretic arm facilitated temporary motor recovery [##REF##15634731##12##]. In addition, anodal stimulation of the left motor cortex in healthy subjects improved right-hand performance [##REF##16603933##13##]. A very recent study demonstrated that anodal stimulation to the supramarginal gyrus enhanced tone memory performance in musical novices [##REF##16791101##14##].</p>", "<p>In the context of these findings the question arises, whether the feeling of presence can be influenced by applying tDCS to brain areas known to be involved in the control of presence. In this study we focus on the dorso-lateral prefrontal cortex (dlPFC), which is known to be involved in controlling many higher-order behaviors. Typically it has been shown that this area is involved in selecting a possible range of responses and suppressing inappropriate ones [##REF##12563285##15##]. In addition, it has been shown that this area is critically involved in the inhibition (and control) of impulsive behavior controlled by other brain-regions (e.g. the brainstem, basal ganglia; this system is sometimes called the „impulsive system\") [##REF##16251988##16##].</p>", "<p>Thus, we anticipate that the dlPFC will be involved in the modulation of presence experience. If the dlPFC is activated there will be strong top-down control available inhibiting the automatically evoked presence feeling by the \"impulsive system\". When the dlPFC is deactivated the \"impulsive system\" can unfold its bottom-up activation with less top-down control of the dlPFC. If the dlPFC is indeed the critical area modulating presence feeling during the exposure of virtual environments the differential presence experience in kids, adolescents, and adults can be explained on the basis of the late maturing dlPFC [##REF##15148381##17##]. The late myelination of the dlPFC can partly explain why adolescents' behavior is characterized by motivational difficulties, impulsivity and addiction (also in the context of video games and virtual scenes) [##REF##12777258##18##].</p>", "<p>In our study we modulated the right dlPFC with tDCS while participants were watching a virtual roller coaster scene. In order to further evaluate the success of this modulation, we also conducted a classical Go-Nogo task. The performance in this test depends on the functioning of dlPFC [##REF##11170305##19##] and indicates the degree of impulsivity. There is evidence, that the task performance in the Go-Nogo task can be influenced by tDCS application to the left dlPFC [##REF##17166593##20##] and with other methods also on the right dlPFC (transcranial magnetic stimulation (TMS) [##REF##16352348##21##]). In addition, it has been shown that the right dlPFC is involved in controlling risk-taking behavior [##REF##16775134##22##,##REF##18596613##23##] and reciprocal fairness [##REF##17023614##24##].</p>", "<p>We hypothesize that the feeling of being present in the virtual environment is enhanced if the excitability (and thus the activation) of the dlPFC is decreased. In addition, lowered activation within in the dlPFC should also be accompanied by higher impulsiveness as measured with the Go-Nogo-task. On the other hand, if the excitability of the dlPFC (and thus the activation) is increased this should lead to a lowered presence experience and reduced impulsiveness.</p>" ]
[ "<title>Methods</title>", "<title>Subjects</title>", "<p>Thirty-five (17 female, 18 male) subjects participated in the experiment. Most of them being students of the University of Zurich. The mean age was 24.9 yr (standard deviation: ± 3.7 yr). All of the participants were classified as being consistent right-handed (CRH) using the Annett hand preference questionnaire [##REF##5457503##25##]. No subject reported a history of neurological or psychiatric diseases and gave their informed consent for the participation in the experiment.</p>", "<title>tDCS application</title>", "<p>In order to prevent an interaction between the two brain-hemispheres we decided to constrain tDCS to one hemisphere. In pilot experiments in our lab we found slightly stronger correlations between presence experience and brain activation on the right dlPFC than on the left side. Therefore, we only applied tDCS to the right dlPFC. The application side was at the FC3 electrode position of the international EEG 10–20-System. In order to constrain tDCS application to one hemisphere the reference electrode was placed on the ipsilateral mastoid. For tDCS application the \"DC stimulator\" by Eldith<sup>© </sup><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.eldith.de\"/> was used. The constant current was applied using two saline-soaked electrodes with a surface of 35 cm<sup>2</sup>. During the anodal tDCS mode, the anode electrode was positioned on FC3 and the cathode electrode on the ipsilateral mastoid. During the cathodal condition, the two electrodes were switched (cathode over FC3, anode over ipsilateral mastoid). tDCS application lasted 5.5 min at a constant current intensity of 1.5 mA. The system automatically turned off the stimulation when the electrical resistance was too high. For sham stimulation the stimulator was switched off.</p>", "<title>Virtual roller coaster</title>", "<p>The subjects were sitting on a chair while watching three different rollercoaster scenarios on a 22-inch computer screen placed at a distance of 60 cm in front of them. The rollercoaster scenarios were taken from a commercially available rollercoaster simulation software <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.nolimitscoaster.com\"/>. Realistic driving noises were presented on loudspeakers. Every scenario consisted of three different phases. It started with an \"ascending phase\" (30 s) followed by a \"dynamic phase\" with movements in different dimensions and very high speed (60 s) and an \"end-phase\" with low speed and without inclination (Figure ##FIG##0##1##).</p>", "<title>Psychophysiological measures</title>", "<p>During the roller coaster ride electro-dermal activity (EDA) and the electro-myogram (EMG) were registered. The EDA and EMG measurements were conducted using a commercially available device (PAR-PORT; Hogrefe Company, Germany). For EDA recording, electrodes were attached to the thenar and hypothenar areas on the palm of the left hand. EDA activity was quantified using two different measures. First, we measured skin conductance responses (SCR) to the roller coaster scenario. In addition, we measured skin conductance level (SCL) to measure the tonic level of skin conductance during the experimental sessions. SCL was measured as log-transformed mean EDA amplitude (log [EDAsumamp+1]). Log-transformation was used to normalize the SCL data. The EMG electrodes were attached at the left eyebrow muscle (musculus corrugator supercilii) and quantified as mean tonic EMG activity level at this site during the different experimental conditions.</p>", "<title>Go-Nogo task</title>", "<p>The Go-Nogo task was taken from a German standard battery used to test several executive and attentional functions (Testbatterie zur Aufmerksamkeitsprüfung, TAP, [##UREF##1##26##]). This test consisted of 5 types of stimuli including lines in different directions. The subjects were required to press a button if one of the two defined target stimuli were presented. In total 100 stimuli were presented, 40 of them were target stimuli. The number of false alarms (FA, button-press when seeing a non-target stimulus) indicates the degree of impulsivity.</p>", "<title>Questionnaires</title>", "<p>Since presence is a subjective feeling (first person) it is also necessary to use questionnaires asking the subjects for their particular presence feeling during the different conditions. We used an adapted version of the spatial presence questionnaire MEC-SPQ [##REF##15803164##5##]. The questionnaire was presented to the subjects immediately after the rollercoaster ride. Participants indicated their degree of presence on a visual analog scale. Moreover, the SAM (Self Assessment Manikin) was administered after each roller coaster ride in order to control for mood changes during the tDCS application. With the SAM experienced arousal and valence during the roller coaster presentation was measured. Although not being the main focus of this paper we used some subscales (\"thrill and adventure seeking scales\"; TAS) of the „sensation seeking questionnaire\" to control whether this trait might have an influence on presence experience [##REF##4614324##27##,##REF##2198341##28##].</p>", "<title>Design</title>", "<p>We used a repeated measurements design in which every subject was randomly assigned to the three different conditions (anodal, cathodal, sham). During each condition the subjects were exposed to the roller coaster scenario after they have received different tDCS treatments. Each condition comprised the tDCS application, followed by the Go-Nogo task, the roller coaster presentation and the final questionnaire measurement (Figure ##FIG##1##2##). Between each condition there was a break of 3.5 minutes without any task and tDCS application.</p>", "<title>Statistical analysis</title>", "<p>The number of false alarms, SCR, SCL, as well as ratings of valence, arousal, and presence were subjected to one-way repeated measurements ANOVAs with three levels (sham, anodal, and cathodal). Before ANOVA analysis the variances were evaluated for homoscedasticity and we also checked the data for normal distribution. There was no significant deviation from homoscedasticity making it unnecessary to use specific corrections (e.g., Greenhouse Geisser corrections). In addition, the data were also evaluated whether they deviate from normal distribution. Since there were no strong deviations from normal distribution we deemed the ANOVA as an appropriate method to analyze this data set. In case of significant main effects subsequent post-hoc t-tests were conducted using the Bonferroni-Holm procedure [##UREF##2##29##]. A p value &lt; = 0.05 was used as statistical threshold.</p>" ]
[ "<title>Results</title>", "<title>Go-Nogo task</title>", "<p>Figure ##FIG##2##3## shows the results of the Go-Nogo task separately for the three experimental conditions. During cathodal tDCS participants generated more often false alarms indicating a tendency for impulsive behavior. There was no change in performance during anodal stimulation. Subjecting the number of false alarms to a one-way repeated measurements ANOVA revealed a significant between-condition difference for the number of false alarms (F(2,68) = 3.653; p = 0.03). Subsequently conducted post hoc tests revealed significant differences between false alarms obtained during \"sham\" vs. \"cathodal\" (p = 0.032) and \"anodal\"- vs. \"cathodal\" (p = 0.033).</p>", "<title>Psychophysiological measures</title>", "<p>Due to artifact contamination only data of 29 participants could be used for analysis of psychophysiological measures. The EMG measure during the roller coaster ride showed no significant difference during the three tDCS conditions. For SCR a significant between-condition difference emerged. Figure ##FIG##3##4## shows a clear SCR at the start of the virtual roller coaster ride. In the first 30 seconds of the rollercoaster ride (ascending phase), subjects showed stronger SCR during cathodal tDCS (F(2,56) = 3.237; p = 0.047; cathodal &gt; sham: p = 0.021). The one-way ANOVA conducted for the SCL data did not reveal significant differences (F(2,56) = 3.016; p = 0.057).</p>", "<p>Figure ##FIG##4##5## shows the mean peak SCL measured during the first 12 seconds of the roller coaster ride. Peak SCLs were significantly different in the three conditions (F(2,56) = 4.958 p = 0.01) with a higher peak during cathodal stimulation vs. anodal stimulation (p = 0.005) and vs. sham stimulation (p = 0.012).</p>", "<title>Presence- and personality questionnaires</title>", "<p>The questionnaire data showed no significant differences between the different tDCS conditions. Interestingly, there was no significant correlation between the subscale \"thrill and adventure seeking\" (TAS) and the SCR measures (p &gt; .33). The self-assessment-manikin (SAM) showed no differences with respect to the experienced valence of roller coaster scenarios during the different conditions (F(2,66) = 1.617; p = 0.206). However, there was a tendency for slightly increased subjective arousal levels during tDCS application compared to sham stimulation (F(2,66) = 2.532; p = 0.087).</p>", "<title>Correlation between Go-Nogo task performance and SCR</title>", "<p>There was also a significant correlation between the number of false alarms (taken as a measure for impulsiveness) and the SCR measures (r = 0.42, p &lt; 0.02). Thus, if participants act more impulsively in the Go-Nogo task, they also show stronger SCR measures in the ascending phase of the roller coaster ride.</p>" ]
[ "<title>Discussion</title>", "<p>Our study demonstrates that the application of cathodal tDCS to the right dlPFC modulates the degree of impulsivity (as measured with the number of false alarms in the Go-Nogo task). It follows from the current interpretation of the effect of cathodal tDCS on the neural system underlying the cathode that cathodal tDCS downregulates the dlPFC, with a resultant reduction in neural activation in this area. In line with this interpretation, we suggest that the dlPFC exerts less top-down control over the \"impulsive system\", increasing the likelihood therefore of impulsive behavior [for a summary see [##UREF##3##30##]]. Applying anodal tDCS to the dlPFC did not affect impulsiveness as indicated by Go-Nogo performance. We hypothesized at the beginning of the study that this kind of tDCS application would lead to increased neural activation of the dlPFC, this in turn would facilitate increased top-down regulation of the \"impulsive system\" in the form of reduced impulsiveness. The reason that we did not obtain this result is probably due to the fact that the task was too easy with too few false alarms, even in the sham condition. Thus, there was a kind of \"floor effect\" without any opportunity to decrease the number of false alarms. This might also explain the different findings in previous studies using a more difficult and slightly different versions of the Go-Nogo task [##REF##17166593##20##,##REF##16352348##21##].</p>", "<p>Besides the differential effect of tDCS on the number of false alarms, we also obtained different results for the skin conductance responses (SCR) used to indicate the reactivity of the vegetative nervous system. Application of cathodal tDCS to the dlPFC elicited increased SCRs while the subjects were exposed to the roller coaster scenario. This differential SCR was only present in the first phase of the roller coaster ride during which the virtual cab was ascending to the top of the roller coaster course (ascending phase). The strong skin conductance response during the ascent phase of the roller coaster ride might be associated with the anticipation of the following dynamic phase with its ups and downs and with the experience or expectation of bodily arousal in a real roller coaster.</p>", "<p>The correlation between the number of false alarms in the Go-Nogo task and the SCR measures indicates that impulsive behavior and autonomic responses can be influenced by tDCS application, and that both reactions might depend on the activation in the right dlPFC. However, further investigation is needed to develop a better understanding of the relationship between the inhibition of impulsive behavior and vegetative reactions.</p>", "<p>The fact that the personality trait TAS (thrill and adventure seeking) had no impact on our measures (e.g., skin conductance or number of false alarms) indicates that the application of tDCS is independent of the \"sensation seeking\" personality. Nevertheless, there might still be different effects on patients as found in patients with major depression [##REF##17166593##20##].</p>", "<p>A further important result of the present study is that there are significant differences in vegetative reactions in the hypothesized direction associated with tDCS application, but that the subjective reports (measures with questionnaires) did not differ for the different conditions. This shows that subjective measures might not be reliable in the context of presence research (especially because the involvement in a VE requires low cognitive control) and that brain stimulation can lead to a change in bodily reactions without influencing subjective reports.</p>" ]
[ "<title>Conclusion</title>", "<p>Application of tDCS to the right dlPFC can influence the vegetative reactions while watching a virtual roller coaster scene as well as the number of false alarms in a standard Go-Nogo discrimination task commonly used as a behavioral measure of impulsivity. The measured vegetative effects during viewing of the virtual roller coaster ride and concomitant tDCS application had no impact on self-reported experience of presence. The cathodal (inhibiting) condition leads to enhanced impulsivity and higher skin conductance responses. There was no effect on skin conductance and impulsivity during the anodal (exciting) condition.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>\"The feeling of being there\" is one possible way to describe the phenomenon of feeling present in a virtual environment and to act as if this environment is real. One brain area, which is hypothesized to be critically involved in modulating this feeling (also called presence) is the dorso-lateral prefrontal cortex (dlPFC), an area also associated with the control of impulsive behavior.</p>", "<title>Methods</title>", "<p>In our experiment we applied transcranial direct current stimulation (tDCS) to the right dlPFC in order to modulate the experience of presence while watching a virtual roller coaster ride. During the ride we also registered electro-dermal activity. Subjects also performed a test measuring impulsiveness and answered a questionnaire about their presence feeling while they were exposed to the virtual roller coaster scenario.</p>", "<title>Results</title>", "<p>Application of cathodal tDCS to the right dlPFC while subjects were exposed to a virtual roller coaster scenario modulates the electrodermal response to the virtual reality stimulus. In addition, measures reflecting impulsiveness were also modulated by application of cathodal tDCS to the right dlPFC.</p>", "<title>Conclusion</title>", "<p>Modulating the activation with the right dlPFC results in substantial changes in responses of the vegetative nervous system and changed impulsiveness. The effects can be explained by theories discussing the top-down influence of the right dlPFC on the \"impulsive system\".</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>GB participated in the design of the study, performed the statistical analysis and drafted the manuscript. GC participated in the design, carried out the experiments and performed the statistical analysis. TB participated in the design. LJ participated in the design, the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.</p>" ]
[ "<title>Acknowledgements</title>", "<p>This work is funded under the European Union FET project PRESENCCIA Contract Number 27731.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Example still figures of the used rollercoaster scenario. Ascending phase (left, 30 s), dynamic phase (middle, 60 s), end phase (right, 12 s).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Experimental design. Sequence of the different tasks and tDCS applications. The time scale is in seconds. This sequence was repeated three times per subject for the three stimulating conditions (sham, anodal, cathodal).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Number of false alarms (FA) in the different conditions in the Go-Nogo task. Applying cathodal tDCS to the right dlPFC led to an enhanced number of FA (p &lt; .03) compared to sham and anodal-Stimulation. Depicted are means of FA (± SE).</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>Skin conductance level of the first 30 seconds of the rollercoaster ride. Cathodal tDCS application (inhibition) to the right dlPFC led to an enhanced skin conductance response (SCR).</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p>Peak of skin conductance level (maximum SCL) in the first 12 seconds of the roller coaster ride. Cathodal tDCS application (inhibition of the dlPFC) leads to significantly enhanced maximum SCL (p &lt; .01) during the ascending phase in the virtual roller coaster compared to sham and anodal stimulation. Depicted are means of the SCL (± SE).</p></caption></fig>" ]
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[{"surname": ["Vorderer", "Wirth", "Gouveia", "Biocca", "Saari", "J\u00e4ncke", "B\u00f6cking", "Schramm", "Gysberg", "Hartmann", "Klimmt", "Laarni", "Ravaja", "Sacau", "Baumgartner", "J\u00e4ncke"], "given-names": ["P", "W", "FR", "F", "T", "F", "S", "H", "A", "T", "C", "J", "N", "A", "T", "P"], "article-title": ["MEC Spatial Presence Questionnaire (MEC-SPQ): Short documentation and Instructions for Application. Report to the European Community, Project Presence: MEC (IST-2001-37661)"], "year": ["2004"]}, {"surname": ["Zimmermann", "Fimm", "Leclercq M, Zimmermann P"], "given-names": ["P", "B"], "article-title": ["A test battery for attentional performance"], "source": ["Applied Neuropsychology of Attention Theory, Diagnosis and Rehabilitation"], "year": ["2002"], "fpage": ["110"], "lpage": ["151"]}, {"surname": ["Holm"], "given-names": ["S"], "article-title": ["A simple sequentially rejective multiple test procedure"], "source": ["Scand J Stat"], "year": ["1979"], "volume": ["6"], "fpage": ["65"], "lpage": ["70"]}, {"surname": ["Knoch"], "given-names": ["D"], "article-title": ["Funktionelle Hemisph\u00e4renasymmetrie der Selbstkontrolle"], "source": ["Z Neuropsych"], "year": ["2007"], "volume": ["18"], "fpage": ["183"], "lpage": ["192"], "pub-id": ["10.1024/1016-264X.18.3.183"]}]
{ "acronym": [], "definition": [] }
30
CC BY
no
2022-01-12 14:47:25
Behav Brain Funct. 2008 Aug 4; 4:33
oa_package/b0/4e/PMC2529286.tar.gz
PMC2529287
18681960
[ "<title>Background</title>", "<p>Massive hemoptysis is a life threatening condition associated with a mortality rate exceeding 50% in the absence of adequate treatment [##REF##3425580##1##, ####REF##619806##2##, ##REF##5652400##3####5652400##3##]. A standardized and prompt management is required in emergency to improve survival, as well as a careful search of both the location and the etiology of bleeding. More than one hundred causes of massive hemoptysis have been described [##REF##11894445##4##]. Nevertheless, no cause is identified in about 15% of the cases, despite a complete investigation including fiberoptic bronchoscopy and CT-scan [##REF##9266882##5##,##REF##10208199##6##].</p>", "<p>We have recently reported a few subset of patients with so-called cryptogenic hemoptysis, in whom an unexpected vascular abnormality was demonstrated at pathological examination of the pulmonary resection, characterized by a bronchial artery running within the submucosa and called Dieulafoy disease of the bronchus [##REF##17332480##7##]. Additionally, only eight cases of bronchial Dieulafoy disease proved on histological data in six case reports have been reported to date [##REF##16122511##8##, ####REF##16141709##9##, ##REF##15842649##10##, ##REF##11157620##11##, ##REF##7638820##12##, ##REF##10325926##13####10325926##13##]. To our knowledge, no case series have been published from a tertiary referral center managing massive hemoptysis.</p>", "<p>The aim of our study was to report the clinical presentation of the patients who underwent surgery for massive hemoptysis in our center and in whom a Dieulafoy disease of the bronchus was eventually diagnosed and to provide detailed information on the approach to pathological diagnosis in this field.</p>" ]
[ "<title>Patients and methods</title>", "<title>Study design</title>", "<p>All the patients were recruited in a respiratory intensive care unit of a 800-bed tertiary university hospital in Paris, France, between May 1995 and July 2006. During the study period, 810 patients were admitted to our unit for hemoptysis, 111 of whom underwent surgery. Part of those patients has already been reported from our group [##REF##17332480##7##,##REF##17302979##14##]. The study was conducted according to the French law which judged unnecessary ethical approval and patient consent for such a retrospective analysis of medical records.</p>", "<title>Diagnosis of Dieulafoy disease of the bronchus</title>", "<p>The diagnosis of Dieulafoy disease of the bronchus was clinically suspected in case of the need for surgery for massive hemoptysis with no identifiable cause after fiberoptic bronchoscopy and CT-scan. Dieulafoy disease of the bronchus was pathologically confirmed by the evidence of an isolated and localized area of hemorrhage with no underlying lung disease known to be associated with bronchial systemic hypervascularization (such bronchiectasis) on macroscopic inspection plus the evidence of an unusual large superficial bronchial artery located into the submucosa, possibly extended through the mucosa into the bronchus lumen with no vasculitis, aneurysm or arteriosclerosis on microscopic examination.</p>", "<title>Pathologic lung study</title>", "<p>The lung samples (lobectomy) were processed in an uniform way by the same pathologist (MA), when Dieulafoy disease was suspected. First, the fresh lung resection was macroscopically examined. Macroscopic inspection confirmed the focal nature of the hemorrhage. Nodular lesions, blebs, areas of induration and subpleural hypervascularization were checked. Segmental and sub segmental bronchi were subsequently opened with chisel in the absence of obvious etiology. The presence and the location of blood cloths were identified. After careful washing, bronchiectasis, bronchial inflammatory aspect and tumoral obstruction were searched as well as mucosal abnormalities, <italic>i.</italic>e. minute defect and ulceration. Second, after formalin fixation, 3 mm thick serial sections were performed perpendicularly to the axis of the suspected bronchus previously identified by the presence of cloths, focal areas of hemorrhage or mucosal abnormalities; the same procedure was applied to the other bronchi for comparison. Samples of fixed tissue were processed into paraffin block, perpendicularly to the bronchial axes. Third, serial sections were performed on the paraffin block focusing on the suspected bronchus until lesions were found. Sections were stained with hematoxylin-eosin-safran, and elastic stain (Miller stain) for vessel identification.</p>", "<title>Collection of patients' data</title>", "<p>The following prospectively collected clinical data were extracted from our data base and controlled with the review of the medical charts: baseline demographics, drug intake, comorbid conditions, severity of hemoptysis, clinical presentation, laboratory tests, chest radiography, fiberoptic bronchoscopy and CT scan, ICU management and vital status at ICU discharge.</p>" ]
[ "<title>Results</title>", "<p>Seven patients fulfilled the aforementioned criteria during the 11-year study period.</p>", "<title>Patients' characteristics</title>", "<p>The patients (5 males) were aged 54.3 ± 11.5 years (range, 38 to 69 years). They were current heavy smokers (49 ± 28.5 packs/years); all but one were alcohol abusers. Two had mild to moderate chronic obstructive pulmonary disease. One patient had a history of pulmonary tuberculosis. Two patients were treated for systemic hypertension, 1 of whom had a history of a transient ischemic stroke and unexplained intestinal bleeding (Additional file ##SUPPL##0##1##). A previous episode of hemoptysis had occurred in 5 patients, 3 of whom had previously received a bronchial artery embolization (BAE). Of note, 1 patient (patient n°4) had been managed in our center 19 months ago for a first episode of massive hemoptysis (amount of 400 ml). Four fiberoptic bronchoscopies were necessary to locate the bleeding in the left upper lobe, as CT scan showed bilateral ground glasses. A successful BAE was performed. The patient refused a secondary scheduled surgery despite the staff decision.</p>", "<p>The cumulative amount of bleeding ranged from 350 ml to more than 1000 ml on admission to our unit. There were, however, mild clinical and biological consequences of the bleeding. Bedside chest X-ray was unremarkable. High resolution (n = 5) or multidetector (n = 2) CT-scan angiography showed ground glass opacities in all patients, that were isolated or associated with alveolar opacities (n = 4). The upper lobes were mainly involved (n = 5). Of note, a frank contrast media extravasation within the lumen of the bronchus related to the focal hemorrhagic area was evidenced using multidetector CT-scan angiography (patient n°4). There was no lung parenchyma abnormality suggestive of carcinoma, bronchiectasis or tuberculosis. A flexible fiberoptic bronchoscopy was performed within the first 24 hours of admission, to locate both the side and site of the bleeding. Overall, 10 bronchoscopic procedures were performed. A bilateral bronchial flooding by blood was evidenced in all patients. The location of the bleeding was successfully performed after bronchoscopic techniques in 5 patients.</p>", "<title>Management</title>", "<p>The therapeutic management was standardized, as described elsewhere [##REF##17302979##14##]. Bronchoscopic techniques were attempted to control the bleeding, combining blood aspiration and local instillation of cold saline lavage. Vasoconstrictive agents were delivered bronchoscopically (adrenalin, n = 4), intravenously (terlipressin, n = 1) or both (n = 1). A BAE was first attempted in all patients totaling 10 sessions before surgery (Figure ##FIG##0##1##). Technical failure of bronchial arteriography was related to failure of canulation in 2 patients (patients n°1 &amp; n°3) and anatomical consideration in 2 others (patients n°5 &amp; n°6). All bronchial arteries draining the bleeding site were enlarged without systemic to pulmonary artery shunting. A frank contrast media extravasation into the bronchial lumen was evidenced in 2 patients (patients n°2 &amp; n°7). Altogether, BAE was completed in 5 cases (4 patients) and controlled the bleeding in 2 cases (2 patients). According to the high initial amounts of bleeding, all the patients were secondary referred for surgery after 6.7 ± 5.8 days (median time 5 days). The lobe to remove, from which the bleeding originated, was identified by the combination of clinical examination, chest X-ray, CT scan and bronchoscopic findings. Bronchial arteriography was not used for locating the bleeding.</p>", "<title>Follow-up after surgery</title>", "<p>All patients were alive at hospital discharge. Follow-up data were available for a mean duration of 35 ± 30 months (range, 6 to 96 months) after surgery. All but one patient (patient n°4) remained free of bleeding recurrence during follow-up. This latter patient was treated successfully with BAE. The patient n°7 died from an ischemic stroke 12 months later.</p>", "<title>Pathological findings</title>", "<p>Lung macroscopic examination located the pathological area in all patients by showing a focal hemorrhagic area within the parenchyma and identifying a localized clot in the corresponding segmental (n = 5) or sub segmental bronchus (n = 2) (Additional file ##SUPPL##1##2##). Moreover, a minute bronchial mucosal defect was eventually observed (n = 3) (Figure ##FIG##1##2A##). There was no evidence of bronchial or vascular chronic disease. The macroscopic inspection was unremarkable, except blebs (patients n°1 &amp; n°2) and nodular mass (patient n°6) that were distant from the pathological hemorrhagic area.</p>", "<p>Microscopic examination revealed a large and dysplasic superficial bronchial artery in the submucosa in all patients beneath the cartilage (Figure ##FIG##1##2B##). Artery ulceration with rupture into the bronchial lumen was observed in 3 patients (Figure ##FIG##1##2C##). The structure of the bronchial artery appeared dysplastic with irregular thickness of the wall, which was either fibrotic or rich in elastic fibrils and had a tortuous appearance when cut at various angles. These arteries appeared on Miller stain with a thick internal lamina and a thin external one, in favor of the bronchial nature of the artery (Figure ##FIG##1##2D##). Finally, we observed (patient n°4) the material of embolization in the lumen of the vessel in 1 patient (Figure ##FIG##1##2B##). The respiratory epithelium was either clearly or slightly eroded and appeared sometimes metaplastic, although the structure of the bronchus remained normal. Of note, the nodular mass aforementioned (patient n°6) was related to a granulomatous disease consistent with an old sarcoidosis. In another patient (patient n°1) a deposit of amyloidal structure was identified. Otherwise, histological examination was unremarkable. No evidence of chronic bronchial or other vascular disease was identified. No microorganisms were grown, after special staining and culture for mycobacteria and fungi.</p>" ]
[ "<title>Discussion</title>", "<p>Our study aimed at better describing the process for diagnosing the Dieulafoy disease of the bronchus, from the clinical suspicion to the pathological confirmation, based on a series of 7 patients who underwent surgery for massive hemoptysis in a referral center over a 11-year period. The condition was clinically suspected in heavy smokers with recurrent and unexplained episodes of massive hemoptysis, characterized by amounts of bleeding both high and rather disproportioned, as compared with those usually reported in patients presumed to have a cryptogenic hemoptysis. Although there were no specific CT-scan or angiographic criteria, the frequent findings of both the direct and frank contrast media extravasation within the suspected bronchial lumen and the enlarged aspect of the bronchial artery without systemic to pulmonary artery shunting were suggestive of the vascular anomaly. A subsequent structured and rigorous pathological examination of the surgical lung resection confirmed definitively the diagnosis.</p>", "<p>Dieulafoy disease is a vascular anomaly characterized by the presence of a dysplastic artery in the submucosa. It was first reported in the gastrointestinal tract, accounting for up to 2% of the bleedings [##REF##11419815##15##]. The disease has been recently described in the respiratory tract [##REF##7638820##12##]. However, the incidence of the Dieulafoy disease of the bronchus is unknown and probably slightly underestimated regarding to the rigorous pathological procedure needed for establishing the diagnosis. In our experience, Dieulafoy disease of the bronchus accounted for at least 6% of the patients undergoing surgery for hemoptysis overall and up to 55% of the patients undergoing surgery for hemoptysis presumed to be cryptogenic [##REF##17332480##7##].</p>", "<p>The diagnosis of Dieulafoy disease of the bronchus should be suspected on the combination of history, clinical features and imaging investigations in order to consider surgical treatment and alert the lung pathologist. First, our patients were heavy smokers, similarly to the previous published isolated case reports. Second, respiratory past history was unremarkable, except unexplained and severe episodes of hemoptysis in 5 patients, 3 of whom had received a BAE (Additional file ##SUPPL##0##1##). Third, hemoptysis was massive and presumed to be cryptogenic, since no cause was identified after physical examination, fiberoptic bronchoscopy and CT-scan. Conversely to the gastrointestinal disease for which the endoscopic findings are diagnostic, we did not use bronchoscopic criteria to diagnose the vascular disease because the source of bleeding may be difficult to assess during active massive hemoptysis [##REF##10205720##16##, ####REF##8200191##17##, ##REF##10834728##18####10834728##18##], the bronchial abnormalities may be sub segmental and therefore not accessible and the small size of the bronchial lesion (usually less than 10 mm) may be difficult to detect when surrounded by clots. However, a few mucosal abnormalities have been bronchoscopically described in this setting, such as a smooth elevated non pulsating lesion [##REF##10325926##13##] or a nodular lesion within a normal overlying mucosa [##REF##16141709##9##]. It should be emphasized that these later bronchoscopic findings are not specific and may be related to bronchial artery aneurysms, arteriovenous malformations or small cancers [##REF##3829738##19##, ####REF##1486979##20##, ##REF##8563125##21####8563125##21##]. As the amount of bleeding related to Dieulafoy disease may be massive, bronchial biopsies should be avoided in this setting, even during a period of non active bleeding [##REF##16141709##9##,##REF##10325926##13##]. Moreover, in our opinion, performing biopsy should be not useful in this setting, since the diagnosis of Dieulafoy disease of the bronchus should be based on the pathological examination of a large surgical lung resection. Fourth, the bronchial arteriography findings did neither evidence systemic to pulmonary shunts nor aneurysms. Additionally, the arteries appeared all enlarged and frank contrast media extravasation in the bronchial lumen was frequent, when combining the findings of both BAE (n = 2) and multidetector CT-scan angiography (n = 1). These findings are in accordance with those reported by <italic>Durham et al </italic>in gastrointestinal Dieulafoy disease [##REF##2305095##22##]. Conversely, little angiographic data are available regarding to the bronchial artery disease [##REF##16122511##8##, ####REF##16141709##9##, ##REF##15842649##10####15842649##10##,##REF##7638820##12##,##REF##14530826##23##,##REF##11933825##24##]. Dilated vessels have been described [##REF##16141709##9##,##REF##15842649##10##,##REF##14530826##23##], especially in association with specific parenchymal diseases [##REF##15842649##10##,##REF##14530826##23##]. Last, although no firm conclusions can be drawn regarding to the small size of our population, our study highlights the poor efficacy of BAE in this clinical setting, as compared with the usual 80% to 90% successful rate of bleeding control using this procedure [##REF##8200191##17##]. Nevertheless, owing to the morbidity and the mortality related to emergency surgery performed during active bleeding, we recommend to attempt BAE as the first-line therapeutic approach [##REF##17332480##7##]. Additionally, some patients with a non-diagnosed Dieulafoy disease may have probably been treated with BAE.</p>", "<p>To our knowledge, our series is the first to carefully describe the pathological investigation to diagnose the Dieulafoy disease of the bronchus on surgical lung resection. The main pathological criterion is the evidence of a large and superficial bronchial artery located within the sub mucosa [##REF##16122511##8##, ####REF##16141709##9##, ##REF##15842649##10##, ##REF##11157620##11##, ##REF##7638820##12##, ##REF##10325926##13####10325926##13##]. In our series, the macroscopic analysis was crucial to detect a minute mucosal defect, as usually observed in the Dieulafoy disease of the gastrointestinal tract. As ectopic bronchial arteries have also been described during chronic pulmonary diseases [##REF##8200191##17##,##REF##1989820##25##,##REF##11348980##26##], a special attention was made to exclude chronic lung diseases, such bronchiectasis and other inflammatory processes or carcinoma. Additionally, CT scan demonstrated no parenchymal abnormalities, except ground glass or alveolar opacities reflecting the severity of bleeding [##REF##16916805##27##]. Although a few parenchymal abnormalities (blebs, nodular mass and amyloidal deposit) were pathologically evidenced, those later were actually distant from the focal hemorrhagic area and consequently not considered as the cause of bleeding.</p>", "<p>The pathogenesis of Dieulafoy disease remains unclear. Whether the origin of the anomaly is congenital, acquired or merely a variation of normal is not known. As suggested in our study, age and/or tobacco use may be predisposing states to the occurrence of the disease. Even if the dysplastic artery wall change may contribute to its weakness, the trigger factor of the vessel rupture is not known. Does the vessel rupture occur after a mucosal injury or does the vessel pressure induce a mucosal defect is unclear. Furthermore, the nature of the vessel that is bleeding remains controversial. Some have suggested that the artery belongs to the pulmonary vasculature owing to the failure of BAE, while others identified the vessel as originating from the systemic vasculature [##REF##15842649##10##,##REF##10325926##13##]. In our series, the angiographic data are supporting this latter hypothesis, as well as the pathological findings of the vessel with elastic stain and the intravascular evidence of material of embolization (patient n°4) (Figure 3B). Last, Dieulafoy disease might be a part of a general disease as suggested by the clinical history and outcome of the patient n°4.</p>", "<p>The limitations of our study are related to its retrospective nature and to the fact that it was conducted on patients undergoing surgery in a referral center with an extensive experience of severe hemoptysis.</p>", "<p>In summary, Dieulafoy disease of the bronchus should be considered in a heavy smoker patient with unexplained and recurrent massive hemoptysis, after a rigorous confrontation of both clinical and radiological findings. Useless and dangerous bronchial biopsies should be avoided. The therapeutic approach should be surgical and the pathological examination should be structured and meticulous.</p>" ]
[]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>There are limited series concerning Dieulafoy disease of the bronchus. We describe the clinical presentation of a series of 7 patients diagnosed with Dieulafoy disease of the bronchus and provide information about the pathological diagnosis approach.</p>", "<title>Patients and methods</title>", "<p>A retrospective review of patients who underwent surgery for massive and unexplained recurrent hemoptysis in a referral center during a 11-year period.</p>", "<title>Results</title>", "<p>Seven heavy smoker (49 pack years) patients (5 males) mean aged 54 years experienced a massive hemoptysis (350–1000 ml) unrelated to a known lung disease and frequently recurrent. Bronchial contrast extravasation was observed in 3 patients, combining both CT scan and bronchial arteriography. Efficacy of bronchial artery embolization was achieved in 40% of cases before surgery. Pathological examination demonstrated a minute defect in 3 cases and a large and dysplasic superficial bronchial artery in the submucosa in all cases.</p>", "<title>Conclusion</title>", "<p>Dieulafoy disease should be suspected in patients with massive and unexplained episodes of recurrent hemoptysis, in order to avoid hazardous endoscopic biopsies and to alert the pathologist if surgery is performed.</p>" ]
[ "<title>Abbreviations</title>", "<p>BAE: Bronchial artery embolization.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Financial support</title>", "<p>None</p>", "<title>Authors' contributions</title>", "<p>AP had full access to the data and takes responsibility for the integrity of the data at the accuracy of the data analysis. MA performed the pathological analysis. All authors read and approved the final manuscript.</p>", "<title>Supplementary Material</title>" ]
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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Bronchial arteriography efficacy</bold>. ASA = anterior spinal artery; RBICT = right broncho-intercostal trunk.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Pathological Findings</bold>. Macroscopic view showing a minute defect within the bronchial tree (arrow) (A). Low-power view showing the location of the vessel in the sub-mucosa beneath the cartilage plate, and the presence of the material of embolization in the lumen (arrow) (B). High-power view revealing the protrusion of the superficial vessel in the lumen with an ulceration and a squamous metaplasia of the epithelium (C). High power-view showing a dysplastic artery with elastic stain (Miller stain) (D).</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p><bold>Table S1.</bold> Patients' characteristics, management and outcome. Comparison with the literature cases.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional file 2</title><p><bold>Table 2.</bold> Pathological findings. Comparison with the literature cases.</p></caption></supplementary-material>" ]
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[ "<graphic xlink:href=\"1465-9921-9-58-1\"/>", "<graphic xlink:href=\"1465-9921-9-58-2\"/>" ]
[ "<media xlink:href=\"1465-9921-9-58-S1.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>", "<media xlink:href=\"1465-9921-9-58-S2.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>" ]
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{ "acronym": [], "definition": [] }
27
CC BY
no
2022-01-12 14:47:25
Respir Res. 2008 Aug 5; 9(1):58
oa_package/89/a6/PMC2529287.tar.gz
PMC2529288
18699987
[ "<title>Background</title>", "<p>SLPI is an efficient inhibitor of NE and other serine proteinases[##REF##3462719##1##,##REF##3977840##2##] and is found in high concentrations in secretions such as respiratory mucus[##REF##881164##3##]. <italic>In vitro </italic>it has antibacterial [##REF##8890201##4##, ####REF##11053013##5##, ##REF##12023766##6####12023766##6##] and antifungal[##REF##9291323##7##] properties and has been shown to prevent viral infection[##REF##10799472##8##,##REF##9089405##9##]. In addition it has anti-inflammatory properties distinct from inhibition of extracellular NE, that are potentially important in host defence and auto-immune conditions [##REF##12084717##10##, ####REF##10456890##11##, ##REF##16352738##12##, ##REF##15155685##13##, ##REF##10449524##14####10449524##14##]. These properties suggest that SLPI may be important in diseases such as bronchiectasis and COPD that are characterized by neutrophilic inflammation and infection. SLPI should be protective in these conditions, and indeed patients with chronic bronchitis (CB) have a higher concentration of SLPI in lung secretions than healthy controls[##UREF##0##15##], probably because of submucosal gland hypertrophy and increased serous cell secretion. However the amount of SLPI found in the sputum of patients with CB and bronchiectasis decreased during exacerbations [##REF##10588615##16##, ####REF##3698703##17##, ##REF##12885984##18####12885984##18##] and increased again in the stable state[##REF##10588615##16##,##REF##12885984##18##]. It was also lower in the stable state in sputum from patients with frequent exacerbations compared to those with infrequent exacerbations [##REF##11120902##19##], suggesting that low concentrations of SLPI increase the risk of developing an exacerbation. In addition it was lowest in those patients with the greatest neutrophilic inflammation in the stable state[##REF##10471615##20##], and lower in those who remained colonized in the stable state compared to those who cleared their infection[##REF##12885984##18##,##REF##10996579##21##]. This relation to infection has been confirmed in studies showing that SLPI was also reduced in vaginal secretions in the presence of bacterial infection [##REF##11084573##22##].</p>", "<p>In general, SLPI and NE concentration appear to be inversely related in sputum in COPD and CB[##REF##1385052##23##] but the reason for this relationship <italic>in vivo </italic>is not understood. The genetics of patients with early-onset COPD have been investigated and no mutations, deletions or disease-associated polymorphisms in the SLPI gene have been described[##REF##1674946##24##]. In our studies over 20 years, we have not identified any patients with an absence of SLPI in sputum. Therefore at present the concept that a primary deficiency of SLPI initiates inflammation or infection is not well supported by the available data.</p>", "<p>A number of studies have described an inverse relationship between SLPI and NE <italic>in vitro </italic>using tissues ranging from primary nasal and bronchial epithelial cells, through a variety of cell lines, to isolated tracheal submucosal glands [##REF##11133497##25##, ####REF##7946401##26##, ##REF##10979241##27##, ##REF##9384232##28####9384232##28##]. Since cell culture supernatant from cells exposed to NE has a lower concentration of SLPI protein than supernatant from cells not exposed to NE, neutrophilic inflammation may directly predispose to low SLPI <italic>in vivo</italic>. Previous <italic>in vitro </italic>work also included measurement of SLPI gene expression in cells exposed to NE and high levels were found to increase gene expression [##REF##7946401##26##, ####REF##10979241##27##, ##REF##9384232##28##, ##REF##8105697##29####8105697##29##]. The fall in protein must therefore relate to alterations in post-transcriptional events. Immunohistochemical studies of primary nasal epithelial cells showed greater SLPI protein in cells treated with NE than those not treated, suggesting failure of secretion or redistribution of the protein rather than failure of synthesis[##REF##9384232##28##]. Studies of NE-treated cells lysed using a detergent-based solution after removal of media demonstrated that the SLPI not found in cell culture media was in the cell lysate [##REF##10979241##27##].</p>", "<p>One mechanism that might explain the NE-induced redistribution of SLPI protein hinges on the positive charge of the NE molecule, which may enable it to associate with cell membranes [##UREF##1##30##]. Once associated with epithelial cell membranes, it could bind SLPI, although not as efficiently as in free solution[##REF##7593196##31##], and would hence remove SLPI from cell supernatant. The known structures of NE[##REF##2911584##32##] and SLPI[##REF##3366116##33##] suggest that charged residues may remain exposed on the outside of each molecule when complexed with each other, so the NE-SLPI complex is likely to have a more potent positive charge and therefore is more likely than SLPI alone to associate with cell membranes and negatively-charged proteins.</p>", "<p>Other possible mechanisms include failure to secrete, or binding of SLPI to other cell surface proteins exposed by NE. For example, SLPI has been shown to bind specifically to annexin II[##REF##15545357##34##] and to scramblase[##REF##10869562##35##], and these may potentially be upregulated or exposed in the presence of proteinases. It is known that there is a receptor specific for the complex of NE and α1 antitrypsin (α1 AT) [##REF##2160076##36##], and therefore it is also possible that the SLPI-NE complex binds to a specific receptor. Finally, there may be a mechanism to import SLPI actively into cells. Studies with neutrophils[##REF##9201260##37##], megakaryocytes and platelets[##REF##15315966##38##] show SLPI inside cells, and a recent study indicated that macrophages can import exogenous SLPI into both the cytoplasm and nucleus[##REF##16352738##12##].</p>", "<p>In the present study, we have investigated the hypothesis that charge-related association of the SLPI-NE complex with cell membranes is the primary mechanism reducing SLPI concentration in cell culture supernatants from SLPI producing lung epithelial cells in the presence of NE. We have conducted experiments investigating the nature of the effect of NE on SLPI protein levels in cell culture supernatant, and pursued preliminary studies into the localization of SLPI in these cells when exposed to NE.</p>" ]
[ "<title>Methods</title>", "<title>Proteinases and inhibitors</title>", "<p>NE was purified from empyema fluid using the method of Martodam et al[##REF##441048##39##]. Absence of endotoxin contamination was confirmed with the Limulus amoebocyte assay (E-TOXATE, Sigma, UK). Activity of NE was measured using the synthetic chromogenic substrate N-succinyl-(ala)<sub>3</sub>-<italic>p</italic>-nitroanilide (Sigma, UK). Cathepsin G (CG) was similarly obtained from empyema fluid and activity was measured using N-succinyl-phe-pro-phe-<italic>p</italic>-nitroanilide (Bachem, UK). Porcine pancreatic elastase (PPE) was obtained from Sigma and the activity was measured using N-succinyl-(ala)<sub>3</sub>-<italic>p</italic>-nitroanilide. Trypsin was cell culture grade (Invitrogen) and activity was measured using N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) (Sigma). Human mast cell chymase and tryptase were obtained from Elastin Products Co. and activity was measured using N-succinyl-val-pro-phe-<italic>p</italic>-nitroanilide and Z-gly-pro-arg-<italic>p</italic>-nitroanilide respectively (both from Bachem). ZD0892, a small synthetic peptidyl trifluoromethylketone NE inhibitor with an affinity for NE similar to that of SLPI[##REF##9379436##40##], was a gift from Zeneca Pharmaceuticals (Wilmington, USA) and inhibitory function was measured against a known amount of active NE using N-succinyl-(ala)<sub>3</sub>-<italic>p</italic>-nitroanilide. Recombinant human SLPI (rhSLPI) was a gift from Amgen and the concentration was confirmed using the SLPI ELISA (see below). Inhibitory activity was determined as for ZD0892.</p>", "<title>Measurement of SLPI and total protein</title>", "<p>SLPI concentration was measured using the R&amp;D Systems ELISA, a sandwich ELISA consisting of a mouse monoclonal antibody for capture and a horseradish peroxidase-conjugated polyclonal antibody for detection. Total protein was measured in microplates using the Bio-Rad assay (Bio-Rad, USA). The ELISA was validated using mixtures of SLPI and NE or CG in varying proportions to ensure that the proteinases did not interfere with quantification of SLPI. Briefly, recombinant human SLPI supplied as standard in the ELISA kit was mixed at varying molar ratios with NE and CG, incubated at 37°C for 30 minutes and the amount of SLPI recoverable was measured using the ELISA.</p>", "<title>Cell culture</title>", "<p>A549 cells, a lung epithelial cell line derived from lung carcinoma, were obtained from the ECACC and cultured in 50/50 F10/DMEM (both from Gibco, UK) with 10% fetal calf serum (FCS) (Gibco). HepG2 cells, a liver epithelial cell line derived from liver carcinoma, were obtained from the ATCC. They were cultured in DMEM containing 10% FCS, antibiotic antimycotic solution, 150 mmol L-glutamine and MEM non-essential amino acids (all from Sigma). Primary human bronchial epithelial cells (PBEC) were obtained from Cambrex (UK) and cultured in the same supplier's recommended basal media with growth supplements according to their instructions. Experiments were carried out with cells between passage 3 and 8, using basal media only.</p>", "<p>Most experiments were performed on A549 cells and selected experiments were repeated on PBEC or HepG2 cells in standard submerged monolayer culture. All experiments were performed in at least triplicate wells, and results are expressed as mean ± standard error of the mean (SEM) of at least 3 experiments. Cells were cultured to confluence in T75 or T25 flasks (Gibco) and plated out into 12 or 24 well plates (Gibco). Once confluent, the media were changed to serum free media (SFM) (50/50 F10/DMEM for A549 cells, DMEM alone for HepG2 cells and basal media without additives for PBEC). A549 cells and HepG2 cells were cultured in SFM for a further 24 h before experiments were performed. After treatment with the experimental conditions, supernatants were aspirated and stored at -70°C for later analysis for SLPI and protein concentrations. Cells were rinsed with PBS and harvested using trypsin to detach them and viability was assessed where appropriate using trypan blue exclusion. Statistical analysis was performed using SPSS version 12 for Windows.</p>", "<title>Experiments</title>", "<title>1. Dose response</title>", "<p>A dose response experiment was undertaken to examine the relationship of NE concentration to the reduction of SLPI concentration in cell culture supernatants. The initial concentration of SLPI in the cell supernatants was not known at the outset of an experiment. Preliminary experimental data showed that the typical concentration of SLPI in supernatants of A549 cells after 24 h culture in SFM was between 0.5 and 2 nM (5–23.4 ng/ml). Cells were therefore incubated in SFM alone as control, or SFM containing NE from 0.5 to 5 nM. Initial experiments also included a control consisting of the buffer used to suspend the NE, in SFM at the same concentration as the highest concentration of NE used. Dose response experiments were repeated with CG and using PBEC with both proteinases.</p>", "<title>2. Effect of other proteinases</title>", "<p>Proteinases such as PPE have, like NE, been shown to increase SLPI expression[##REF##8105697##29##], although the effect on protein secretion has not been reported. PPE (which does not bind SLPI), human mast cell chymase, trypsin and human mast cell tryptase (which bind SLPI with varying affinity but do not carry a high positive charge) were used to assess whether the degree of binding to SLPI by the proteinase or its ability to associate with cell membranes by charge would modify the ability of the proteinase to reduce SLPI in the supernatant. The effect on SLPI secretion was compared for PPE, trypsin, chymase, tryptase and CG by incubating cells with varying concentrations of each proteinase for 24 h.</p>", "<title>3. Time course</title>", "<p>The hypothesis of binding of the NE-SLPI complex to cell membranes because of positive charge suggested that addition of NE to conditioned media would cause removal of SLPI that had already been secreted. Confluent PBEC were cultured for 24 h in SFM and at the end of this period an aliquot was removed for baseline analysis and replaced with either SFM or NE at a concentration sufficient to achieve 10 nM in each well. After gentle mixing of the contents of the wells once with a 1 ml pipette, cells were incubated for a further 24 h and aliquots of 25 μl were taken at various time points within this period and stored at -70°C for analysis by ELISA. Similar experiments were repeated on A549 cells using both NE and CG, and with 1 nM rhSLPI in DMEM added to HepG2 cells followed by NE or SFM control. HepG2 cells were used as a control cell line that does not express or secrete SLPI, to demonstrate the effect of proteinases on SLPI concentration without any possibility of secreted SLPI contaminating the results.</p>", "<title>4. Effect of a synthetic NE inhibitor</title>", "<p>Addition of NE inhibitors to culture media containing NE has been shown to abrogate the fall in SLPI protein[##REF##10979241##27##]. If binding of NE to SLPI is important in the mechanism of the fall in SLPI protein, then the timing of the addition of the inhibitor will be equally important. NE was incubated for 30 min at 37°C with a tenfold excess of ZD0892. The mixture of NE and ZD0892 was added to the supernatant of A549 cells that had been serum-starved for 24 h, to achieve a final concentration of 10 nM NE and 100 nM ZD0892. This was compared with NE 10 nM added 10 minutes prior to the addition of 100 nM ZD0892, and ZD0892 100 nM added 10 minutes prior to the addition of NE 10 nM. Control cells were treated with NE 10 nM alone, ZD0892 100 nM alone, or SFM alone. All mixtures were incubated for a further 10 minutes and the supernatants were then aspirated and stored at -70°C for analysis by ELISA.</p>", "<title>5. Localization of SLPI in cell culture system</title>", "<p>Given that many proteins associate non-specifically with charged surfaces such as cell culture plastic, we speculated that the NE-SLPI complex may associate more with cell culture materials than SLPI alone. This was tested by comparing the concentration of SLPI in solutions added to empty tissue culture plates with the concentration of SLPI in solutions added to wells containing confluent cells, with and without NE. SLPI was added to SFM to achieve a concentration of 1 nM, and the solution was incubated in 12 well plates for 10 minutes, a baseline aliquot was taken for measurement of SLPI and replaced with SFM or NE to achieve a final enzyme concentration of 10 nM. After a further period of incubation the contents of the wells were harvested for measurement of SLPI concentration. In order to block the effect of non-specific binding, both 12 well plates containing cells and 12 well plates without cells were also incubated with media containing 1% w/v human serum albumin (HSA) and SLPI at 1 nM. The effect of 10 nM NE was assessed as before in both sets of plates. Plates were also coated with HSA prior to addition of cells, and the above experiments were repeated using plates with or without A549 cells. Finally in cell-free conditions, the effect of 1% Tween 20 (polyoxyethylene sorbitan monolaurate, Sigma) was assessed in the same way.</p>", "<title>6. Two compartment model</title>", "<p>SLPI is secreted both basally and apically by cells <italic>in vitro</italic>[##REF##8343852##41##], and <italic>in vivo </italic>epithelial and glandular cells might be exposed to NE release from neutrophils at both sites during neutrophil migration. To examine whether this may influence the secretion of SLPI, a two compartment model was studied. Transwells (Transwell PET 0.4 μm pore size 12 mm inserts, Corning Life Sciences) were coated with human placental collagen (Sigma) at 50 μg/ml and dried in air. A549 cells were added to the upper compartment in 500 μl media, with 1.5 ml media in the lower compartment. As soon as the cells appeared as a confluent monolayer, media were replaced by SFM at the same volumes. After a further 24 h incubation, reagents were added to upper and lower compartments. Triplicate wells received either SFM alone in both compartments or NE 10 nM in one compartment with SFM in the other. Supernatants were aspirated after a further 10 minutes incubation, and stored at -70°C for later analysis for total protein and SLPI concentration.</p>", "<title>7. Theoretical modelling of the external charge of the complex between SLPI and proteinases</title>", "<p>The modelling procedure involved the use of the X-ray crystal structure of SLPI with chymotrypsin (structure file kindly provided by Professor Wolfram Bode as described previously[##REF##3366116##33##]) as a template to construct analogous complexes with other proteinases. X-ray crystal structures of NE (PDB ID 1H1B; [##REF##12190311##42##]), CG(PDB ID 1T32;[##REF##15741158##43##]), trypsin (PDB ID 1TX6;[##REF##15381428##44##]), mast cell chymase (PDB ID 1T31;[##REF##15741158##43##]) and mast cell tryptase (PDB ID 2BM2;[##REF##15781396##45##]) were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank[##REF##10592235##46##]<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.pdb.org\"/>. These structures were then superimposed on the proteinase in the SLPI-proteinase complex using SWISSPDB[##REF##9504803##47##]<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.expasy.org/spdbv/\"/>. The values for the original proteinase (chymotrypsin) were then deleted and the new complex saved. SLPI alone was obtained by deleting chymotrypsin from the original structure file. Electrostatic surfaces were generated using SWISSPDB with values of -3.00, 2.00 and 8.00 for the red, white and blue extremes of the spectrum.</p>" ]
[ "<title>Results</title>", "<p>The ELISA confirmed SLPI concentration was fully recoverable at the range of concentrations of NE and CG used in these experiments. However at very high molar ratios of proteinase to inhibitor (&gt; 100:1), there was a fall in quantification which was attributed to the effects of unopposed proteinase activity on the antibody used in the ELISA (Figure ##FIG##0##1##).</p>", "<title>1. Dose-dependent effect of NE on SLPI in A549 supernatants</title>", "<p>NE at the concentrations used in these experiments did not affect cell viability (by trypan blue exclusion, data not shown). The concentration of SLPI after 24 h showed a dose related fall with NE treatment from 1.36 ± 0.37 nM at 0.5 nM to 0.20 ± 0.14 nM at 5 nM, whilst media control contained 1.63 ± 0.31 nM (p = 0.006, one way ANOVA) (Figure ##FIG##1##2##). Similar results were obtained when the experiments were repeated with A549 cells using CG and with PBEC using NE (data not shown).</p>", "<p>Using a dose range from 0–2.5 nM NE, a linear relationship was seen between the difference in SLPI concentration between treated and control cells, and the amount of NE (Figure ##FIG##2##3##). This relationship suggests about 1.4 molecules of NE are required to remove 1 molecule of SLPI from cell culture media.</p>", "<title>2. Effect of other proteinases</title>", "<p>At high concentrations of proteinases, all cells detached from the wells although cell viability was not affected during the duration of the experiment as assessed by trypan blue exclusion (data not shown). At these higher proteinase concentrations, SLPI concentration fell sharply. CG caused a dose-dependent fall in SLPI concentration (79.9 ± 8.2% of control values (p &lt; 0.05) at 1 nM, 4.7 ± 4.3%, 0.02 ± 0.0% and 0.07 ± 0.1%, (p &lt; 0.001) at 10 nM, 100 nM and 1000 nM respectively), but had no effect at 0.1 nM (96.2 ± 0.8% control) while SLPI concentration fell significantly only at high concentrations for PPE (9.0 ± 3.5% at 100 nM, 2.6 ± 1.3% at 1000 nM, p &lt; 0.005) and trypsin (4.5 ± 0.8% at 1000 nM, p &lt; 0.005), at which cell morphology was affected. Mast cell chymase caused a significant reduction in SLPI secretion at concentrations from 10–100 nM. This was associated with a marked change in cell morphology: loss of cell-cell contact but no detachment. At lower concentrations, with normal cell morphology, there was no effect on SLPI secretion. Mast cell tryptase did not affect either SLPI concentration or cell morphology at any dose (Figure ##FIG##3##4##).</p>", "<title>3. Time course of the effect of NE</title>", "<p>Several groups have demonstrated that the presence of NE reduces the amount of SLPI in cell supernatants after periods of culture ranging from 1 h to 48 h[##REF##7946401##26##,##REF##10979241##27##]. When NE was added to wells containing A549 cells following 24 hours incubation (therefore containing substantial amounts of SLPI), a rapid fall in SLPI concentration was seen at the earliest time point tested (10 min). The results were similar with PBEC (Figure ##FIG##4##5a##), A549 cells (data not shown) and HepG2 cells (Fig ##FIG##4##5b##). CG also caused a decrease in supernatant SLPI from media of A549 cells within 10 min (control wells 102.36 ± 3.75% of baseline, wells treated with CG 10 nM 1.50 ± 0.27% baseline at 10 minutes). Subsequent experiments using A549 cells showed that NE already caused a detectable decrease in SLPI within 2 min (data not shown).</p>", "<title>4. Effect of synthetic NE inhibitor</title>", "<p>ZD0892 alone did not affect SLPI concentration in A549 supernatants (99.12 ± 4.7% of media control). The concentration of SLPI in NE-treated wells fell to 12.05 ± 2% of control values (p &lt; 0.001 compared to media control). When NE and ZD0892 were pre-incubated, the effect of NE on SLPI was abrogated (100.2 ± 3.7% media control, p &lt; 0.005 compared to NE alone). Addition of NE after ZD0892 caused some decrease in SLPI concentration (to 63.0 ± 8.5% of media control, p &lt; 0.05 compared to media control) but the fall in SLPI concentration was less than with NE alone (p &lt; 0.05). Addition of NE before ZD0892 caused a fall in SLPI concentration (to 28.7 ± 13.7% control, p &lt; 0.05 compared to media control), which was not significantly different from NE alone. The difference between the results for NE and ZD0892 added in either order was not statistically significant. These experiments are summarised in Figure ##FIG##5##6##.</p>", "<title>5. Attachment of SLPI to plastic surfaces</title>", "<p>We next explored whether the loss of SLPI in the presence of NE required the presence of cells, or whether it would occur in cell-free conditions. We added SLPI to empty tissue culture plate wells with or without NE. There was a marked loss of SLPI from the empty tissue culture plates as only about 1% of the expected amount was recovered from control wells and therefore an additional effect of NE could not be identified. Further investigation demonstrated that there was a large loss of SLPI when diluted in SFM in standard 20 ml polystyrene tubes, and a further loss in tissue culture plates, which was attributed to charge-related non-specific binding to plastic (data not shown). This may have explained the observed decrease in SLPI in cells treated with high concentrations of trypsin or PPE that caused detachment, since this would have exposed culture plastic which would be capable of binding the previously secreted SLPI, resulting in a fall in SLPI concentration in the supernatant.</p>", "<p>Use of polypropylene plates and siliconised glass culture systems did not offer more than partial protection against this loss of SLPI (data not shown) and cells did not grow satisfactorily without a substrate on siliconised glass. Addition of HSA 1% w/v to culture media, or coating of tissue culture plates with HSA or human placental collagen prior to plating out cells, partially reduced the non-specific loss of SLPI from culture media (data not shown). Furthermore the presence of HSA in solution partially inhibited the effect of NE in reducing SLPI concentration in cell-free systems (Figure ##FIG##6##7##), and similarly in plates coated with HSA prior to plating out cells (data not shown). Addition of 1% Tween to cell-free systems completely prevented loss of SLPI when incubated without NE, but did not prevent almost total loss of SLPI in the presence of NE (data not shown).</p>", "<title>6. Two compartment model</title>", "<p>Supernatant from the upper compartments of Transwells on which A549 cells were cultured contained about fourteen times as much SLPI as the lower compartments. The difference may be due to polarized secretion of SLPI, or might be accounted for by SLPI binding to the plastic of the cell culture plates in the lower compartment, which had not been treated to prevent non-specific binding and contained no cells. Addition of NE to the upper compartment caused reduction in SLPI concentration to 10.6 ± 0.3% of control values, and in the lower compartment it caused a reduction to 8.6 ± 1.9% of control. SLPI concentrations in the control-treated compartment were not affected (Figure ##FIG##7##8##).</p>", "<title>7. Theoretical modelling of SLPI in complex with proteinases</title>", "<p>Figure ##FIG##8##9## shows the SLPI molecule alone (Fig ##FIG##8##9a##) and SLPI-proteinase complexes (Fig ##FIG##8##9b–f##). Using colour to indicate charge (red for negative, blue for positive), it can be seen that both NE and CG produced a highly positively-charged complex with SLPI. Mast cell chymase and trypsin varied in charge on the two aspects shown whereas mast cell tryptase was highly negatively-charged on both aspects. PPE was not modelled for this experiment because it does not bind SLPI. The results suggest that SLPI in complex with CG and NE would be predicted to associate more with negatively-charged surfaces than either free SLPI, or SLPI complexed with other proteinases, because of the greater positive charge.</p>" ]
[ "<title>Discussion</title>", "<p>The results from the present study show that both NE and CG decrease SLPI released into culture supernatants from lung epithelial cells. The dose response of the proteinases was linear, and the effect was almost immediate. Experiments using a synthetic NE inhibitor showed that when the inhibitor was added together with NE, the NE-mediated decrease in SLPI was completely prevented. In contrast, when the inhibitor was added after NE had been applied to the cells it only partially prevented a reduction in SLPI. Experiments using a Transwell model showed that the effect of NE on SLPI required direct contact between these molecules, suggesting that direct binding of NE or CG to SLPI was involved.</p>", "<p>Only the strongly cationic proteinases NE and CG were able to reduce SLPI concentration at almost molar equivalent doses. The other proteinases only caused reduction in SLPI when used at concentrations sufficient to alter cell morphology and/or cause detachment, which probably leads to non-specific binding to the tissue culture plastic. Theoretical modelling of surface charge supported the concept that an NE-SLPI or CG-SLPI complex would have a greater positive charge than SLPI alone or in complex with the other proteinases studied. Therefore it seems likely that the reduction in SLPI concentration in the presence of proteinases was mediated by charge-related binding to other structures.</p>", "<p>Previous studies on the effect of NE on SLPI <italic>in vitro </italic>have shown a decrease in SLPI concentration with a variety of cells and tissues [##REF##11133497##25##, ####REF##7946401##26##, ##REF##10979241##27####10979241##27##], but only two studies have attempted to explore this further. The study by Marchand <italic>et al</italic>[##REF##9384232##28##] used immunohistochemistry to examine nasal explanted epithelial tissue exposed to NE and found greater signal than in the control tissues not exposed to NE. The study by van Wetering <italic>et al</italic>[##REF##10979241##27##] used PBEC and again found a dose-dependent effect, present from a few hours after exposure of cells to NE, which could be inhibited by co-administration of an inhibitor (α1 AT). Analysis of cell lysates indicated that SLPI became cell-associated in the presence of NE rather than in the culture media, and hence suggested a block in secretion or redistribution of the protein rather than an effect on synthesis. These studies did not pursue more detailed analysis of the relationship between NE and SLPI concentration and the possible mechanism responsible for this relationship. In addition, none of the studies in the literature has controlled for non-specific binding of SLPI to tissue culture materials. Studies on the ability of SLPI to protect fibronectin from degradation by NE showed that SLPI could associate with this substrate through ionic interactions [##REF##7912452##48##] and examination of human lung tissue showed that SLPI was associated with elastin fibres[##REF##2764383##49##], again suggesting non-specific binding. SLPI binds non-specifically to many large molecules such as mucins[##REF##1346959##50##] and DNA[##REF##2467900##51##], however, to our knowledge no studies have examined whether the NE-SLPI complex is more likely to associate with cells or substrates than SLPI alone.</p>", "<p>Our initial validation studies demonstrated that in the presence of a molar excess of NE or CG, detection of SLPI by ELISA was not affected. The studies described here did not examine the possibility of cleavage or degradation of SLPI by excess of proteinase, but even if this happened, the cleavage products would appear to be detected fully by the ELISA, suggesting that the monoclonal capture antibody recognises an epitope not affected by proteolysis, and that the polyclonal detection antibody also recognises preserved sites. Incubation with samples containing excessive amounts of NE may however expose the capture antibody to proteolytic degradation, and this may be the explanation for the fall in detection at high concentrations of NE, rather than degradation of SLPI by NE. Since the validation studies used recombinant rather than endogenous SLPI and were conducted in assay buffer rather than conditioned cell culture media, it cannot be guaranteed that the ELISA would perform as reliably in experimental conditions. However in the published studies previously mentioned, the same effect of dose-dependent reduction of SLPI in the cell culture environment was shown by systems using a variety of different antibodies for detection of SLPI, suggesting that the effect is real rather than a systematic measurement error. Possible effects on the ELISA from complex formation or cleavage of SLPI therefore cannot fully explain the complete loss of signal of SLPI in the presence of cells treated with NE. Nevertheless, our experiments do not conclusively demonstrate that SLPI bound to cells in presence of NE is biologically active. Furthermore, it needs to be noted that the difficulties in avoiding non-specific binding of SLPI to the plastic components of the cell culture system are not fully countered in these studies. If the hypothesis that binding of SLPI to NE causes adherence to cells by charge interactions is valid, then the same mechanism could apply to any negatively-charged surface, such as plastic cell culture components which were also shown to bind free SLPI avidly. NE did appear to increase non-specific binding of SLPI to plastic, because when detergent was used as a blocking agent, SLPI concentration fell only in the presence of NE (suggesting that the higher ionic charge of the NE-SLPI complex was able to overcome the concentration of detergent used). The ELISA system contains strong buffering agents that must be presumed to protect against loss of free SLPI. The validation studies suggest that they are sufficient to protect also against loss of the NE-SLPI complex. However it cannot be presumed that there was not loss of SLPI to plastic during the cell culture process, harvesting of supernatant and storing of the samples prior to preparation of the ELISA, and that this loss of SLPI was not enhanced in the presence of NE solely by increased binding to plastic rather than to cell membranes. Proof of the concept of binding of SLPI to cells would require a different experimental approach, and therefore the experiments presented here cannot claim to prove that the effect is biological and relevant to the <italic>in vivo </italic>situation. The location of the lost SLPI protein remains uncertain, although preliminary immunofluorescence studies in our laboratory suggested at least in part that the SLPI does become internalised (data not shown). Further studies, using SLPI bound to a fluorescent or radioactive label that does not impede binding to NE, will be required to confirm this.</p>", "<p>There is a likely biological relevance of the phenomenon of free SLPI associating with cell membranes and extracellular matrix proteins because this would allow the lung to establish a protective antiproteinase coat over structures that would be at most risk of damage by free NE. The major function of NE is probably intracellular killing of bacteria within the phagolysosome. Where neutrophils are migrating through the lung, some local NE activity (from a released azurophil granule or released from cell membrane association) may possibly be beneficial to this process. Free NE in lung secretions however is believed to be uniformly deleterious. Where this occurs, it would be neutralized to some extent by the presence of SLPI bound to vulnerable structures. There may be some value in the SLPI-NE complex associating with these structures. If the NE-SLPI complex prevented further association of NE with extracellular matrix substrates more effectively than SLPI alone (because of its higher positive charge), it would leave the free NE in epithelial lining fluid to be inactivated by α1 AT, which the latter cannot do once NE is bound to substrate[##REF##3492198##52##]. There are no published data on the avidity of binding of the NE-SLPI complex to cell membranes or matrix substrates. However it is known that there is a strong association between NE and neutrophil cell membranes[##UREF##1##30##], and between SLPI and fibronectin[##REF##7912452##48##], based on the ionic strength or pH required to dissociate them. The ionic charge of the NE-SLPI complex has never been measured, because the binding of these two strongly cationic molecules is sufficiently reversible that standard electrophoretic methods cause them to dissociate. It is also unknown how stable the complex would be when membrane-bound. It is possible that membrane-bound complex might be able to dissociate, or be passively internalised during membrane cycling, or actively internalised by endocytosis or phagocytosis, probably requiring a specific receptor. Internalisation of the complex might generate biological responses central to lung inflammation. Further studies will clearly be required to explore these possibilities.</p>" ]
[ "<title>Conclusion</title>", "<p>In summary, the experiments described in this paper support the hypothesis that the association of SLPI in complex with NE to negatively-charged structures leads to its removal from cell culture media, and that this effect is a passive biochemical process, probably dependent on charge. It is likely that this effect occurs with cell membranes as well as non-biological components, but this has yet to be confirmed. Future experiments should include measures to counter the ability of SLPI to bind non-specifically to a variety of structures, particularly in the presence of NE or CG.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Secretory leukoproteinase inhibitor (SLPI) is an important inhibitor of neutrophil elastase (NE), a proteinase implicated in the pathogenesis of lung diseases such as COPD. SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes <italic>in vitro</italic>.</p>", "<title>Methods</title>", "<p>Respiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling.</p>", "<title>Results</title>", "<p>We observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration.</p>", "<title>Conclusion</title>", "<p>These data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon <italic>in vitro</italic>, which may correlate with changes observed <italic>in vivo</italic>.</p>" ]
[ "<title>Abbreviations</title>", "<p>SLPI: Secretory leukoproteinase inhibitor; NE: Neutrophil elastase; COPD: Chronic obstructive pulmonary disease; CB: Chronic bronchitis, α1 AT: alpha 1 antitrypsin inhibitor; CG: Cathepsin G, PPE: Porcine pancreatic elastase; ELISA: enzyme linked immunosorbent assay; ECACC: European Collection of Cell Cultures; DMEM: Dulbecco's modified Eagle's medium; FCS: Fetal calf serum, ATCC: American Type Culture Collection; MEM: Minimum essential medium; PBEC: primary bronchial epithelial cells; PBS: Phosphate-buffered saline; SEM: Standard error of the mean; SFM: Serum free media, HSA: Human serum albumin, DNA: deoxyribonucleic acid.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>AS carried out the experimental work and wrote the manuscript. TD carried out the molecular modelling studies and produced the illustrations. PH conceived the hypothesis, advised on experimental work and assisted in drafting the manuscript. RS supervised the experimental work and assisted in drafting the manuscript. All authors read and approved the final manuscript.</p>", "<title>Availability &amp; requirements</title>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.pdb.org\"/></p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.expasy.org/spdbv/\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>Grateful thanks to Mr Darren Bayley for preparation of some of the enzymes used, and to Amgen and AstraZeneca for supplying recombinant human SLPI and ZD0892 respectively. Dr Sullivan was supported by a Research Training Fellowship from the Wellcome Trust. Dr Dafforn is supported by a Medical Research Council Fellowship.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Validation of the ELISA for measuring SLPI with proteinases</bold>. Recombinant human SLPI was mixed with varying concentrations of NE and assayed by ELISA to assess the recovery of SLPI in complex with the proteinase and in the presence of excess proteinase. The molar ratio of proteinase to SLPI is given along the x axis, and the percentage of SLPI recovered is shown on the y axis. The figure shows the results obtained with NE, CG, PPE and trypsin. The ELISA was affected by a large molar excess of proteinase, but not at the molar ratios relevant to this paper (&lt; 10:1).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Effect of neutrophil elastase (NE) on SLPI in supernatants from cultured A549 cells</bold>. A549 cells were cultured for 24 h with NE at the concentrations shown, with an equivalent concentration of the buffer alone, or in a serum free media control. The y axis shows the average of the SLPI secreted in each condition as a percentage of the SLPI secreted by control cells (% SLPI release). Error bars represent standard error of the mean (SEM). N = 3–5 separate experiments for all except buffer control (N = 2). A dose-related reduction in SLPI supernatant concentration was seen with NE (statistically significant from 2.5 nM) (p = 0.006, one way ANOVA).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Relationship between NE and SLPI decrease in the supernatant of A549 cells</bold>. The total NE-induced decrease in SLPI concentration, calculated from the experiment shown in Figure 2, was plotted against the concentration of NE used and a linear relationship found (Pearson correlation coefficient 0.846, p &lt; 0.001).</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Effect of other proteinases on SLPI concentration in A549 cells</bold>. A549 cells were cultured with varying concentrations of each proteinase for 24 h. The concentration of SLPI, expressed as a percentage of the concentration in serum free media, is shown on the <italic>y </italic>axis with the SEM represented by error bars. P indicates that the cells exhibited morphological changes of partial detachment, 'rounding up' and losing attachment to each other but remaining adherent to the culture plate, whilst D indicates that the cells detached completely from the culture plate. The effect on SLPI concentration was only independent of cell morphology for CG. Values given are mean ± SEM for at least 3 experiments.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Time course of the effect of NE on SLPI concentration</bold>. Figure 5a: PBEC were cultured in basal media for 24 h, and then an aliquot of media was removed as baseline and replaced with the same volume of media control or media containing NE at a concentration sufficient to achieve a concentration of 10 nM in the wells. Further aliquots were taken at the times shown, and SLPI concentration was measured by ELISA. The figure shows the results from 3 experiments. The y axis gives the concentration of SLPI as % of the baseline value for triplicate wells treated with basal media or NE. (Error bars indicate SEM.) The concentration of SLPI fell at the earliest time point studied in wells treated with NE but remained stable in the media controls. Over 24 h the concentration in media controls rose in accordance with the steady state concentrations predicted, but remained low in NE-treated wells. At 24 h, some SLPI was present in the media of NE-treated cells but this was substantially lower than the media controls (p &lt; 0.001 Wilcoxon signed ranks test). b: HepG2 cells were cultured in 12 well plates and, after rinsing with PBS, media containing 1 nM SLPI was added to the cells. After a few minutes equilibration, a baseline aliquot was removed and replaced with either media control or NE to achieve a final concentration of 10 nM. Further aliquots were taken at the time points shown. In the media controls it remained stable throughout, but the concentration in NE-treated wells fell significantly at 10 minutes and remained low thereafter (p &lt; 0.001 Wilcoxon signed ranks test).</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Timing of the addition of inhibitor to NE affects concentration of SLPI</bold>. The diagram shows the concentration of SLPI in the media of wells treated according to the conditions shown on the <italic>x </italic>axis. The concentration of SLPI is expressed as % of the concentration in control wells. * indicates significant difference from control wells, $ indicates significant difference from NE-treated wells. Values shown are mean ± SEM for 3 experiments. Prior incubation of NE with a tenfold excess of ZD0892 completely abrogated the effect of NE on SLPI concentration. When NE and ZD0892 were added separately, there was a fall in SLPI concentration which was less pronounced than that seen with NE alone, but still significantly different from both media control and NE alone. Although the average fall was greater when NE was added before rather than after ZD0892, this difference was not statistically significant.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p><bold>Protection by albumin against non-specific binding of SLPI to plastic</bold>. The diagram shows the concentration of SLPI in SFM added to tissue culture plates without cells, before addition of NE or control (baseline) and 10 min afterwards. Parallel wells used SFM containing human serum albumin at 1 mg/ml. Data shown are the mean results from 3 experiments with SEM represented by the error bars. Without albumin, almost all the SLPI was lost regardless of whether NE was added. Albumin provided partial protection against loss of SLPI at baseline and after addition of NE.</p></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Figure 8</label><caption><p><bold>The effect of NE on SLPI concentration in media of cells grown on Transwells</bold>. The diagram shows the concentration of SLPI in each compartment of A549 cells grown in Transwells, relative to the concentration in control wells. Wells were treated with either 10 nM NE or serum free media (SFM) alone for 10 minutes, and the concentration of SLPI was then determined by ELISA. Upper and lower compartment results for the same well are adjacent; the solid bars indicate upper compartments and the open bars indicate lower compartments. SFM/NE indicates that NE was applied to the lower compartment only whilst NE/SFM indicates that NE was applied to the upper compartment only. The concentration of SLPI in the NE-treated compartments fell significantly (p = 0.001 by independent t-test) from 100% to 10.6 ± 0.3% (NE added to upper compartment) and 8.6 ± 1.9% (NE added to lower compartment) whilst there was no difference in concentration of the paired SFM-treated compartments (99.9 ± 1.5% and 90.31 ± 7.8% of control wells respectively).</p></caption></fig>", "<fig position=\"float\" id=\"F9\"><label>Figure 9</label><caption><p><bold>Predicted charge of the complex of SLPI with various proteinases</bold>. Modelling of the SLPI molecule alone (A) and in complex with various proteinases (B-F): blue indicates positive charge, white is neutral and red indicates negative charge. All models are shown in the same orientation with views of opposite sides. The SLPI molecule was predominantly neutral with some positively-charged areas. Cathepsin G (D) formed the most positively-charged complex, followed by NE (C). Chymase (F) was strongly positively-charged on one surface only. Trypsin (B) had weak positive and negative charge on different surfaces, and tryptase (E) was strongly negatively-charged.</p></caption></fig>" ]
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[{"surname": ["Stockley", "Morrison"], "given-names": ["RA", "HM"], "article-title": ["Elastase inhibitors of the respiratory tract"], "source": ["Eur Respir J"], "year": ["1990"], "volume": ["3 Suppl 9"], "fpage": ["9s"], "lpage": ["15s"]}, {"surname": ["Owen", "Campbell", "Boukedes", "Campbell"], "given-names": ["CA", "MA", "SS", "EJ"], "article-title": ["Cytokines regulate membrane-bound leukocyte elastase on neutrophils: a novel mechanism for effector activity"], "source": ["Am J Physiol Lung Cell Mol Physiol"], "year": ["1997"], "volume": ["272"], "fpage": ["L385"], "lpage": ["L393"]}]
{ "acronym": [], "definition": [] }
52
CC BY
no
2022-01-12 14:47:25
Respir Res. 2008 Aug 12; 9(1):60
oa_package/c0/6f/PMC2529288.tar.gz
PMC2529289
18699999
[ "<title>Background</title>", "<p>Current provision of complementary therapy services in the NHS is patchy, sporadic and under threat [##UREF##0##1##,##UREF##1##2##]. However, their adoption may be more likely with a service model that is designed to the specifications of clinicians and Primary Care Trust (PCT) managers. Although many models of complementary therapy services exist, the details of service design have rarely been comprehensively studied, with one notable exception [##UREF##2##3##]. Most of the existing literature tends to focus on evaluations of particular services [##REF##7619572##4##, ####UREF##3##5##, ##UREF##4##6####4##6##] or ways in which doctors, nurses and complementary practitioners might work together [##REF##15165426##7##, ####UREF##5##8##, ##REF##15353031##9##, ##UREF##6##10##, ##REF##12738703##11##, ##UREF##7##12##, ##REF##15230977##13####15230977##13##]. The aim of this study was to specify the features of a 'NHS friendly' complementary therapy service – that is a service that would be accepted, endorsed and supported by NHS stakeholders such as clinicians and PCT managers – to help those who are considering ways to design such services in NHS primary care.</p>", "<p>Research on healthcare professionals' attitudes and referral behaviours regarding complementary therapies should offer some guidance on the design features of state funded complementary therapy services. These studies are abundant, particularly for doctors and nurses [##REF##9811281##14##, ####REF##16583281##15##, ##UREF##8##16##, ##REF##9827781##17##, ##REF##11033653##18##, ##REF##17002085##19##, ##REF##16120892##20##, ##REF##7503598##21##, ##REF##11478564##22##, ##REF##16835530##23####16835530##23##]. But they take self-reported behaviour at face value, assuming that people do as they say, so unsurprisingly, different studies have given different results [##REF##15847969##24##]. Moreover, relying on studies of doctors and nurses alone neglects the views of commissioners, such as PCT managers, who allocate funding for NHS healthcare. With recent national policy initiatives, this group is becoming increasingly powerful within the NHS [##REF##17823186##25##]. The few studies of commissioners that do exist have reported that evidence of effectiveness of complementary therapies is strongly persuasive [##REF##9820250##26##, ####REF##15561517##27##, ##REF##15022663##28####15022663##28##]. But this might only partially explain the absence of complementary therapy services in the NHS, as complementary therapy treatments such as spinal manipulation (osteopathy and chiropracty) for mechanical neck disorders [##UREF##9##29##], acupuncture for headaches [##REF##15023828##30##,##REF##15023830##31##] and herbal remedies for benign prostate hypoplasia [##UREF##10##32##,##UREF##11##33##] are not widely available in the NHS, despite having relatively robust evidence of clinical, and in some cases, cost effectiveness.</p>", "<p>As there appeared to be little practical guidance available on service design for those interested in developing NHS complementary therapy services, we undertook a study to address this. We have identified the features of an 'ideal' NHS complementary therapy service in primary care. But this comes with two caveats. First, NHS local health systems are highly context specific and therefore a 'one size fits all' blueprint is not realistic. Thus, the design features detailed in this study are suggestions only. Second, this paper does not address the question of 'should complementary therapy services be mainstreamed within the NHS?' Instead, our question was – what are the features of a NHS service offering complementary therapies that would be more acceptable to primary care doctors, nurses and PCT managers? Or, in other words, what does a 'NHS friendly' complementary therapy service look like?</p>" ]
[ "<title>Methods</title>", "<title>Study design and case site selection</title>", "<p>This study was undertaken by three health services researchers, two with clinical backgrounds (DS is a GP; LW is a complementary therapist) and one with a social science background (AS). We chose a case study approach, as data can be collected from and compared across multiple sources. Because previous studies had over-relied on interviews, we wanted to include other forms of data such as documentation to check reported behaviour against documented behaviour. This research was part of a larger study exploring the changes necessary for the incorporation of complementary therapies into NHS primary care.</p>", "<p>We chose two sites where complementary therapy services were provided on NHS premises. We focused on NHS premises, as we believed clinicians and PCT managers who had direct experience of complementary therapy services had greater knowledge of such services and would be better placed to comment knowledgeably on service design features. Site selection was based on:</p>", "<p>• Funding source (e.g. NHS or other government funding).</p>", "<p>• The degree to which the service appeared to be valued by NHS professionals (as determined through initial contact in recruitment).</p>", "<p>• Willingness to take part (two other sites declined).</p>", "<p>The first case site included a complementary therapy service that was funded by New Deals for Communities money from the Office of the Deputy Prime Minister. This was part of a national urban regeneration programme, in which local communities bid for funds to set up a range of projects in health, education, crime and safety and housing. In this particular community, local residents opted to spend some of the funding on creating a low cost, local complementary therapy service. Fifteen therapists offered a range of different therapies including reflexology, osteopathy and acupuncture. Two local GP surgeries provided treatment rooms at no cost. Patients could be referred to the service through any local health professional (GP, practice nurse, health visitor, midwife, addictions counsellor) and self-referrals were also accepted. As the service was subsidised by New Deals for Communities, patient fees were minimal, initially £5 for the employed and £3 for the unemployed. After nearly four years of funding, New Deals for Communities money finished as it was time limited. In 2006, the local PCT (NHS) took over the funding of a radically modified complementary therapy service that offered three therapies (osteopathy, chiropracty and physiotherapy) for musculoskeletal conditions only. Instead of eight sessions, patients were now only entitled to two and self-referrals were no longer accepted.</p>", "<p>Having completed fieldwork at the first case site and concluded that the service was not popular with some of the doctors, nurses or PCT managers that we had interviewed, we purposefully selected a second case site with a complementary therapy service that appeared better utilised and more highly regarded by referring clinicians. This complementary therapy service was part of a city-wide women's health service that provided treatments for women with pre-menstrual syndrome and menopause. As an adjunct, the women's health service also provided homeopathy, reflexology and aromatherapy treatments which were delivered by two medically trained professionals (a doctor and a nurse) and one professional therapist. To receive complementary therapy treatments, any patients with menopausal or pre-menstrual syndrome symptoms could self-refer into the women's health service or be referred by any NHS clinician across the city (e.g. GP, practice nurse, district nurse, health visitor). After initial assessment by one of the three specialist doctors in the women's health service, these doctors then referred on patients who could not have or did not want pharmacological treatments to the complementary therapy service. Only these three specialist doctors could refer patients to complementary therapists within the complementary therapy service.</p>", "<p>The complementary therapy service at this case site had been funded and line managed by the NHS since 1998. It was located in a former community trust in an inner city area in England. All therapists had NHS contracts. Shortly after beginning fieldwork for this study, the reflexology/aromatherapy service was discontinued as the therapist retired and so fieldwork focused primarily on the homeopathy service. More unexpectedly, NHS funding for the homeopathy service ceased in the summer of 2006, just months after completing fieldwork, as a result of local and national financial cutbacks on 'non-essential' services.</p>", "<title>Data collection and analysis</title>", "<p>We used purposeful sampling techniques to select a range of participants for interviews at each case site [##UREF##12##34##]. A key criterion was professional background (doctor, nurse, administrator or PCT manager). Although their views are valuable, patients and therapists were not included in this particular study, as we were not focusing on the features that would make such a service acceptable to therapists and patients, but instead wanted to find out what features would be acceptable to NHS doctors, nurses and PCT managers. Further criteria guiding sampling were frequency of referral to the complementary therapy service (high or low for clinicians only), current or past key role in developing, maintaining or delivering the service (administrators, PCT managers, clinicians) or senior managerial positions such as Chief Executive (PCT managers). One administrator (practice manager) refused to be interviewed at the first case site, but all those approached at the second case site agreed. Sampling selection at the first case site stopped when no new themes were arising and at the second when all eligible study participants had taken part.</p>", "<p>In total, we interviewed 20 NHS professionals across the two case sites. These included: five PCT managers, nine doctors (six GPs and three women's health specialist doctors), four nurses (two practice nurses, one health visitor and one women's health specialist nurse) and two administrators (one practice manager and the administrator for the women's health service). All of the PCT managers interviewed were based in the local PCT and included a Chief Executive, a Chair, two Public Health specialists and a pharmacist. The doctors, nurses and administrators were based at the GP surgeries (case site one) or community trust (site two). The NHS professionals we interviewed were either influential in decisions about funding the complementary therapy service (PCT managers), provided administrative support to the service (administrators) or were eligible to refer into the service (doctors and nurses).</p>", "<p>We devised a semi-structured topic guide, which we reviewed before every interview. Topics included: knowledge and personal use of complementary therapies, attitudes towards complementary therapies and influences on those attitudes, experiences and opinions of NHS complementary therapy services, perceived referral behaviour (clinicians only), funding considerations (PCT managers only) and possible improvements to the service. Interviews lasted between 15 and 75 minutes and were audio-taped and transcribed verbatim.</p>", "<p>We used descriptive content analysis to analyse interview data [##REF##10940958##35##,##REF##16204405##36##]. Aided by Atlas-ti software, we coded the interview transcripts using both anticipated codes from previous literature and more emergent codes that arose from the data. We coded transcripts in batches following completion of a series of interviews (e.g. PCT managers at site one, doctors at site two etc.). The coding framework developed as the interviews and analysis progressed, with codes being modified or merged, as appropriate, to account for new data. We re-coded all transcripts with the final coding framework, once fieldwork ended, to ensure more recently developed codes were universally applied. In addition to coding within Atlas-ti, we summarised each transcript into a single document noting significant points and quotations to better understand the overall 'story' of each interview.</p>", "<p>From these two processes (that of Atlas-ti coding and summarising each transcript), we developed key themes. To elaborate our understanding of these themes, and check reported behaviour against observed behaviour, we also collected and analysed documentary data.</p>", "<p>Documentary data included: service evaluations, funding bids, minutes of meetings (e.g. of the steering group overseeing the development of the service at site one), e-mails from PCT managers and referral databases of the services. We read service evaluations, funding bids, meeting minutes and e-mails. Information pertinent to service design were highlighted (e.g. referral pathways, service improvements, service design ideas, funding conditions and decisions). With these data, we identified both consistencies with interview data (e.g. indications for referral) and inconsistencies (e.g. the role of research evidence in funding decisions), which will be elaborated in the results section.</p>", "<p>In terms of referral data, the first service held an Excel database that generated data on referrals resulting in treatment from 2001 to November 2006. Details included name of referrer, role (e.g. doctor, nurse etc.), therapy referred to and number of patients referred. At the second service, data were recorded manually from March 2004 – June 2006 and we collected details on name of referrer, therapy referred to and date. To analyse both these datasets, we calculated the frequency of referrals and type of therapy referred to per referrer. We used these data to check the reported behaviour of clinicians against their documented behaviour. For example, during interviews the two practice nurses stated that they were in favour of NHS funded complementary therapy services and were enthusiastic about the complementary therapy service located in their surgeries. Yet on checking against the referral database, neither had referred a single patient to the service. This then fed into interpretation of their interview data in teasing out what service features encourage clinicians to refer.</p>", "<p>We further interrogated emergent findings from interview and documentary data by searching for negative cases, drawing mind maps and 'brainstorming' (see Riley 2000 for an explanation of brainstorming qualitative data [##UREF##13##37##]). To enhance reflexivity, the lead researcher made journal entries weekly or fortnightly. We tested 'face validity' [##REF##14556426##38##] in seminars and conferences as well as individual meetings with a therapist, a PCT manager, a GP and professionals from the Foundation for Integrated Health.</p>", "<p>The study received ethical approval from the London Multi-centre Research Ethics Committee in 2004. We began fieldwork in July 2004 and continued until June 2006. We assured all study participants that their responses would be anonymous and confidential and all gave written consent to participating in the study.</p>" ]
[ "<title>Results</title>", "<p>In identifying the characteristics of a 'NHS friendly' complementary therapy service, the service design features identified have been grouped into three areas, as detailed in the following diagram. Each will be discussed in turn in the results section (see Figure ##FIG##0##1##).</p>", "<title>Specific therapies for specific conditions</title>", "<p>During interviews, PCT managers and NHS clinicians appeared to be more favourably disposed towards a service model whereby specific treatments were provided for specific conditions (e.g. acupuncture for pain or chiropracty for low back pain). This 'specific condition' service model existed at the second case site, which was funded by the NHS, as three therapies (aromatherapy, reflexology and homeopathy) were provided for two types of female hormonal conditions. Once the NHS took over funding the complementary therapy service at the first case site, this type of model also was put in place with three therapies offered for musculo-skeletal conditions only. Thus, a service model in which any complaint would be treated (such as the service at case site one when it was funded by New Deal for Communities) appeared less popular. As a senior PCT manager said,</p>", "<p>I think any introduction of complementary therapies has to be incredibly disciplined. And so we need very clear protocols that say this is the sort of case, this is the sort of need that we're going to meet through this service. Not a' come all ye'. (PCT manager BC site 1)</p>", "<p>Another PCT manager gave an example of how a targeted complementary therapy service could work in practice.</p>", "<p>Acupuncture, we know that in other agencies it's used in helping people over drug misuse and alcohol misuse. It's known to be, or it's been shown to be, quite effective. So if it was being targeted at those people, then we'd say well that is something we would want to support.... But we're not likely to develop an enhanced service for complementary therapy. It would be an enhanced service for a specific condition which may include complementary therapy as part of the service. (PCT manager CC site 1)</p>", "<p>However, selection of the 'right' therapies and specific conditions is crucial. Our data suggest that one criterion for the selection of therapies and conditions in a 'NHS friendly' complementary therapy service is that NHS professionals believe little or nothing else is available.</p>", "<p>I mean as a medic, it's sometimes quite hard to understand it [homeopathy] ....But I'm prepared to go with it and just, because I know I can't do much more from the normal medicine point of view. (Doctor Y site 2)</p>", "<p>This has been coined as an 'effectiveness gap' [##REF##15353018##39##,##UREF##14##40##]. But such a gap is not sufficient in and of itself. According to the study participants interviewed, the 'gap' condition chosen should impact on a substantial numbers of patients. For example, a PCT manager partly attributed the success of the mainstreaming of counselling to the perception that counselling is appropriate for large patient volumes.</p>", "<p>PCT managers and NHS clinicians in this study stated that another criterion for therapy and condition selection is 'good' evidence of clinical effectiveness. But interestingly, this appeared to be based more on perceptions of research evidence than actual knowledge. For example, only three of the nineteen NHS professionals interviewed had directly accessed the research literature on complementary therapies. Their opinions on which complementary therapies had good evidence appeared to be based on collective, unchallenged perceptions, rather than grounded in fact based knowledge.</p>", "<p>For example, data from steering meeting minutes and interviews at case site one indicated that therapies should be chosen on the basis of robust evidence. Herbal medicine was rejected, because clinicians believed that there was not any research evidence, while other therapies such as reflexology and aromatherapy were adopted. Yet, the evidence base for herbal medicine has been identified as the strongest amongst complementary therapies [##REF##12899818##41##], while the research evidence for reflexology and aromatherapy is less robust. Moreover, a doctor at the first site, who claimed during interview that her decisions were based on evidence and she believed that there was insufficient evidence for complementary therapies, was the third highest referrer of the 24 doctors to the complementary therapy service, according to the referral database.</p>", "<p>We also found discrepancies in PCT managers' reported positions that research evidence was paramount in the decision process. For example, although PCT managers claimed that research evidence was an essential precursor to NHS funding of the complementary therapy service, the successful PCT funding bid for the revamped service in 2006 included no reference to evidence of therapeutic or cost effectiveness. An e-mail from the relevant commissioning manager confirmed that such evidence was not needed. Similarly, the funding bid for the service at the second case site in 1998 cited an audit study on homeopathy for one type of female hormonal condition [##REF##9432430##42##] and a more general systematic review on homeopathy [##UREF##15##43##]. No specific randomised controlled trials on homeopathy for menopause or pre-mentrual syndrome or any research on aromatherapy or reflexology were referenced as research evidence in this bid. Thus, factors other than research literature appeared to be influencing both perceptions of 'good' evidence and referral and funding decisions for clinicians and PCT managers.</p>", "<title>NHS management priorities</title>", "<p>In addition to developing a 'specific condition' model, a 'NHS friendly' complementary therapy service design should address NHS management priorities. One current NHS management priority is targeting high health need populations. A doctor at the second case site acknowledged during interview that the service had targeted a low NHS priority group (women with pre-menstrual syndrome and menopause symptoms).</p>", "<p>Everyone in the NHS, they're looking much more at teenage pregnancies and all those figures that are figures, whereas the menopausal lady doesn't come high in the profile in the NHS.... She's not a target, she's not a number, she's not anything. She's very important to the family and everything else at home, but not in the NHS, so in the scheme of things we're very much out on a limb really. We're low priority is the [women's health] clinic, and so homeopathy is probably even lower in that it's just an extra to our clinic. (Doctor Y site 2)</p>", "<p>This may have contributed to the decommissioning of the complementary therapy service at the second site, although we cannot be sure as these events took place after fieldwork terminated.</p>", "<p>Another crucial NHS management priority identified in interviews is reducing costs.</p>", "<p>Well, the PCT is in a very difficult financial position at the moment. We have what's called a local delivery planning group, which is the sort of first sound bite where bids for funding would go. And the criteria we use will be – how much money is this going to save? Basically, it has to pay for itself in terms of hospital admissions or even make savings over and above the cost of running the service. (PCT manager CC site 1)</p>", "<p>So ideally, a 'NHS friendly' complementary therapy service should either pay for itself (cost neutral) or demonstrate that less money can be spent elsewhere in the NHS (ideally on hospital admissions), as a result of the complementary therapy service (cost saving). By demonstrating a cost saving such as reduced medication, GP consultation or hospital usage, complementary therapy services can help shift the allocation of financial resources into community services.</p>", "<p>We can only invest if we find things we can disinvest in. Now that mainly is disinvesting in hospital interventions, whether it be outpatient clinics or diagnostics...And if we can reduce those because we're doing an earlier intervention in primary care, then we can for the first time probably take money out of the acute hospital system and bring it into the community. (PCT manager CB site 1)</p>", "<p>But, according to PCT managers, to save costs, NHS complementary therapy services should only target patients who are already receiving NHS treatments rather than those currently outside the system.</p>", "<p>If we had the respiratory nurse we'll probably find more people with wheezes and so being seen that wouldn't otherwise have been done because they wouldn't have been serious enough to get a hospital appointment.... Now that's an issue for us about whether we're expanding the boundaries of NHS capability and NHS priorities because we're making it more available. That's already an issue for us and I think there is a concern that complementary therapies would take that even further. (PCT manager BC site 1)</p>", "<p>So, for example, a patient with low back pain having physiotherapy is a legitimate focus for a NHS complementary therapy service, as hospital costs for the out-patient physiotherapy service are incurred and a NHS complementary therapy service may be able to treat the same complaint as effectively and more cheaply. But an individual with the same condition who is 'self-managing' is not a candidate for a 'NHS friendly' complementary therapy service, as he or she does not currently make demands on hospital services or GP practices and thus incurs no costs. Ideally, complementary therapy services should treat the former (i.e. those requesting hospital and GP practice services), but avoid the latter (i.e. those who are self-managing).</p>", "<p>Known as 'picking up unmet need', the widening of the NHS net is a major concern to PCT managers given its financial and resource constraints. Thus, PCT managers need reassurance that a 'NHS friendly' complementary therapy service will either be cost neutral or cost saving and reduce demand on existing services, rather than create a new pool of patients that incur additional costs. PCT managers received this reassurance with the service at case site two, as patients were either under the care of the specialist women doctors or received complementary therapy treatments. However, patients at the first site could access both conventional and complementary therapy services in tandem.</p>", "<p>An additional NHS management concern is that demand for complementary therapy services will outstrip supply. Consequently, a 'NHS friendly' complementary therapy service will have mechanisms to regulate demand. These mechanisms were incorporated at the services in both case sites. Funding bids showed that patients could have up to eight treatments at the complementary therapy service at the first case site and patients could have up to six at the service at the second case site. Consultation length was also limited. Therapists at the service at the first case site offered appointments of 30–60 minutes for all consultations, while the homeopaths at the service at the second case site spent an hour with new clients and 15–20 minutes in follow ups. At the service at the second case site, an additional control mechanism was the utilisation of doctors as gatekeepers to the complementary therapy service.</p>", "<p>I think it works, I think it's essential. It's a screening process in that it does control who gets to the homeopathist [sic] and there's some feedback, so we've got a history and some idea of what's going on. (Doctor Y site 2)</p>", "<title>Other service features</title>", "<p>As well being a 'specific condition' service model and meeting NHS management priorities, a 'NHS friendly' complementary therapy service will have other notable characteristics.</p>", "<p>For example at the services at both sites, evaluations had been conducted. Other research we have conducted found that the impact of complementary therapy service evaluations on funding decisions was limited [##REF##16911903##44##]. Nonetheless, during interviews, study participants stated that they believed regular service evaluation was important.</p>", "<p>I think [this] work has been appreciated only because we do the evaluation and [the therapist] continues to do the evaluation. And I think that's very, very powerful. (Doctor NP site 2)</p>", "<p>In addition, doctors and nurses, in particular, stressed that a 'NHS friendly' complementary therapy service should be affordable. At the service at the first site, clinicians were \"put off\" referring (Doctor PS site 1) when patient contributions increased from £3 and £5 (unwaged and waged respectively) to £5 and £10–£15, as New Deals for Communities funding drew to a close.</p>", "<p>Clinicians also stated in interviews that they were unlikely to refer if they did not know they could. With constant NHS staff turnover and a service based across two surgeries, ensuring a high profile for the service at the first site was challenging. Moreover, some potential referrers to the service at the first site indicated they needed personal contact as well as promotional literature to feel confident about referring patients.</p>", "<p>Now it may be because of my part time role at [surgery], but I haven't met any of the [service] therapists and I like to meet people and then I think I'd feel much more comfortable about saying, \"You know what I think? You should go and see [X] about this. Why don't we arrange a referral and this is how we can do it.\" (Doctor PS site 1)</p>", "<p>This was more manageable at the second site partly because only six individuals were involved (three referring women's health specialist doctors and three therapists) unlike the service at the first site where there were 15 therapists and over 100 potential referrers. As one doctor from site one explained during interview,</p>", "<p>You know we refer but part of the problem is that there are tons and tons of therapists many of which do few hours. It's hard for us to get to know any of them particularly well and build up a professional relationship. (Doctor DF site 1)</p>", "<p>Meeting therapists might also go some way to address NHS professionals' concerns about safety. During interviews, concerns about safety were usually expressed in terms of 'safe therapists' rather than 'safe therapies'.</p>", "<p>I would just like to know that hopefully they are not abusive relationships. And you can have an abusive relationship with a mainstream clinician, so I don't excuse general practitioners from that either. But I would hope that on the whole a lot of these people are very vulnerable and it worries me if they are going to see somebody who has had no training and no registration. (Doctor PS site 2)</p>", "<p>To address the safety issue, data from minutes of meetings at site one and interview data from both sites indicated that only trained therapists registered with a recognised professional body would be employed at the services.</p>", "<p>Concerns about safe therapists may also have been addressed at the second site by employing a doctor as a therapist. As one doctor at the second site stated during interview,</p>", "<p>I'm a little bit more comfortable with [the medical homeopath] having a medical background as well as a homeopathy background. (Doctor Y site 1)</p>", "<p>The induction process at the second site also helped to reduce fears around safety and cultivate inter-professional relationships, as all new doctors and nurses to the women's health service observed homeopathy consultations.</p>", "<p>Whenever I first came along to do this [women's health] work, I actually spent time with [homeopath] and sat in with her, seeing her patients and I think it is really valuable that because it gives you an understanding of actually how the homeopath works, you know, how they really go through their decision making process. (Doctor WL Site 2)</p>", "<p>Doctors and nurses at the service at the first site, where inter-professional relationships were less well developed, did not observe complementary therapy consultations. Nor, after nearly four years of complementary therapy service provision, did these NHS clinicians have much more understanding of complementary therapies.</p>", "<p>I don't think I actually know any more about complementary therapies really. (Doctor BM Site 1)</p>", "<p>So to sum up, a 'NHS friendly' complementary therapy should incorporate regular evaluation and be affordable and well-advertised. PCT managers and NHS clinicians need to know that the therapists working in a NHS complementary therapy service are 'safe'. This type of reassurance may come about through employing trained and insured therapists, as well as employing doctors as therapists, and through personal contact between therapists and potential referrers. Inter-professional relationships are more likely to flourish if the numbers of therapists offering treatments and the numbers of referrers are small and if there are opportunities for informal contact and formal interactions (e.g. observation of consultations).</p>" ]
[ "<title>Discussion</title>", "<p>In summary, a 'NHS friendly' complementary service would provide a limited number of therapies for specific conditions for patient populations of high priority where current treatments are limited, ineffective or non-existent. The therapies chosen would be perceived by NHS professionals as having 'good' evidence of effectiveness. In addition, services would be precisely targeted without picking up previously unmet need, cover their own costs and (ideally) demonstrate an appreciable impact on reducing NHS costs elsewhere. The service would be regularly evaluated, affordable and well-advertised. Furthermore, inter-professional relationships would flourish between 'safe' therapists and NHS clinicians, ideally through informal personal contact and more formal exchanges when doctors and nurses observed complementary therapy consultations.</p>", "<title>Strengths and limitations</title>", "<p>This study has addressed a neglected issue by examining the ideal characteristics of a 'NHS friendly' complementary therapy service in primary care. However, a limitation of this study is that at the second site no PCT managers were interviewed, as service providers feared this would jeopardise the funding of the service by raising its profile. An additional limitation is that the study drew on only two case sites. But by focusing in-depth on two services, we were able to carry out detailed examination of reported and actual behaviour as well as expressed views. Further research, possibly through analysis of the Westminster mapping study [##UREF##0##1##], would clarify the extent of this 'NHS friendly' complementary therapy service model nationally.</p>", "<title>Implications</title>", "<p>However, there are numerous difficulties with the 'NHS friendly' complementary therapy service model.</p>", "<p>First, PCT managers and NHS clinicians insisted, when questioned, that 'good' evidence should determine funding allocation, therapy selection and referral practices, yet their behaviour contradicted this. This suggests that, in practice, factors other than actual evidence may have influenced the decisions of our study participants, as has been found in other studies [##REF##11701576##45##, ####UREF##16##46##, ##REF##12823426##47####12823426##47##]. Nonetheless, because the calls for evidence are so strident, the perception of 'good' evidence appears important. Thus, those designing a complementary therapy service have two choices: they can either work to foster the perception of 'good' evidence or select complementary therapies for which NHS professionals already believe 'good' evidence exists, such as acupuncture, osteopathy and chiropracty. Once clinicians and PCT managers are committed to developing a complementary therapy service, relatively little solid evidence may be required. For example, in our study an audit and a general systematic review were sufficient at the second case site to support the bid for homeopathy services. Elsewhere, in bidding for NHS funding in 2006, the Glastonbury Health Centre cited the UKBEAM trial [##REF##15556955##48##,##REF##15556954##49##] and this was enough to convince PCT managers to continue funding osteopathy and acupuncture for musculo-skeletal conditions [##UREF##17##50##].</p>", "<p>A second difficulty is that a 'NHS friendly' model seriously challenges the holistic tenet common to so many complementary therapies. Working 'holistically' means attempting to balance individuals in their inter-related emotional, mental, physical and spiritual dimensions rather than focusing on alleviating a narrow set of predominantly physical (and occasionally mental) symptoms. In doing so, therapists see themselves as generalists. But in the 'specific treatments for specific conditions' service model illustrated in this study, therapists would be specialists, who use selected techniques and remedies to target particular complaints. So how can therapies be both holistic and selective? In Gibson's study of alternative practitioners working within the NHS, an osteopath recounted her difficulties in managing this conflict.</p>", "<p><italic>The forms were divided up into neck problems, thoracic problems, lower back problems. We can't work like that, it's not osteopathic. For example, I'd get a patient with frozen shoulder [in addition to the referral problem] so start working on the shoulder – well you can't do that, that's a separate referral, that's breaking the rules </italic>[##UREF##18##51##].</p>", "<p>This troubled the osteopath sufficiently that she left NHS employment. Targeting treatments for specific conditions essentially conflicts with holistic healthcare philosophies.</p>", "<p>A third difficulty with the service model is that of 'unmet need' and promoting self-care. Broadly speaking, study participants defined 'unmet need' as:</p>", "<p>• Those suffering potentially dangerous conditions without knowing it</p>", "<p>• Those aware of their condition but not seeking treatment either because they were managing it themselves or because they were ignoring their condition</p>", "<p>• Those aware of their condition and seeking treatment but no services were available</p>", "<p>• Those aware of their condition and seeking treatment, but available services were substandard or ineffective</p>", "<p>For many therapists, those people who are self-managing (definition 2) are just the type of individuals for whom complementary therapies, with their emphasis on prevention and self-care, offer the greatest benefit. Instead of playing to the strengths of complementary therapies and offering these patients the extra resources they may require, a 'NHS friendly' service model discourages important self-management behaviours, which ironically are those currently promoted in national policies [##UREF##19##52##,##UREF##20##53##].</p>", "<p>A fourth difficulty in a 'NHS friendly' service model is that the conditions treated should fill an effectiveness gap, defined as an area where current interventions are inadequate or non-existent (definitions 3 and 4). But the stipulation is that only those patients already within the system should be treated, and treated more effectively. If current NHS services are inadequate, patients may already be within the system, but if services are non-existent, they are likely to be outside. Likewise, those who are self-managing or unaware of their condition (definitions 1 and 2) are also outside the health system, but may be included once appropriate services are in place. PCT managers (such as BC in this study) are concerned that the funding of complementary therapy services will extend the already over-stretched boundaries of the NHS, as have other community service initiatives. In brief, the boundaries of the NHS would encompass patients meeting all four definitions of unmet need.</p>", "<p>So how can complementary therapy services plug 'effectiveness gaps' while simultaneously only treating those already within the system? Providing more effective treatment for conditions currently not well treated will, by definition, create demand. This is obvious to everyone, including the pharmaceutical industry. In the Cooksey report on research funding, which was commissioned partly to review incentives to keep pharmaceutical industries within the UK, researchers are urged to identify and close \"unmet needs\" with medicines that open up new markets [##UREF##21##54##]. This implies that addressing \"unmet needs\" may be acceptable, as long as they are met with a pharmaceutical agent.</p>", "<p>A final difficulty is that the 'NHS friendly' service model requires that complementary therapy interventions reduce costs elsewhere in the NHS. How many biomedical treatments are likely to meet this stringent criterion? By setting, or more importantly enforcing, such challenging standards, some NHS professionals are clearly indicating their reluctance to incorporate complementary therapies.</p>" ]
[ "<title>Conclusion</title>", "<p>The inherent contradictions in a 'NHS friendly' complementary therapy service model, and between this service model and the philosophies and approaches of many complementary therapies, make it problematic. Those seeking to establish a NHS complementary therapy service might be well-advised to meet as many of the stipulations of a 'NHS friendly' model as possible, recognising that its full realisation is inherently impossible. However, during periods of innovation and financial security, some relaxation of standards may occur and inroads made.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Provision of complementary therapy services within the NHS is scarce and contested. However, their adoption may be more likely in a service model that is designed to the specifications of clinicians and Primary Care Trust (PCT) managers. Our objective was to identify the features of a 'NHS friendly' service to inform service designers who wish to develop NHS complementary therapy services.</p>", "<title>Methods</title>", "<p>Using a case study approach, two sites offering complementary therapies on NHS premises were studied using interview and documentary data. We conducted interviews with 20 NHS professionals, including PCT managers and clinicians. We used descriptive content analysis to analyse interview data. We collected and analysed documentation, such as referral data, funding bids and evaluations, to compare reported and documented behaviour.</p>", "<title>Results</title>", "<p>Ideally, a 'NHS friendly' complementary therapy service should offer a limited number of therapies for a specific condition for high priority patient populations (e.g. acupuncture for addictions). In this service model, the therapies should be perceived to have 'good' evidence for conditions where there are 'effectiveness gaps' (i.e. current treatments are limited). The service should be evaluated and regularly promoted. Inter-professional relationships would flourish through opportunities for informal contact and formal interactions, such as observations of consultations. However, the service should include gatekeeper mechanisms to control demand and avoid picking up 'unmet need' (i.e. individuals currently not accessing NHS services). The complementary therapy service should pay for itself and reduce NHS costs elsewhere, such as hospital admissions.</p>", "<title>Conclusion</title>", "<p>The service design model identified in this study is problematic. For example, it is contradictory to provide specific interventions for specific conditions within a holistic healthcare framework. It is difficult to avoid providing for 'unmet need' while concurrently filling 'effectiveness gaps'. In addition, demonstrating the impact of a community service on reducing hospital admissions is challenging. Those seeking to establish a NHS complementary therapy service might be well-advised to meet as many of the criteria of a 'NHS friendly' model as possible, recognising that its full realisation may be impossible. However, during periods of innovation and financial security, some relaxation of expectations may occur.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>Funding for the study was obtained by AS and DS. The study was conceived by LW. The majority of the data was collected and analysed by LW, with assistance from AS and DS, especially at the interpretative stages. LW drafted the manuscript and AS and DS made substantive revisions. All engaged in the design process, read and approved the final manuscript and gave approval for the final version.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1472-6963/8/173/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>We are grateful to all the study participants at the two sites. Thanks to the Bristol Homeopathic Hospital Research Group, Clare Arvidsson, Tim Wye and Melanie Macintosh for individual meetings to discuss findings. Thanks also to Eve Douglas, Charlotte Paterson and Michael Dixon for commenting on previous drafts.</p>", "<p>Funding for LW and AS was provided by the National Co-ordinating Centre for Research Capacity Development.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Service model characteristics of a 'NHS friendly' complementary therapy service.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"1472-6963-8-173-1\"/>" ]
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{ "acronym": [], "definition": [] }
54
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no
2022-01-12 14:47:26
BMC Health Serv Res. 2008 Aug 12; 8:173
oa_package/be/13/PMC2529289.tar.gz
PMC2529290
18700979
[ "<title>Background</title>", "<p>The Institute of Medicine (IOM) report <italic>To Err is Human </italic>[##UREF##0##1##] has fundamentally changed the landscape of American medicine by challenging us to examine and improve the safety and quality of the health care system in this country [##REF##16192489##2##]. Seven years after the release of this seminal report, \"the quality chasm in health care remains wide [##UREF##1##3##].\" A recent study from RAND documented that adults in the United States receive only 55% of recommended care [##REF##12826639##4##]. In its blueprint for \"Crossing the Quality Chasm\", the IOM recommended the development of performance and outcome measurements [##UREF##2##5##]. In response, the Agency for Healthcare Research and Quality (AHRQ) has constructed Patient Safety Indicators (PSI) [##UREF##3##6##] to serve as the \"state-of-the art in measuring the safety of hospital care through the analysis of inpatient discharge data [##UREF##3##6##].\"</p>", "<p>The AHRQ PSIs provide information on risk-adjusted rates for potentially preventable adverse events such as decubitus ulcers, postoperative hemorrhage, and postoperative sepsis. In theory, a hospital can benchmark its performance using these safety indicators and target specific areas of poor performance for improvement. This 'global' approach to quality improvement is more likely to be effective than the traditional approach of narrowly focusing on the unsafe actions of a few physicians [##REF##10720363##7##]. Traditional quality assurance (QA) efforts, which are triggered by individual case reviews, may divert limited hospital resources to clinical areas where overall hospital performance is not problematic, while ignoring system-level problems. Using PSIs to guide QI efforts may force hospitals to focus their efforts on the development of systems which will reduce the incidence and mitigate the impact of dangerous medical errors [##REF##10720363##7##].</p>", "<p>The Patient Safety Indicators were designed to be used with administrative data and are currently being used by public and non-public organizations to assess hospital quality of care [##UREF##4##8##]. Basing the PSIs on administrative data makes it possible for virtually any hospital to examine its safety performance. Hospitals can download the PSI software from the AHRQ web site and use their own administrative data to construct risk-adjusted PSI rates and compare themselves to a national benchmark. Because these indicators are based on administrative data, there is no added data collection burden. However, administrative data have well recognized limitations, including the lack of precise definitions and coding differences across hospital [##REF##9382378##9##]. Several investigators have questioned the accuracy of using administrative data to identify complications [##REF##10929998##10##,##REF##12153067##11##], while others have concluded they may be useful for identifying surgical complications [##REF##10929991##12##].</p>", "<p>One of the primary problems with using administrative data to identify adverse events is the failure of most ICD-9-CM codes to distinguish between pre-existing conditions and complications that occur after hospital admission. Counting pre-existing conditions as adverse events may falsely elevate adverse event rates and bias these safety indicators against hospitals which care for sicker patients. In designing the PSIs, AHRQ researchers attempted to exclude ICD-9-CM codes that could represent either a pre-existing condition or a complication [##REF##16148964##13##]. In theory, the use of a present-on-admission (POA) modifier would have made it possible to avoid mis-classifying pre-existing conditions as adverse events. Several states (California, New York State, Florida, and Wisconsin) require hospitals to include a POA modifier in their administrative data. However, AHRQ researchers were initially constrained to develop a set of indicators which could be used by hospitals in all States, and thus were not able to incorporate the POA indicator in the PSI algorithms [##UREF##3##6##]. The latest version (2007) of the AHRQ PSIs incorporates the POA indicator and gives users the options of using this information to calculate the AHRQ PSIs [##UREF##5##14##].</p>", "<p>Using the POA modifier to differentiate pre-existing conditions from complications, we will examine the accuracy of the AHRQ PSIs using discharge abstracts for patients undergoing coronary artery bypass graft (CABG) surgery in the California State Inpatient Database. Previous studies have shown that the AHRQ PSIs over-estimate the number of patient safety events when pre-existing conditions are incorrectly counted as complications [##REF##17667313##15##,##REF##18419045##16##]. One of these studies, by Houchens and colleagues [##REF##18419045##16##], specifically examined the impact of counting pre-existing conditions as complications on hospital PSI rates based on all patient admissions. However, both studies are based on a broad range of patient diagnoses and procedures. Although it is likely that there is some overlap in the care processes that prevent certain complications across different surgical procedures and medical conditions, it is also likely that specific best practices for preventing complications may be unique to different patient populations. For example, it is unlikely that one hospital-wide approach to preventing postoperative hemorrhage will be equally effective in patients undergoing hysterectomy compared to CABG patients, because the causes of postoperative hemorrhage in these two groups are so different. Just as procedure-specific mortality rates (e.g. CABG) are more informative, and possibly more useful, than overall hospital mortality rates, we believe that diagnosis-specific PSI rates may prove more useful that hospital-wide PSI rates.</p>", "<p>We have previously shown that the addition of date stamp information to administrative data has a substantial impact on the ranking of hospital quality based on risk-adjusted mortality rates [##REF##16899015##17##,##REF##18219238##18##]. In the current study, our goal is to examine whether the AHRQ PSIs accurately measure the rates of adverse events, using the data with the POA modifier as the gold standard for comparison. Under the Deficit Reduction Act of 2005, the Centers for Medicare and Medicaid Services will require hospitals to add POA indicators to Medicare claims starting in 2007 [##UREF##6##19##]. The results from this study will help to inform policy-makers as to the value of requiring the inclusion of the POA modifier in non-Medicare claims as well.</p>" ]
[ "<title>Methods</title>", "<title>Data</title>", "<p>This analysis of patients undergoing isolated CABG surgery was conducted using the 1998–2000 California State Inpatient Database, which contains 100 percent of the state's inpatient discharge records. The data were obtained from the Healthcare Cost and Utilization Project (HCUP). Each patient record has ICD-9-CM coding slots for up to 30 diagnoses. Except for E-codes, each ICD-9-CM code is modified by a POA code that specifies whether a diagnosis was present at the time of hospital admission. Although not fully validated, previous work suggests that the POA field has clinical validity [##REF##16899015##17##,##UREF##6##19##, ####REF##17200477##20##, ##REF##17290649##21##, ##REF##16430609##22##, ##REF##16799364##23####16799364##23##].</p>", "<p>The CABG study population was identified using ICD-9-CM codes 36.10 – 36.19 and 36.2. We excluded 557 patients with missing hospital identifiers, age less than 18 years, missing gender, or missing discharge status. In order to eliminate hospitals which may be coding the POA modifier inaccurately, we excluded 13 hospitals (n = 2593 patient discharges) that coded POA = yes or POA = no for every record, hospitals for which greater than 10% of the POA codes were missing, and hospitals whose percent of ICD-9-CM codes coded as present-on-admission was outside of the 95% confidence interval for the CABG patients in this data set (0.67, 0.91). The final patient population consisted of 82,063 patients from 111 hospitals.</p>", "<title>Identification of adverse events</title>", "<p>The AHRQ Patient Safety Indicator (PSI) software [##UREF##3##6##] was used to calculate event rates for adverse events. These indicators were developed by the AHRQ Evidence-Based Practice Center at the University of California and Stanford [##UREF##3##6##]. The selection and grouping of ICD-9-CM codes into PSIs was based on the Complications Screening Program [##REF##8028405##24##], developed by Iezzonni, and a comprehensive review of existing ICD-9-CM codes [##UREF##3##6##]. The validity of these indicators was evaluated using the RAND/UCLA Appropriateness method [##UREF##7##25##] and empirical analyses to assess the frequency, variance, and bias of these indicators [##UREF##3##6##]. Risk adjustment is performed by the PSI software using logistic regression models which are based on the 29 states in the 2000 HCUP State Inpatient Databases. These models adjust for differences in casemix as defined by age, sex, modified DRGs, and the AHRQ Elixhauser comorbidity diagnostic categories [##REF##16116352##26##,##REF##9431328##27##].</p>", "<p>We selected eight PSIs which had relatively high event rates and were clinically relevant for patients undergoing CABG surgery:</p>", "<title>Decubitus Ulcer</title>", "<p>Patient records with secondary diagnosis of decubitus ulcer and length-of-stay greater than 4 days. Patient exclusions include any diagnosis of hemiplegia, paraplegia, or quadriplegia; and patients admitted from a long-term facility or transferred from an acute care facility [##UREF##3##6##].</p>", "<title>Failure-to-Rescue</title>", "<p>Patient records in which the discharge disposition is death and which indicate a potential complication of care during the hospitalization (i.e., pneumonia, DVT/PE, sepsis, acute renal failure, shock/cardiac arrest, or GI hemorrhage). Patient exclusions include age greater than 75 years; and patients admitted from a long-term facility or transferred from an acute care facility [##UREF##3##6##].</p>", "<title>Infection due to Medical Care</title>", "<p>Patient records with secondary diagnosis of infectious complication of medical care; or infection due to other vascular device, implant or graft. Patient exclusions include length-of-stay less than 2 days, any diagnosis code for immunocompromised state or cancer, or cancer DRG [##UREF##3##6##].</p>", "<title>Postoperative Hemorrhage or Hematoma</title>", "<p>Records of patients with secondary diagnosis of postoperative hemorrhage or hematoma who required postoperative control of bleeding or a drainage procedure. Cases were excluded if a procedure for postoperative control of bleeding or a drainage procedure was the only procedure in the record, or occurred prior to the operative procedure [##UREF##3##6##].</p>", "<title>Postoperative Physiologic and Metabolic Derangement</title>", "<p>Patient records with secondary diagnosis of physiologic and metabolic derangements. Cases were excluded it the records included ICD-9-CM codes for chronic renal failure; acute renal failure where dialysis occurs prior to or on the same day as the operative procedure; or a primary diagnosis of diabetes and a secondary diagnosis code for ketoacidosis, hyperosmolarity, or coma [##UREF##3##6##].</p>", "<title>Postoperative Pulmonary Embolism (PE) or Deep Venous Thrombosis (DVT)</title>", "<p>Patient records with secondary diagnosis of DVT or PE. Cases were excluded if a procedure for interrupting the vena cava is (1) the only operative procedure or (2) occurs before or on the same day as the operative procedure [##UREF##3##6##].</p>", "<title>Postoperative Sepsis</title>", "<p>Patient records with secondary diagnosis of sepsis. Cases were excluded if the principal diagnosis was infection, if any of the diagnoses included immunocompromised state or cancer, or if the length-of-stay was less than 4 days [##UREF##3##6##].</p>", "<title>Accidental Puncture of Laceration</title>", "<p>Patient records with secondary diagnosis of accidental cut, puncture, perforation, or laceration [##UREF##3##6##].</p>", "<title>Impact of POA indicator</title>", "<p>In order to analyze the impact of the POA indicator on the PSIs, we modified the AHRQ PSI algorithm to make use of the information from the POA indicator to exclude ICD9 codes that were present-on-admission and thus could not be complications. With the exception of the Failure-to-Rescue PSI, only the numerator for the PSIs was modified. For failure-to-rescue, only the denominator was modified since the numerator equals the number of deaths, whereas the denominator represents complications (only cases where the POA indicator indicated that the condition was not present on admission were included in the denominator for the modified PSIs). We also modified the AHRQ Elixhauser comorbidity algorithm by only including secondary diagnoses present-on-admission for risk adjustment. We then compared the PSI obtained using the \"modified\" PSI algorithms versus the \"standard\" PSI algorithm which ignored information contained in the POA modifier. This analysis was performed first at the level of individual patients, and then at the hospital-level. For the hospital level analysis, we calculated the intra-class correlation coefficient to assess the level of agreement between the point estimates of the risk-adjusted PSI rates obtained with and without the information contained in the POA indicator [##UREF##8##28##].</p>", "<p>The AHRQ PSI algorithms were run using SAS version 8.2 (SAS Corp., Cary, NC). This study was exempted from review by the University Of Rochester School Of Medicine Research Subjects Review Board.</p>" ]
[ "<title>Results</title>", "<p>Overall, the false positive error rate for the PSIs, defined as one minus the positive predictive value, ranged between 7% for \"infection due to medical care\" to 41% for \"decubitus ulcer\" (Table ##TAB##0##1##). The false positive error rate was greater than or equal to 20% for four of the eight PSIs: decubitus ulcer, failure-to-rescue, postoperative physiologic and metabolic derangement, and postoperative pulmonary embolism or DVT. The observed rates per 1000 discharges at risk differed significantly (P-value ≤ 0.05) for six of the eight PSIs, and was marginally significant for one of the eight (P-value = 0.06).</p>", "<p>The comparisons of the hospital risk-adjusted PSI rates obtained using the <italic>standard algorithm </italic>(does not use the POA field to distinguish pre-existing conditions from complications) and the <italic>modified algorithm </italic>(uses the POA field to distinguish between pre-existing conditions and complications) is shown in Figure ##FIG##0##1##. Pairwise comparison of these risk-adjusted PSI rates, with and without POA information, demonstrated almost perfect agreement for five of the eight PSI's (intraclass correlation coefficient: 0.81–1.00). In the case of \"decubitus ulcer\", \"failure-to-rescue\", and \"postoperative pulmonary embolism or DVT\", there was significant deviation of the regression line (standard versus modified risk-adjusted PSI rate) from the 45-degree line, and the intraclass-correlation coefficient ranged between 0.63 to 0.79 (Table ##TAB##1##2## and Figure). With the exception of failure-to-rescue, adding the POA indicator generally decreases a hospital's PSI rates because the original PSI algorithm is flagging conditions as complications that were pre-existing conditions. In the case of failure-to-rescue (FTR), adding POA information lowers the PSI rate because the risk pool for FTR is made up of patients with complications, and fewer patients are flagged as having complications using the POA indicator.</p>" ]
[ "<title>Discussion</title>", "<p>In this study we find that the present-on-admission (POA) indicator has a significant impact on some of the AHRQ Patient Safety Indicators (PSI) rates in patients undergoing CABG surgery. The PSIs are one component of the quality toolbox developed by AHRQ to facilitate quality improvement and provide hospitals with the opportunity to benchmark their performance [##REF##16898983##29##,##REF##12674418##30##].</p>", "<p>In practice, quality assurance is usually triggered by case reviews and focuses on the perceived failures of individual physicians and providers. Medical errors are attributed to \"aberrant mental processes such as forgetfulness, inattention, poor motivation, carelessness, negligence, and recklessness [##REF##10720363##7##,##REF##9801008##31##].\" Since individual cases selected for examination are often reviewed in isolation, as opposed to being reviewed as part of a cohort of similar cases, the critical role of health care systems in causing medical errors is frequently ignored. Because PSI rates are, by construction, a measure of global hospital performance, they shift the focus of error analysis from the individual provider to the level of the health care system. For example, a high rate of postoperative sepsis after CABG surgery across cardiac surgeons is more likely to improve with better adherence to patient safety practices, such as the use of maximum sterile barriers during catheter insertion or the use of antibiotic-impregnated catheters [##REF##12132984##32##], than by the act of \"disciplining\" a single physician. Thus, PSIs may provide the impetus for a hospital's leadership to examine the \"latent conditions\" that lead to medical errors – production pressure, inadequate staffing, fatigue – and help set the stage for the adoption of a true \"systems approach\" to reducing medical error and improving health care quality.</p>", "<p>The AHRQ PSI rates have the advantage of being based on administrative data, which are collected by virtually all hospitals in computerized form, and thus is readily available at low cost. Furthermore, the availability of the AHRQ PSI software in the public domain provides all hospitals with the opportunity to benchmark and track their PSI rates. However, the use of administrative data to monitor complications also has important limitations. In particular, the under-reporting of complications using ICD-9-CM codes, in addition to variability in coding practices across institutions, raises questions regarding the validity of using ICD-9-CM codes to report complications [##REF##15746590##33##] and creates concerns that public reporting of PSI rates may unfairly penalize those hospitals with more accurate reporting practices. The primary limitation of this study is the assumption that the POA indicator accurately distinguishes complications from pre-existing conditions. Parker and colleagues [##REF##16799364##23##] recently examined the accuracy of administrative data from California, using the POA indicator to exclude complications, with a clinical registry for CABG patients. Using the clinical data as the gold standard, the sensitivity of the risk factors in the administrative data ranged between 22% to 95%, with most above 50%. For most risk factors, specificity exceeded 90%. AHRQ has recently released a report summarizing the evidence supporting the value and validity of the POA indicator [##UREF##6##19##].</p>", "<p>However, these well recognized limitations of administrative data for error reporting should not prevent individual hospitals or hospital systems from using non-public reports based on the AHRQ PSI to facilitate quality improvement. Despite the inherent limitations of risk-adjustment for \"leveling the playing field\" [##REF##9370507##34##,##REF##15064036##35##], public and non-public reporting of hospital mortality rates have been associated with significant decreases in mortality for cardiac [##REF##8114213##36##,##REF##1907669##37##] and non-cardiac surgery [##REF##9790339##38##].</p>", "<p>Our findings in this study examine one of the known limitations of administrative data for error reporting, namely, the inability of administrative data to effectively distinguish between pre-existing conditions and complications. Despite the fact that the AHRQ PSI were designed to \"emphasize specificity over sensitivity\", we found significant numbers of false positives for some of the PSIs. The planned expansion of the use of the POA indicator to all Medicare claims, beginning in 2007, could improve the validity of the AHRQ PSIs if the AHRQ algorithm were revised to include the POA indicator. Recent research in the private sector has led to the development of a system to identify Potentially Preventable Complications (PPC) [##REF##17290649##21##]. By incorporating the POA indicator into its algorithms for the PPC groups, it was possible to expand the number of diagnoses that could be considered complications without sacrificing specificity. This expansion in scope of error monitoring, predicated on the use of the POA indicator to distinguish complications from pre-existing conditions, may be the \"next step\" in the evolution of the AHRQ PSI. This \"next-generation\" complication reporting system may provide greater opportunities for reducing medical errors and improving health care quality. Our study, by showing significant number of false positives using the AHRQ PSIs, further reinforces the need for the widespread adoption of the POA indicator which will make it possible for revised PSI systems, such as the PPC system, to be widely adopted.</p>", "<p>Two recent studies have investigated the impact of the POA indicator on patient safety events. The first, by Naessens and colleagues [##REF##17667313##15##], was based on hospital discharges from the Mayo Clinic Rochester hospitals. This study found that after eliminating secondary diagnoses that were present on admission, the overall rate of patient safety events decreased by nearly 50%. The second, by Houchens and colleagues [##REF##18419045##16##], used data from California and New York State Inpatient Databases to examine the impact of the POA indicator. This study found that three of the 13 PSIs greatly over-estimated the number of patient safety events when information from the POA indicator was not used to differentiate pre-existing conditions from complications. For these three PSIs, there were significant discrepancies between hospital risk-adjusted PSI rates before and after excluding pre-existing conditions. Our study adds to the existing literature by focusing on a single medical condition, CABG surgery, as opposed to basing the analysis on all inpatient admissions. We believe that PSIs will be useful only insofar as they allow physicians and hospitals to identify problems and, then to focus QI efforts for specific hospital departments, as opposed to solely providing hospitals with a global measure of patient safety events. In this light, studies evaluating the validity of the AHRQ PSIs should assess disease-specific performance, in addition to global performance. In addition, our previous work has shown that the extent to which complications are mis-identified as pre-existing conditions varies substantially across patient populations (e.g. CABG, abdominal aortic aneurysm repairs, stroke patients) [##REF##16430609##22##]. Thus, it is likely that the accuracy of PSIs would also vary across patient groups. The accuracy of the AHRQ PSI in CABG patients may be of particular interest to hospitals seeking to improve CABG outcomes that only have access to administrative data without the POA indicator.</p>", "<p>Increasingly, private payers and Medicare are promoting the use of financial incentives to improve the quality of care through pay for performance initiatives. Nationally, over fifty-percent of Health Maintenance Organizations covering greater than 80% of enrolled patients, have pay-for-performance programs in place [##REF##17079763##39##]. Under the Deficit Reduction Act of 2005, the reduction in hospital Medicare payments to hospitals not reporting quality data will increase five-fold from 0.4 percent to 2 percent, and infectious complications will no longer entitle hospitals to higher reimbursement rates [##UREF##9##40##]. However, the actual impact of pay-for-performance on quality is largely unknown [##UREF##10##41##], although recent work suggests that financial incentives has a relatively modest effect on adherence to process measures [##UREF##11##42##]. Even if financial incentives were found to significantly improve adherence to process measures, recent work suggests that adherence to \"best practices\" has only a relatively modest impact on risk-adjusted 30-day mortality rates for patients with acute myocardial infarctions (0.6%), heart failure (0.1%) and pneumonia (0.1%) [##REF##17164455##43##]. In light of the weak association between many processes of care and outcome, direct outcome measures, such as PSI and related measures of adverse events, may have an important role in future efforts to improve health care quality.</p>" ]
[ "<title>Conclusion</title>", "<p>For some of the Patient Safety Indicators, there are significant differences in the rates of adverse events depending on whether the POA indicator is used to distinguish between pre-existing conditions and complications. The use of the POA indicator will increase the accuracy of the AHRQ PSIs as measures of adverse outcomes, and will make the future implementation of more comprehensive measures of complications more feasible.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The Agency for Healthcare Research and Quality (AHRQ) Patient Safety Indicators (PSIs) provide information on hospital risk-adjusted rates for potentially preventable adverse events. Although designed to work with routine administrative data, it is unknown whether the PSIs can accurately distinguish between complications and pre-existing conditions. The objective of this study is to examine whether the AHRQ PSIs accurately measure hospital complication rates, using the data with present-on-admission (POA) codes to distinguish between complications and pre-existing conditions</p>", "<title>Methods</title>", "<p>Retrospective cohort study of patients undergoing isolated CABG surgery in California conducted using the 1998–2000 California State Inpatient Database. We calculated the positive predictive value of selected AHRQ PSIs using information from the POA as the gold standard, and the intra-class correlation coefficient to assess the level of agreement between the hospital risk-adjusted PSI rates with and without the information contained in the POA modifier.</p>", "<title>Results</title>", "<p>The false positive error rate, defined as one minus the positive predictive value, was greater than or equal to 20% for four of the eight PSIs examined: decubitus ulcer, failure-to-rescue, postoperative physiologic and metabolic derangement, and postoperative pulmonary embolism or deep venous thrombosis. Pairwise comparison of the hospital risk-adjusted PSI rates, with and without POA information, demonstrated almost perfect agreement for five of the eight PSI's. For decubitus ulcer, failure-to-rescue, and postoperative pulmonary embolism or DVT, the intraclass-correlation coefficient ranged between 0.63 to 0.79.</p>", "<title>Conclusion</title>", "<p>For some of the AHRQ Patient Safety Indicators, there are significant differences in the risk-adjusted rates of adverse events depending on whether the POA indicator is used to distinguish between pre-existing conditions and complications. The use of the POA indicator will increase the accuracy of the AHRQ PSIs as measures of adverse outcomes.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>LGG conceived and designed the study, performed the statistical analysis, and drafted the manuscript. YL modified the computer algorithm used to calculate the AHRQ PSIs and contributed to the drafting of the manuscript. TMO, DBM, and AWD participated in the design of the study and contributed to the drafting and revision of the manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1472-6963/8/176/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>The authors wish to gratefully acknowledge the support of the Agency for Healthcare and Quality (AHRQ R01 HS 13617).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Comparison of hospital risk-adjusted PSI rates based on the <italic>standard </italic>PSI algorithm versus <italic>modified </italic>PSI algorithm</bold>. <italic>standard PSI algorithm </italic>– does not use information from the POA field to distinguish pre-existing conditions from complications <italic>modified PSI algorithm </italic>uses information from the POA field to distinguish between pre-existing conditions and complications identity line is a 45-degree line which corresponds to perfect agreement for risk-adjusted PSI rates based on the 'standard' and 'modified' PSI algorithms.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Impact of the POA indicator on observed rates of adverse events.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Patient Safety Indicators</td><td align=\"center\" colspan=\"2\">No. of Events</td><td align=\"center\" colspan=\"2\">Risk Pool</td><td align=\"center\">1-PPV</td><td align=\"center\" colspan=\"2\">Observed Rates per 1000 Discharges at Risk</td></tr><tr><td/><td colspan=\"4\"><hr/></td><td/><td colspan=\"2\"><hr/></td></tr><tr><td/><td align=\"center\">No POA</td><td align=\"center\">POA</td><td align=\"center\">No POA</td><td align=\"center\">POA</td><td/><td align=\"center\">No POA</td><td align=\"center\">POA</td></tr></thead><tbody><tr><td align=\"left\">Decubitus Ulcer</td><td align=\"center\">228</td><td align=\"center\">135</td><td align=\"center\">49,463</td><td align=\"center\">49,463</td><td align=\"center\">0.41</td><td align=\"center\">4.61†</td><td align=\"center\">2.73†</td></tr><tr><td align=\"left\">Failure to Rescue</td><td align=\"center\">403</td><td align=\"center\">324</td><td align=\"center\">3298</td><td align=\"center\">2,095</td><td align=\"center\">0.20</td><td align=\"center\">122.2†</td><td align=\"center\">154.7†</td></tr><tr><td align=\"left\">Infection due to Medical Care</td><td align=\"center\">374</td><td align=\"center\">349</td><td align=\"center\">72,954</td><td align=\"center\">72,954</td><td align=\"center\">0.07</td><td align=\"center\">5.13</td><td align=\"center\">4.78</td></tr><tr><td align=\"left\">Postop Hemorrhage or Hematoma</td><td align=\"center\">524</td><td align=\"center\">455</td><td align=\"center\">82,046</td><td align=\"center\">82,046</td><td align=\"center\">0.13</td><td align=\"center\">6.39†</td><td align=\"center\">5.55†</td></tr><tr><td align=\"left\">Postop Physiologic &amp; Metabolic Derangement</td><td align=\"center\">191</td><td align=\"center\">153</td><td align=\"center\">35,003</td><td align=\"center\">35,003</td><td align=\"center\">0.20</td><td align=\"center\">5.46†</td><td align=\"center\">4.37†</td></tr><tr><td align=\"left\">Postop Embolism or DVT</td><td align=\"center\">512</td><td align=\"center\">343</td><td align=\"center\">82,040</td><td align=\"center\">82,040</td><td align=\"center\">0.33</td><td align=\"center\">6.24†</td><td align=\"center\">4.18†</td></tr><tr><td align=\"left\">Postop Sepsis</td><td align=\"center\">247</td><td align=\"center\">207</td><td align=\"center\">30,054</td><td align=\"center\">30,054</td><td align=\"center\">0.16</td><td align=\"center\">8.22¥</td><td align=\"center\">6.89¥</td></tr><tr><td align=\"left\">Accidental Puncture or Laceration</td><td align=\"center\">961</td><td align=\"center\">848</td><td align=\"center\">82,050</td><td align=\"center\">82,050</td><td align=\"center\">0.12</td><td align=\"center\">11.71†</td><td align=\"center\">10.34†</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Extent of agreement of hospital risk-adjusted PSI rates based on 'standard' versus 'modified' PSI algorithm.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Patient Safety Indicators</td><td align=\"center\">Intraclass Correlation Coefficient</td></tr></thead><tbody><tr><td align=\"left\">Decubitus Ulcer</td><td align=\"center\">0.666</td></tr><tr><td align=\"left\">Failure to Rescue</td><td align=\"center\">0.791</td></tr><tr><td align=\"left\">Infection due to Medical Care</td><td align=\"center\">0.988</td></tr><tr><td align=\"left\">Postoperative Hemorrhage or Hematoma</td><td align=\"center\">0.935</td></tr><tr><td align=\"left\">Postoperative Physiologic &amp; Metabolic Derangement</td><td align=\"center\">0.871</td></tr><tr><td align=\"left\">Postoperative Embolism or DVT</td><td align=\"center\">0.628</td></tr><tr><td align=\"left\">Postoperative Sepsis</td><td align=\"center\">0.931</td></tr><tr><td align=\"left\">Accidental Puncture or Laceration</td><td align=\"center\">0.954</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><p>† P-value ≤ 0.05</p><p>¥ P-value = 0.06</p><p>DVT – deep venous thrombosis, POA – present-on-admission indicator; PPV – positive predictive value; obs – observed; postop – postoperative</p></table-wrap-foot>", "<table-wrap-foot><p>DVT – deep venous thrombosis</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1472-6963-8-176-1\"/>" ]
[]
[{"surname": ["Kohn LT, Corrigan JM and Donaldson MS"], "source": ["To Err is Human: Building a Safer Health System"], "year": ["2000"], "publisher-name": ["Washington, D.C., National Academy Press"]}, {"collab": ["Institute of Medicine"], "source": ["Performance Measurement: Accelerating Improvement"], "year": ["2007"], "publisher-name": ["Washington, D.C., National Academies Press"]}, {"source": ["Crossing the Quality Chasm"], "year": ["2001"], "publisher-name": ["Washington, D.C., National Academy Press"]}, {"article-title": ["Guide to Patient Safety Indicators."], "year": ["2006"]}, {"surname": ["Hussey", "Mattke", "Morse", "Ridgely"], "given-names": ["PS", "S", "L", "S"], "source": ["Evaluation of the Use of AHRQ and Other Quality Indicators"], "year": ["2006"], "publisher-name": ["AHRQ"]}, {"article-title": ["AHRQ Quality Indicatdors: Patient Safety Indicators. Technical Specifications."], "year": ["2007"]}, {"surname": ["Coffey", "Milenkovic", "Andrews"], "given-names": ["R", "M", "RM"], "source": ["The Case for the Present-on-Admission (POA) Indicator."], "year": ["2006"], "publisher-name": ["U.S. Agency for Healthcare Research and Quality"]}, {"surname": ["Fitch", "Bernstein", "Aguilar MD", "LaCalle", "Lazario"], "given-names": ["K", "J", "BB", "JR", "P"], "source": ["The Rand/UCLA Appropriateness Method User's Manual"], "year": ["2001"], "publisher-name": ["RAND"]}, {"surname": ["Fleiss"], "given-names": ["JL"], "article-title": ["Reliability of Measurement."], "source": ["Design and Analysis of Clinical Experiments"], "year": ["1999"], "publisher-name": ["New York, John Wiley & Sons, Inc."], "fpage": ["1"], "lpage": ["31"]}, {"article-title": ["Deficit Reduction Act of 2005"], "year": ["2005"], "comment": ["S.1932 Section 5001 Public Law No. 109-171"]}, {"surname": ["Epstein"], "given-names": ["AM"], "article-title": ["Pay for Performance at the Tipping Point"], "source": ["N Engl J Med"], "year": ["2007"]}, {"surname": ["Lindenauer", "Remus", "Roman", "Rothberg", "Benjamin", "Ma", "Bratzler"], "given-names": ["PK", "D", "S", "MB", "EM", "A", "DW"], "article-title": ["Public Reporting and Pay for Performance in Hospital Quality Improvement"], "source": ["N Engl J Med"], "year": ["2007"]}]
{ "acronym": [], "definition": [] }
43
CC BY
no
2022-01-12 14:47:26
BMC Health Serv Res. 2008 Aug 13; 8:176
oa_package/a1/73/PMC2529290.tar.gz
PMC2529291
18664300
[ "<title>Background</title>", "<p>Access to healthcare providers is a significant factor in improving public health and helping poor households escape from poverty [##REF##12219150##1##]. In developing countries, out-of-pocket payments for health services have catastrophic economic effects on individuals and their ability to seek and receive adequate healthcare [##REF##15694962##2##,##REF##11532823##3##]. The choice of healthcare service depends on the various characteristics of potential providers (e.g., area of expertise or quality of service), as well as of the patients themselves (e.g., economic status, health status, education, age, and gender). Such factors can influence accessibility to healthcare, even where services exist [##REF##10187600##4##].</p>", "<p>This paper discusses (a) the current pattern of healthcare in a rural district of Vietnam in relation to the choice of provider, and (b) household expenditure for different providers following the transition from a socialist system to a market economy in Vietnam from 1989 until 2002. During this period, the Vietnamese economy grew rapidly and the living standards of both urban and rural areas improved significantly.</p>", "<p>Coupled with this growth was a rapid widening of the economic gap between the rich and poor [##UREF##0##5##]. Vietnam is still a poor country. While the annual income per capita has increased by 5–6%, the average annual income remained around USD370 in 2002 [##UREF##1##6##,##UREF##2##7##]. The national budget allocated for healthcare is still limited to approximately USD5.7 (VND91,100) per capita per year in 2002 [##UREF##3##8##]. Therefore, the government has implemented a number of measures to mobilize new resources for the heath sector. Among the most important measures have been the introduction of user fees at public hospitals, health insurance schemes, the legalization of the pharmaceutical industry, and the deregulation of the retail trade in drugs [##UREF##0##5##,##UREF##4##9##,##UREF##5##10##].</p>", "<p>Under these measures, the government has given considerable autonomy to healthcare providers, and has relaxed commercial sales of pharmaceuticals and fee-for-health service. These changes have led to significant improvements in the quality of Vietnam's healthcare [##UREF##6##11##,##UREF##7##12##] such as the use of different providers and increased spending for healthcare. However, they have also lead to an increase the out-of-pocket health expenditures as a proportion of total health expenditures from 59% in 1989 to 84% 1998 and 80% in 2001 [##UREF##5##10##,##REF##11861587##13##] which has resulted in a portion of the population being unable to afford utilize healthcare services [##UREF##8##14##].</p>", "<p>Some surveys show that self-treatment and the use private of providers are very common among the rural households. The poor report \"no treatment\" or the use of commune health centers more often than the non-poor. They used less public care and less care at higher levels than the rich do, and paid as much as the better-off did when visiting public healthcare facilities [##REF##12173499##22##,##UREF##15##24##].</p>", "<p>The purpose of this paper is to assess the affect of these various policy changes on (i) the pattern of medical provider choice for rural Vietnamese residents, and (ii) the factors that affect the choice of healthcare provider and the allocation of household expenditure to different (types of) healthcare provider for different expenditure groups.</p>", "<p>These findings should be of interest to policy-makers and health professionals in formulating and implementing intervention policies.</p>" ]
[ "<title>Methods</title>", "<p>This study was conducted in the Bavi district of Hatay province in Vietnam. Bavi district is situated in the Northwest part of Vietnam, which about 60 km west of Hanoi. It has a population of 235,000 comprising three major ethnic groups: Kinh (91%), Muong (8%) and Dao (1%). There are also some families of the Tay, Hoa, and Khmer tribal groups. The district is divided into 32 communes including one small town. Farming is the predominant occupation [##REF##11861587##13##,##UREF##8##14##].</p>", "<p>There is a demographic surveillance site in Bavi, the Epidemiological Field Laboratory for the Health Systems Research Project (Filabavi) in Vietnam. FilaBavi is a joint project between Hanoi Medical University, Karolinska Institute, University of Umeå, and the Nordic School of Public Health in Sweden. The goals of the project are to implement a longitudinal epidemiological surveillance system to collect basic health and health care data, to supply information for health planning, to serve as a background and a sampling frame for specific studies (especially intervention studies), and to constitute a setting for epidemiological training of research students. In 1999 a baseline household survey was conducted followed by quarterly surveillance of vital events and complete re-surveys every two years [##UREF##8##14##].</p>", "<p>The FilaBavi infrastructure was utilised for the study presented in this paper. The total sample consists of 11,089 households which were selected using a multistage sampling procedure. In the first stage, 67 population clusters were selected using a probability proportional to size. These clusters had 11,089 households and 51,024 individuals [##UREF##8##14##]. Assuming an α level of 5% and a 50% probability that a household will have an episode of illness in a year, the give a sample size of 576. To ensure adequate sample size, one out of every 18 households was randomly selected from the original sample. The procedure generated a sample of 629 households.</p>", "<p>The study units of the FilaBavi project are households. The heads of households were interviewed at monthly intervals during July 2001 to June 2002. If the head of the household could not be contacted, another adult was interviewed. These household representatives provided information on each member of household's health situation, healthcare utilisation, household health expenditures as well as total incomes and expenditures each month for 12 months. For information on illnesses, the respondents were specifically asked if the household member in question had seen a medically trained person (doctor, nurse, health worker or such) and if so, what diagnosis had been made. For all questions related to female-specific diagnoses, the interviewers were instructed to interview the patient directly.</p>", "<p>Households kept daily notes of their health situation and healthcare payments. Such notes included illness events for every person in the household as well as household incomes and expenditures. During the first week of each month, the interviewer conducted an interview based on the daily notes from the previous month. The interviews were carried out by 42 qualified interviewers employed by the larger FilaBavi project. All interviewers had completed high school and were inhabitants of the Bavi district. The interviewers used a structured questionnaire and were given special training on data collection strategies for collecting information on income, expenditure and illnesses. A systematic random sampling approach was applied in the study; about five to ten per cent of the questionnaires were randomly selected for re-interviews and for checks by researchers before the data entry.</p>", "<p>Microsoft ACCESS™ was used for data entry. Data analyses were done with STATA (Stata Corporation, College Station, TX, USA) software.</p>", "<p>In the analysis, statistical significance was deemed to be at a 95% confidence level (CI) or p-value &lt; 0.05. A multiple logistic regression method was used to identify the factors influencing the use of drugs or services. The choice of healthcare provider was based on the use of healthcare for multiple episodes of illness on the same individuals. Hence, when analyzing the data using multiple logistic regression method we have adjusted standard errors for correlation between episodes for each person (corrections for clustering).</p>", "<p>The expenditure quintile (hereinafter referred to as quintile) is ranked by equalized per capita household expenditure (eqexp<sub>h</sub>). The following analysis utilizes expenditure groups when comparing indicators by income category. To identify the quintile we use the methodology developed by Xu. (2005): eqexp<sub>h </sub>= exp<sub>h</sub>/eqsize<sub>h</sub>;</p>", "<p></p>", "<p><italic>exp</italic><sub><italic>h </italic></sub>is household expenditures; <italic>eqsize</italic><sub><italic>h </italic></sub>is the equivalence scale, <italic>hhsize</italic><sub><italic>h </italic></sub>is the average of household size or number of household members.</p>", "<p>The value of the parameter β (0.56) has been estimated from previous studies based on household survey data from 59 countries [##UREF##12##19##]. The equivalent household size for each household is generated by the formula: eqsize<sub>h </sub>= hhsize<sub><italic>h</italic></sub><sup><italic>0.56</italic></sup>.</p>", "<title>Definitions used in this study</title>", "<p>To be considered in this study, an <italic>illness episode </italic>must meet at least one of the following criteria: the subject stayed in bed; had been restricted from normal activities (e.g. work or school); or had been able to do normal activities but with reduced capacity for at least one day. An illness episode concludes when normal activities resume [##REF##14649642##17##].</p>", "<p><italic>Symptoms </italic>are defined as a perceptible change in the body. The study considered the four most prevalent symptoms to include cough, fever, headache, and \"pain in bones or joints\". During an illness episode, a person might have more than one symptom.</p>", "<p><italic>Diseases </italic>are reported based upon the diagnosis notes collected by the health workers.</p>", "<p><italic>Illness </italic>is the occurrence of at lease one of the above symptoms or diseases during an illness episode.</p>", "<p><italic>Perceived seriousness of illness </italic>is classified into three levels: <italic>\"can work,\"</italic> where the patient was ill but could still be at work; <italic>\"miss work,\"</italic> where they are absent from work but can move around during the illness;<italic>\"confined to bed,\"</italic> where they are bedridden, an invalid, or incapacitated to such an extent that they must depend on a care giver.</p>", "<p><italic>Medical provider </italic>is classified as a traditional healer, a community health station (CHS), district health centre (DHC), province/centre hospital (P/CH), or private healthcare facility.</p>", "<p><italic>Self-treatment </italic>is an action whereby patients treat themselves using medicines available at home, purchased from drug sellers without any medical examination, or taken following the advice given by any person without formal medical background [##REF##14649642##17##].</p>", "<p><italic>Traditional healer treatment </italic>is treatment whereby patients receive healthcare or take traditional medicines from traditional healers.</p>", "<p><italic>Private healthcare </italic>includes healthcare services provided by private clinics, by public health workers when they are practicing privately after regular work or are retired health workers practicing at home, or services/medication purchased following medical examination or advice given by drug sellers.</p>", "<p><italic>Educational level </italic>is classified into three categories: (i) <italic>no-schooling </italic>– never attended school; (ii) <italic>only primary school </italic>– less than 7 years (with the pre-reform education system) or 9 years (with the post-reform system) of education; (iii) <italic>higher education </italic>– high school graduation, university study, graduation, or vocational education after high school.</p>", "<p><italic>Household expenditure </italic>is measured by cash payments for different purposes (including the interest due on borrowed funds but not including payment on the original debt).</p>", "<p><italic>Socio-economic status (SES) </italic>is classified by household total expenditure quintiles. Expenditure, rather than income, is commonly used as a measure of SES in developing countries for three reasons. First, expenditure more accurately reflects the basic purchasing power of the household. Second, households may be less willing to state their true income or may underestimate their total income. Third, expenditure may vary less over time than income, therefore it is easier to measure [##UREF##9##15##,##UREF##11##18##].</p>", "<p>The division into expenditure quintiles is based on data from the sum of twelve months' expenditures. The expenditures recorded in the study are the total financial outlays that the households had each month for food, health care and other means.</p>", "<title>Ethical considerations</title>", "<p>This study was approved by the Scientific and Ethical Committee, Hanoi Medical University, and the Ministry of Health (Decision -QD-BYT-2001). The study was also agreed to by the local authorities, and heads of households.</p>" ]
[ "<title>Results</title>", "<p>Based on the survey data, there were 8,380 illness episodes reported by 2,727 individuals in 621 households. Average household size is 4.4. Health services or drugs were used to treat almost all of the illness episodes (97%).</p>", "<p>The data shows that private providers were the most common form of curative service used by study participants (almost 60% of episodes). Public providers were used in only 10% of the episodes. Services for the remaining illness episodes consisted of self-treatment (approximately 23%) and a few mixed services or use of drugs (less than 5%). Self treatment was more common among the poor (31%) than among the rich (14.5%). For public providers, people in the highest quintile used DHC, P/CH more often than those in the lowest quintile. Similarly, for the perceived seriousness of an illness, the rich more regularly reported their illness to be less critical (<italic>can work </italic>and <italic>miss work</italic>) than the poor. A more serious illness (<italic>confined to bed</italic>) was reported significantly more often (40% vs. 24%) by those in the lowest quintile than by those in the highest quintile (Table ##TAB##0##1##).</p>", "<p>For episodes of illness in which individuals used healthcare services, the rich used services more frequently than the poor (approximately 84%vs.66%). More significantly, self-treatment accounted for 32.6% of the cases in the poorest group, compared to 14.7% in the highest quintile group (see Table ##TAB##1##2##).</p>", "<p>The influence of various socio-demographic variables and perceived seriousness of illness on the decision to use drugs/services and the decision to choose a medical provider versus self-treatment and a private versus a public provider when ill was analysed using multivariate logistic regression. The finding is provided in Table ##TAB##2##3##.</p>", "<p>The number of people using health services or drugs increased from the lower to higher education level (2.10, CI: 1.29–3.42 and 3.59, CI: 1.65–6.79, respectively). Those who perceived their illness to be sufficiently serious to <italic>miss work </italic>(2.7, CI: 1.95–3.73), or those in the <italic>confined to bed </italic>category (4.54, CI: 3.00–6.98) were also more likely to use health services or drugs than those who could still work during their illness episode. Individuals in the highest quintile were almost 5 times more likely to use health services or drugs than those in the lowest quintile.</p>", "<p>Patients who had a higher education were one and half times more likely to use a provider versus self-treatment than those with no schooling (1.51, CI: 1.05–2.16). The number of people making such decisions increased from not at all serious to very serious scales (1.31, CI: 1.15–1.5 and 1.85, CI: 1.59–2.16), and from the third to the highest quintiles groups. Furthermore, when the household size increased by one person, the odds of choosing a provider increased by 5%. (1.05, CI: 1.01–1.11)</p>", "<p>The choice of public versus private provider correlated significantly with age in group 60+ and being confined to bed by the perceived seriousness of illness. Individuals in two highest quintiles were almost two times more likely to use such health services than those in the lowest quintile.</p>", "<p>In addition, the distribution of household curative expenditures for self-treatment and for different providers is presented in Table ##TAB##3##4##. The results show that the highest percentage of healthcare payments was for private services (59.9%). The share of payments was for self-treatment (12.8%); these payments were significantly higher in the lowest quintile (17.3%) group when compared with the highest quintile (8.7%) group. The percentage of payments for treatment in DHC and P/CH were significantly higher in the highest quintile than in the lowest quintile groups (7.6% vs. 6.6% and 22.4% vs. 9.8%, respectively). By contrast, the share of payments for treatment in community health station was significantly lower in the highest quintile than in the lowest quintile group (2.2% vs. 6.3% (Table ##TAB##3##4##).</p>" ]
[ "<title>Discussion and Conclusion</title>", "<p>Monthly data collection and interview-based follow-ups tracked households for the period of one year. We believe this to be a particular strength of the study because we have avoided the inherent bias in 4-week cross-sectional surveys that can be affected by seasonal fluctuations. The head of household, using daily notes (a system used to assure accuracy in reporting), reported illness events and household healthcare expenditures on a monthly basis.</p>", "<p>Despite the safeguards, we must consider the possibility that people neglected to milder illnesses for which there are no need to use medicine. The findings may indicate that poorer individuals tend to report illnesses less reliably. This is unlikely to be objectively true. Most surveys in developing countries that use externally observed measures of morbidity find higher levels of sickness among the poor. It could be suggested that the poor are particularly likely to modify their perception of illness in order to avoid economic costs, such as those of healthcare, associated with illness [##UREF##13##20##,##REF##10163961##21##].</p>", "<p>With respect to the pattern of drugs or service use, our findings illustrate that private health providers and self-treatment are commonly used while public providers are less so. This observation supports the results of previous studies [##REF##12173499##22##,##UREF##14##23##]. Such a pattern is quite different from that found in the years prior to 1989, during which the health sector was subsidized entirely by the state and all people were treated free of charge using public facilities [##UREF##0##5##].</p>", "<p>Based on these findings we can make the following observations. First, with the legalization of private practices, there has been an increase in the number of private healthcare providers and also in the availability of non-prescription drugs at markets or through pharmacies. Second, the decline in government resources for healthcare has led to a decrease in both the availability of subsidized drugs from public providers, and service quality at the low levels such as district health centre (DHC) and community health station (CHS) [##UREF##14##23##]. Finally, the elimination of free services has affected people's decisions regarding using of drugs and services.</p>", "<p>The existence of a private healthcare sector has provided the wealthy with greater access to more and better health services and the poor with better access to medicines via self-medication or service through private pharmacies. However, the increase of self-medication or use of private services also reflects a reduction in the number of people contacting public services. Problems related to unsafe, improper or irresponsible use of drugs have also become more common as is described in a study from Tbilisi, Georgia that Self-treatment is cheaper than visiting a health care provider which explains the preference for self-treatment among the poorest quintile. Furthermore, weak enforcement of pharmaceutical regulations enables people to purchase even prescription drugs directly from pharmacies without a prescription. Thus, on the one hand, the high cost of medical care, the possibility of securing prescription drugs directly from the pharmacy, explain the popularity of the choice to self-treat. [##UREF##13##20##].</p>", "<p>There are differences between the richest and poorest quintiles with respect to their use of medical provider. The poorest usually contact lower public providers like community health station (CHS) only when they get sick, whilst the richest typically contact higher level public providers, such as district health central (DHC) or province/centre hospital (P/CH). With respect to professional services, both poor and non-poor often used private services. Moreover, the number of episodes of self-treatment or no care was higher in the poorest group than in the richest group (see Table ##TAB##0##1## and ##TAB##1##2##). These situations are also consistent with other studies. People in rural areas often choose self-medical or use private services before public services; and the poor use less public care and care at lower levels than the rich [##REF##12173499##22##,##UREF##15##24##]. Such observations could relate to reduced access to State aid (in the form of partial subsidiary by the State to public healthcare centers) amongst the poor compared to the rich. Patients who use these facilities are typically wealthier and benefit from State-subsidized fees.</p>", "<p>Among other factors that we found that might influence a person's choice of healthcare services was that as education level increased, the number of people choosing providers for healthcare also increased. This is similar to findings from other surveys [##UREF##13##20##,##UREF##16##25##], and implies that education has an influence on whether a population is strong and healthy based on their selection and investment in long-term routine healthcare. Education also appears to be a significant factor affecting choices for healthcare management. Women tend to use drugs or services more often than men. We recognize, however, that women and men have different healthcare problems and different perceptions of the importance of selecting and utilizing healthcare services [##UREF##13##20##]. The decision to use drugs or a medical provider is significantly influenced by the seriousness of the illness and by the SES of the household. A larger share of the patients who visit public healthcare providers and higher-level services (P/CHs) belong to the richest quintile or suffer from a serious illness (confined to bed). These analyses helped to realize that if a visit to the local healthcare facility does not help or if the person is diagnosed with a serious illness, they should be encouraged to contact a higher-level public hospital where more extensive diagnoses and appropriate treatment and care are available under doctors' supervision. Our research suggests that public providers still play the decisive role of the healthcare system in Vietnam.</p>", "<p>Our findings also show that the cost of basic healthcare is of critical importance in the decision of when and what services to use. Income for the poor frequently derives from physical labor. When sick or confined to hospital due to illness, the poor are unable to work to earn money. Compounding the problem is the typical lack of savings by the poor. As a result, they will generally choose to ignore their illness or self-treat. When state of health has deteriorated to such an extent that they are incapacitated, they may fall even deeper into poverty as a result of the cost of healthcare and the corresponding loss of income whilst sick [##UREF##17##26##].</p>", "<p>Our results show that the expenditure for a single illness episode treated in public facilities is greater than those in private facilities or by self-treatment (see Table ##TAB##4##5##). Our data also shows that expenditure for self-treatment is only 13% of total curative expenditure, while self-treatment episodes stand for 23% of total episodes where drugs and services were used. These shares are 27% and 10% respectively for public care; and 13% and 2% respectively for higher facility levels (P/CH) (Table ##TAB##0##1## and ##TAB##3##4##). Those results support the discussion above that for more serious illnesses, people often chose public or higher public providers; for minor illnesses, people often undergo self-treatment or seek treatment from private providers. However, the evidence shows that the number of poor who utilize self-treatment (as well as their expenditure on such treatment) is higher than that of the non-poor, whereas use of public healthcare is less. In general, healthcare fees are the same for both the poor and non-poor, which mean that public sources mainly subsidize the rich rather than the poor.</p>", "<p>Taking into account the limitations of the study, we consider the distance to healthcare providers, either in physical units or in time, has generally been found to be associated with utilization of health services. Unfortunately, our data are likely to underestimate this association and are unable to paint a full picture of all factors that might influence a person's choice of healthcare services.</p>", "<p>A major concern in is the substantial difference in access to different healthcare providers between the rich and poor. Part of this difference can be attributed to the transition from a socialist system to a market economy. However, during the time of our study the government has made considerable progress in developing and supporting programs for providing healthcare for the poor. In particular, the Government issued a program providing for and supporting the socio-economic development of the extreme rural areas of the country [##UREF##18##27##]. A major components of the government's program were the introduction of healthcare insurance and the public funding of healthcare expenses targeted specifically at the poor [##UREF##19##28##,##UREF##20##29##]. This progress could be expected to affect our findings. One of the objectives of the Government's program is to reduce the burden healthcare among the poor households in communes with special difficulties and to decrease the gap between poor and rich.</p>", "<p>The results of this study should be of interest to policy-makers and healthcare professionals who are formulating healthcare policies. Of particular importance are the methods to reduce self-treatment and no-treatment. Our research has also identified several other significant issues. These include the management of private practices and maintaining public healthcare providers at all levels, particularly at the basic levels (district, commune) where the poor seek care more than the rich. Healthcare at the basic level is also vital because it is mostly utilized by the poor who find it very difficult and costly to access health facilities at higher levels. If we can improve the quality of such services, then it can help improve the treatment quality for a large portion of people, including the seriously ill in both the poor and non poor groups. The findings of this study could also provide a background for further studies and strategic policy-making on healthcare utilizations and healthcare financing.</p>" ]
[ "<title>Discussion and Conclusion</title>", "<p>Monthly data collection and interview-based follow-ups tracked households for the period of one year. We believe this to be a particular strength of the study because we have avoided the inherent bias in 4-week cross-sectional surveys that can be affected by seasonal fluctuations. The head of household, using daily notes (a system used to assure accuracy in reporting), reported illness events and household healthcare expenditures on a monthly basis.</p>", "<p>Despite the safeguards, we must consider the possibility that people neglected to milder illnesses for which there are no need to use medicine. The findings may indicate that poorer individuals tend to report illnesses less reliably. This is unlikely to be objectively true. Most surveys in developing countries that use externally observed measures of morbidity find higher levels of sickness among the poor. It could be suggested that the poor are particularly likely to modify their perception of illness in order to avoid economic costs, such as those of healthcare, associated with illness [##UREF##13##20##,##REF##10163961##21##].</p>", "<p>With respect to the pattern of drugs or service use, our findings illustrate that private health providers and self-treatment are commonly used while public providers are less so. This observation supports the results of previous studies [##REF##12173499##22##,##UREF##14##23##]. Such a pattern is quite different from that found in the years prior to 1989, during which the health sector was subsidized entirely by the state and all people were treated free of charge using public facilities [##UREF##0##5##].</p>", "<p>Based on these findings we can make the following observations. First, with the legalization of private practices, there has been an increase in the number of private healthcare providers and also in the availability of non-prescription drugs at markets or through pharmacies. Second, the decline in government resources for healthcare has led to a decrease in both the availability of subsidized drugs from public providers, and service quality at the low levels such as district health centre (DHC) and community health station (CHS) [##UREF##14##23##]. Finally, the elimination of free services has affected people's decisions regarding using of drugs and services.</p>", "<p>The existence of a private healthcare sector has provided the wealthy with greater access to more and better health services and the poor with better access to medicines via self-medication or service through private pharmacies. However, the increase of self-medication or use of private services also reflects a reduction in the number of people contacting public services. Problems related to unsafe, improper or irresponsible use of drugs have also become more common as is described in a study from Tbilisi, Georgia that Self-treatment is cheaper than visiting a health care provider which explains the preference for self-treatment among the poorest quintile. Furthermore, weak enforcement of pharmaceutical regulations enables people to purchase even prescription drugs directly from pharmacies without a prescription. Thus, on the one hand, the high cost of medical care, the possibility of securing prescription drugs directly from the pharmacy, explain the popularity of the choice to self-treat. [##UREF##13##20##].</p>", "<p>There are differences between the richest and poorest quintiles with respect to their use of medical provider. The poorest usually contact lower public providers like community health station (CHS) only when they get sick, whilst the richest typically contact higher level public providers, such as district health central (DHC) or province/centre hospital (P/CH). With respect to professional services, both poor and non-poor often used private services. Moreover, the number of episodes of self-treatment or no care was higher in the poorest group than in the richest group (see Table ##TAB##0##1## and ##TAB##1##2##). These situations are also consistent with other studies. People in rural areas often choose self-medical or use private services before public services; and the poor use less public care and care at lower levels than the rich [##REF##12173499##22##,##UREF##15##24##]. Such observations could relate to reduced access to State aid (in the form of partial subsidiary by the State to public healthcare centers) amongst the poor compared to the rich. Patients who use these facilities are typically wealthier and benefit from State-subsidized fees.</p>", "<p>Among other factors that we found that might influence a person's choice of healthcare services was that as education level increased, the number of people choosing providers for healthcare also increased. This is similar to findings from other surveys [##UREF##13##20##,##UREF##16##25##], and implies that education has an influence on whether a population is strong and healthy based on their selection and investment in long-term routine healthcare. Education also appears to be a significant factor affecting choices for healthcare management. Women tend to use drugs or services more often than men. We recognize, however, that women and men have different healthcare problems and different perceptions of the importance of selecting and utilizing healthcare services [##UREF##13##20##]. The decision to use drugs or a medical provider is significantly influenced by the seriousness of the illness and by the SES of the household. A larger share of the patients who visit public healthcare providers and higher-level services (P/CHs) belong to the richest quintile or suffer from a serious illness (confined to bed). These analyses helped to realize that if a visit to the local healthcare facility does not help or if the person is diagnosed with a serious illness, they should be encouraged to contact a higher-level public hospital where more extensive diagnoses and appropriate treatment and care are available under doctors' supervision. Our research suggests that public providers still play the decisive role of the healthcare system in Vietnam.</p>", "<p>Our findings also show that the cost of basic healthcare is of critical importance in the decision of when and what services to use. Income for the poor frequently derives from physical labor. When sick or confined to hospital due to illness, the poor are unable to work to earn money. Compounding the problem is the typical lack of savings by the poor. As a result, they will generally choose to ignore their illness or self-treat. When state of health has deteriorated to such an extent that they are incapacitated, they may fall even deeper into poverty as a result of the cost of healthcare and the corresponding loss of income whilst sick [##UREF##17##26##].</p>", "<p>Our results show that the expenditure for a single illness episode treated in public facilities is greater than those in private facilities or by self-treatment (see Table ##TAB##4##5##). Our data also shows that expenditure for self-treatment is only 13% of total curative expenditure, while self-treatment episodes stand for 23% of total episodes where drugs and services were used. These shares are 27% and 10% respectively for public care; and 13% and 2% respectively for higher facility levels (P/CH) (Table ##TAB##0##1## and ##TAB##3##4##). Those results support the discussion above that for more serious illnesses, people often chose public or higher public providers; for minor illnesses, people often undergo self-treatment or seek treatment from private providers. However, the evidence shows that the number of poor who utilize self-treatment (as well as their expenditure on such treatment) is higher than that of the non-poor, whereas use of public healthcare is less. In general, healthcare fees are the same for both the poor and non-poor, which mean that public sources mainly subsidize the rich rather than the poor.</p>", "<p>Taking into account the limitations of the study, we consider the distance to healthcare providers, either in physical units or in time, has generally been found to be associated with utilization of health services. Unfortunately, our data are likely to underestimate this association and are unable to paint a full picture of all factors that might influence a person's choice of healthcare services.</p>", "<p>A major concern in is the substantial difference in access to different healthcare providers between the rich and poor. Part of this difference can be attributed to the transition from a socialist system to a market economy. However, during the time of our study the government has made considerable progress in developing and supporting programs for providing healthcare for the poor. In particular, the Government issued a program providing for and supporting the socio-economic development of the extreme rural areas of the country [##UREF##18##27##]. A major components of the government's program were the introduction of healthcare insurance and the public funding of healthcare expenses targeted specifically at the poor [##UREF##19##28##,##UREF##20##29##]. This progress could be expected to affect our findings. One of the objectives of the Government's program is to reduce the burden healthcare among the poor households in communes with special difficulties and to decrease the gap between poor and rich.</p>", "<p>The results of this study should be of interest to policy-makers and healthcare professionals who are formulating healthcare policies. Of particular importance are the methods to reduce self-treatment and no-treatment. Our research has also identified several other significant issues. These include the management of private practices and maintaining public healthcare providers at all levels, particularly at the basic levels (district, commune) where the poor seek care more than the rich. Healthcare at the basic level is also vital because it is mostly utilized by the poor who find it very difficult and costly to access health facilities at higher levels. If we can improve the quality of such services, then it can help improve the treatment quality for a large portion of people, including the seriously ill in both the poor and non poor groups. The findings of this study could also provide a background for further studies and strategic policy-making on healthcare utilizations and healthcare financing.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>In Vietnam, the health-sector reforms since 1989 have lead to a rapid increase in out-of-pocket expenses. This paper examines the choice of medical provider and household healthcare expenditure for different providers in a rural district of Vietnam following healthcare reform.</p>", "<title>Methods</title>", "<p>The study consisted of twelve monthly follow-up interviews of 621 randomly selected households. The households are part of the FilaBavi project sample – Health System Research Project. The heads of household were interviewed at monthly intervals from July 2001 to June 2002.</p>", "<title>Results</title>", "<p>The use of private health providers and self-treatment are quite common for both episodes (60% and 23% of all illness episodes) and expenditure (60% and 12.8% of healthcare expenditure) The poor tend to use self-treatment more frequently than wealthier members of the community (31% vs. 14.5% of illness episodes respectively). All patients in this study often use private services before public ones. The poor use less public care and less care at higher levels than the rich do (8% vs.13% of total illness episodes, which decomposes into 3% vs. 7% at district level, and 1% vs. 3% at the provincial or central level, respectively). The education of the patients significantly affects healthcare decisions. Those with higher education tend to choose healthcare providers rather than self-treatment. Women tend to use drugs or healthcare services more often than men do. Patients in two highest quintiles use health services more than in the lowest quintile. Moreover, seriously ill patients frequently use more drugs, healthcare services, public care than those with less severe illness.</p>", "<title>Conclusion</title>", "<p>The results are useful for policy makers and healthcare professionals to (i) formulate healthcare policies-of foremost importance are methods used to reduce self-treatment and no treatment; (ii) the management of private practices and maintaining public healthcare providers at all levels, particularly at the basic levels (district, commune) where the poor more easily can access healthcare services, is also important, as is the management of private practices and (iii) provide a background for further studies on both short and long-term health service strategies.</p>" ]
[ "<title>Competing interests</title>", "<p>We, the authors, declare that there are no financial or non-financial conflicts of interest (political, personal, religious, academic, ideological, intellectual, commercial or any other).</p>", "<title>Authors' contributions</title>", "<p>We are co-authors in this paper. Each author has participated sufficiently in the study to take responsibility for appropriate portions of the content as follow: NTBT: The first author, who designed the questionnaire, was responsible for monitoring the interview process, performing the statistical analysis, drafting and revising the manuscript CL: The second author, who participated in the planning of the study, was involved in drafting and revising the manuscript. LL and NTKC: The third and fourth authors, who participated in the planning of the study, and in the revisions of the manuscript. All co-authors read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1472-6963/8/162/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>This study was conducted in the Epidemiological Field Laboratory for health systems research in Vietnam (FILABAVI), a collaborative research project between Hanoi Medical University (HMU) and the Division of International Health (IHCAR), Karolinska Institute in Stockholm, Umeå International School of Public Health, Umeå, Sweden. Funding from Sida/SAREC, Stockholm, is gratefully acknowledged, however the organisation played no direct role in our study.</p>" ]
[]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>The use of providers for the episodes of illness (%)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"5\">Equalized household expenditure quintiles</td><td align=\"center\">Total</td><td align=\"left\">p-value</td></tr><tr><td/><td colspan=\"5\"><hr/></td><td colspan=\"1\"><hr/></td><td colspan=\"1\"><hr/></td></tr><tr><td/><td align=\"center\">Poorest</td><td align=\"center\">2</td><td align=\"center\">3</td><td align=\"center\">4</td><td align=\"center\">Richest</td><td/><td/></tr></thead><tbody><tr><td align=\"left\">No drug or service used</td><td align=\"center\">76(5.0)</td><td align=\"center\">53(3.3)</td><td align=\"center\">53(2.9)</td><td align=\"center\">56(3.1)</td><td align=\"center\">17(1.0)</td><td align=\"center\">255(3.0)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\">Self treatment</td><td align=\"center\">470(31.0)</td><td align=\"center\">435(27.2)</td><td align=\"center\">480(26.7)</td><td align=\"center\">283(15.7)</td><td align=\"center\">242(14.5)</td><td align=\"center\">1910(22.8)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\">Private providers</td><td align=\"center\">776(51.1)</td><td align=\"center\">887(55.5)</td><td align=\"center\">1060(58.9)</td><td align=\"center\">1170(65.1)</td><td align=\"center\">1105(66.4)</td><td align=\"center\">4998(59.6)</td><td/></tr><tr><td align=\"left\"> Traditional healer</td><td align=\"center\">63(4.2)</td><td align=\"center\">54(3.4)</td><td align=\"center\">107(5.9)</td><td align=\"center\">172(9.6)</td><td align=\"center\">190(11.4)</td><td align=\"center\">586(7.0)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> Privatehealth care</td><td align=\"center\">713(47.0)</td><td align=\"center\">833(52.1)</td><td align=\"center\">953(52.9)</td><td align=\"center\">998(55.5)</td><td align=\"center\">915(55.0)</td><td align=\"center\">4412(52.6)</td><td/></tr><tr><td align=\"left\">Public providers</td><td align=\"center\">129(8.5)</td><td align=\"center\">128(8.0)</td><td align=\"center\">136(7.6)</td><td align=\"center\">220(12.2)</td><td align=\"center\">225(13.5)</td><td align=\"center\">838(10.0)</td><td/></tr><tr><td align=\"left\"> Commune health station</td><td align=\"center\">62(4.1)</td><td align=\"center\">56(3.5)</td><td align=\"center\">71(3.9)</td><td align=\"center\">66(3.7)</td><td align=\"center\">51(3.1)</td><td align=\"center\">306(3.7)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> District health centre</td><td align=\"center\">47(3.1)</td><td align=\"center\">55(3.4)</td><td align=\"center\">35(1.9)</td><td align=\"center\">98(5.5)</td><td align=\"center\">119(7.2)</td><td align=\"center\">354(4.2)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> Province/central hospital</td><td align=\"center\">20(1.3)</td><td align=\"center\">17(1.1)</td><td align=\"center\">30(1.7)</td><td align=\"center\">56(3.1)</td><td align=\"center\">55(3.3)</td><td align=\"center\">178(2.1)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\">Mixed all</td><td align=\"center\">67(4.4)</td><td align=\"center\">96(6.0)</td><td align=\"center\">72(4.0)</td><td align=\"center\">69(3.8)</td><td align=\"center\">75(4.5)</td><td align=\"center\">379(4.5)</td><td/></tr><tr><td align=\"left\">Total number of episodes</td><td align=\"center\">1518(100)</td><td align=\"center\">1599(100)</td><td align=\"center\">1801(100)</td><td align=\"center\">1798(100)</td><td align=\"center\">1664(100)</td><td align=\"center\">8380(100)</td><td/></tr><tr><td align=\"left\">Perceived seriousness of illness</td><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Can work</td><td align=\"center\">495(32.6)</td><td align=\"center\">569(35.6)</td><td align=\"center\">662(36.8)</td><td align=\"center\">702(39.0)</td><td align=\"center\">652(39.2)</td><td align=\"center\">3080(36.8)</td><td/></tr><tr><td align=\"left\"> Missed work</td><td align=\"center\">420(27.7)</td><td align=\"center\">532(33.3)</td><td align=\"center\">635(35.3)</td><td align=\"center\">654(36.4)</td><td align=\"center\">613(36.8)</td><td align=\"center\">2854(34.1)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> Confine to bed</td><td align=\"center\">603(39.7)</td><td align=\"center\">498(31.1)</td><td align=\"center\">504(28.0)</td><td align=\"center\">442(24.6)</td><td align=\"center\">399(24.0)</td><td align=\"center\">2446(29.2)</td><td align=\"left\"><bold>***</bold></td></tr><tr><td align=\"left\">Total number of episodes</td><td align=\"center\">1518(100)</td><td align=\"center\">1599(100)</td><td align=\"center\">1801(100)</td><td align=\"center\">1798(100)</td><td align=\"center\">1664(100)</td><td align=\"center\">8380(100)</td><td/></tr><tr><td align=\"left\">Total number of persons</td><td align=\"center\">479(100)</td><td align=\"center\">558(100)</td><td align=\"center\">577(100)</td><td align=\"center\">558(100)</td><td align=\"center\">555(100)</td><td align=\"center\">2727(100)</td><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Episodes of illness for which a provider was used according to the choice of provider (%)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"5\">Equalized household expenditure quintiles</td><td align=\"center\">Total</td><td align=\"left\">p-value</td></tr><tr><td/><td colspan=\"5\"><hr/></td><td colspan=\"1\"><hr/></td><td colspan=\"1\"><hr/></td></tr><tr><td/><td align=\"center\">Bottom</td><td align=\"center\">2</td><td align=\"center\">3</td><td align=\"center\">4</td><td align=\"center\">Top</td><td/><td/></tr></thead><tbody><tr><td align=\"left\">Used drugs or services</td><td/><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Used service (public/private)</td><td align=\"center\">956(66.3)</td><td align=\"center\">1,088(70.4)</td><td align=\"center\">1,254(71.7)</td><td align=\"center\">1,440(82.7)</td><td align=\"center\">1,385(84.1)</td><td align=\"center\">6,123(75.4)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> Self-treatment</td><td align=\"center\">470(32.6)</td><td align=\"center\">435(28.1)</td><td align=\"center\">480(27.5)</td><td align=\"center\">283(16.2)</td><td align=\"center\">242(14.7)</td><td align=\"center\">1,910(23.5)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> M ixed all</td><td align=\"center\">16(1.1)</td><td align=\"center\">23(1.5)</td><td align=\"center\">14(0.8)</td><td align=\"center\">19(1.1)</td><td align=\"center\">20(1.2)</td><td align=\"center\">92(1.1)</td><td/></tr><tr><td align=\"left\">Total</td><td align=\"center\">1,442(100)</td><td align=\"center\">1,546(100)</td><td align=\"center\">1,748(100)</td><td align=\"center\">1,742(100)</td><td align=\"center\">1,647(100)</td><td align=\"center\">8,125(100)</td><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Multivariate logistic regression showing variables influencing the odds of using services or drugs and the choice of providers when being ill</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"right\">Using vs. not use services or drugs</td><td align=\"right\">Providers vs. Self-treatment</td><td align=\"right\">Public vs. private</td></tr></thead><tbody><tr><td align=\"left\">Explanatory variables</td><td align=\"right\">OR (CI 95%)</td><td align=\"right\">OR (CI 95%)</td><td align=\"right\">OR (CI 95%)</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td/><td align=\"right\">(N = 8380)</td><td align=\"right\">(N = 8125)</td><td align=\"right\">(N = 6123)</td></tr><tr><td/><td align=\"right\">R2: 0.085</td><td align=\"right\">R2: 0.036</td><td align=\"right\">R2:0.049</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\">No of household members (continuous)</td><td align=\"right\">1.07(0.98–1.16)</td><td align=\"right\"><bold>1.05(0.01–1.11)</bold></td><td align=\"right\">0.98(0.88–1.07)</td></tr><tr><td align=\"left\">Perceived seriousness of illness</td><td/><td/><td/></tr><tr><td align=\"left\"> Can work (reference category)</td><td align=\"right\">1.00</td><td align=\"right\">1.00</td><td align=\"right\">1.00</td></tr><tr><td align=\"left\"> Miss work</td><td align=\"right\"><bold>2.70(1.95–3.73)</bold></td><td align=\"right\"><bold>1.31(1.15–1.5)</bold></td><td align=\"right\">1.31(0.89–1.91)</td></tr><tr><td align=\"left\"> Confine to bed</td><td align=\"right\"><bold>4.55(3.0–6.98)</bold></td><td align=\"right\"><bold>1.85(1.59–2.16)</bold></td><td align=\"right\"><bold>3.11(2.23–4.35)</bold></td></tr><tr><td align=\"left\">Male (reference group female)</td><td align=\"right\"><bold>0.49(0.34–0.71)</bold></td><td align=\"right\">0.96(0.81–1.13)</td><td align=\"right\">0.87(0.72–1.04)</td></tr><tr><td align=\"left\">Age group</td><td/><td/><td/></tr><tr><td align=\"left\"> 15–60 (reference category)</td><td align=\"right\">1.00</td><td align=\"right\">1.00</td><td align=\"right\">1.00</td></tr><tr><td align=\"left\"> Under 15</td><td align=\"right\">1.59(94–2.67)</td><td align=\"right\">1.13(0.89–1.43)</td><td align=\"right\">1.41(0.94–2.09)</td></tr><tr><td align=\"left\"> Over 60</td><td align=\"right\">0.71(0.45–1.10)</td><td align=\"right\">1.17(0.94–1.58)</td><td align=\"right\"><bold>0.41(0.25–0.78)</bold></td></tr><tr><td align=\"left\">Education level</td><td/><td/><td/></tr><tr><td align=\"left\"> No schooling (reference category)</td><td align=\"right\">1.00</td><td align=\"right\">1.00</td><td align=\"right\">1.00</td></tr><tr><td align=\"left\"> Only primary school</td><td align=\"right\"><bold>2.10(1.29–3.42)</bold></td><td align=\"right\">1.22(0.94–1.58)</td><td align=\"right\">0.80(0.50–1.28)</td></tr><tr><td align=\"left\"> Higher education</td><td align=\"right\"><bold>3.59(1.65–6.79)</bold></td><td align=\"right\"><bold>1.51(1.05–2.16)</bold></td><td align=\"right\">1.12(0.61–2.05)</td></tr><tr><td align=\"left\">Equalized household expenditure quintiles</td><td/><td/><td/></tr><tr><td align=\"left\"> Poorest (reference category)</td><td align=\"right\">1.00</td><td align=\"right\">1.00</td><td align=\"right\">1.00</td></tr><tr><td align=\"left\"> 2nd</td><td align=\"right\">1.55(0.96–2.46)</td><td align=\"right\">1.22(0.95–1.56)</td><td align=\"right\">1.20(0.68–2.08)</td></tr><tr><td align=\"left\"> 3rd</td><td align=\"right\"><bold>1.67(1.02–2.72)</bold></td><td align=\"right\"><bold>1.31(1.02–1.66)</bold></td><td align=\"right\">1.34(0.79–2.23)</td></tr><tr><td align=\"left\"> 4th</td><td align=\"right\"><bold>1.66(1.11–2.83)</bold></td><td align=\"right\"><bold>2.51(1.92–3.28)</bold></td><td align=\"right\"><bold>1.89(1.11–3.23)</bold></td></tr><tr><td align=\"left\"> Richest</td><td align=\"right\"><bold>4.78(2.42–8.43)</bold></td><td align=\"right\"><bold>2.77(2.10–3.66)</bold></td><td align=\"right\"><bold>2.09(1.22–3.32)</bold></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Household curative expenditures per year by providers and expenditure quintiles in Vietnamese dong (%)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"5\">Equalized household expenditure quintiles</td><td align=\"center\">Total</td><td align=\"left\">p-value</td></tr><tr><td/><td colspan=\"5\"><hr/></td><td colspan=\"1\"><hr/></td><td colspan=\"1\"><hr/></td></tr><tr><td/><td align=\"center\">Bottom</td><td align=\"center\">2</td><td align=\"center\">3</td><td align=\"center\">4</td><td align=\"center\">Top</td><td/><td/></tr></thead><tbody><tr><td align=\"left\">Self-treatment</td><td align=\"center\">44,930(17.3)</td><td align=\"center\">54,707(14.5)</td><td align=\"center\">71,020(14.1)</td><td align=\"center\">68,341(13.6)</td><td align=\"center\">62,575(8.7)</td><td align=\"center\">60,332(12.8)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\">Provider(Public+private)</td><td align=\"center\">714,930(82.7)</td><td align=\"center\">322,547(85.5)</td><td align=\"center\">432,668(35.9)</td><td align=\"center\">434,280(86,4)</td><td align=\"center\">658,559(91.3)</td><td align=\"center\">412,615(87.2)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\">Private providers</td><td align=\"center\">155,922(60.0)</td><td align=\"center\">214,627(56.9)</td><td align=\"center\">292,527(58.1)</td><td align=\"center\">326,895(65.0)</td><td align=\"center\">426,687(59.2)</td><td align=\"center\">283,346(59.9)</td><td/></tr><tr><td align=\"left\"> Traditional healer</td><td align=\"center\">19,962(7.7)</td><td align=\"center\">21,626(5.7)</td><td align=\"center\">45,266(9.0)</td><td align=\"center\">53,294(10.6)</td><td align=\"center\">40,181(5.6)</td><td align=\"center\">36,081(7.6)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> Private healthcare</td><td align=\"center\">135,960(52.3)</td><td align=\"center\">193,001(51.2)</td><td align=\"center\">247,261(49.1)</td><td align=\"center\">273,602(54.4)</td><td align=\"center\">386,506(53.6)</td><td align=\"center\">247,266(52.3)</td><td/></tr><tr><td align=\"left\">Public providers</td><td align=\"center\">58,938(22.7)</td><td align=\"center\">107,920(28.6)</td><td align=\"center\">140,141(27.8)</td><td align=\"center\">107,385(21.4)</td><td align=\"center\">231,872(32.2)</td><td align=\"center\">129,269(27.3)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> Commune health station</td><td align=\"center\">16,360(6.3)</td><td align=\"center\">19,848(5.3)</td><td align=\"center\">46,813(9.3)</td><td align=\"center\">19,328(3.8)</td><td align=\"center\">15,961(2.2)</td><td align=\"center\">23,699(5.0)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> District health centre</td><td align=\"center\">17,034(6.6)</td><td align=\"center\">65,697(17.4)</td><td align=\"center\">52,600(10.4)</td><td align=\"center\">38,198(7.6)</td><td align=\"center\">54,522(7.6)</td><td align=\"center\">45,621(9.6)</td><td align=\"left\"><bold>**</bold></td></tr><tr><td align=\"left\"> Province/central hosp ital</td><td align=\"center\">25,544(9.8)</td><td align=\"center\">22,375(5.9)</td><td align=\"center\">40,728(8.1)</td><td align=\"center\">49,859(9.9)</td><td align=\"center\">161,389(22.4)</td><td align=\"center\">59,948(12.7)</td><td align=\"left\"><bold>***</bold></td></tr><tr><td align=\"left\">Total curative</td><td align=\"center\">259,790(100)</td><td align=\"center\">377,255(100)</td><td align=\"center\">503,689(100)</td><td align=\"center\">502,622(100)</td><td align=\"center\">721,134(100)</td><td align=\"center\">472,947(100)</td><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Average household expenditure per episode by providers and expenditure quintiles in Vietnamese dong</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"5\">Equalized household expenditure quintiles</td><td align=\"center\">Total</td></tr><tr><td/><td colspan=\"5\"><hr/></td><td colspan=\"1\"><hr/></td></tr><tr><td/><td align=\"center\">Poorest</td><td align=\"center\">2</td><td align=\"center\">3</td><td align=\"center\">4</td><td align=\"center\">Richest</td><td/></tr></thead><tbody><tr><td align=\"left\">Self treatment</td><td align=\"center\">11,854</td><td align=\"center\">15,595</td><td align=\"center\">18,495</td><td align=\"center\">29,945</td><td align=\"center\">32,063</td><td align=\"center\">19,616</td></tr><tr><td align=\"left\">providers (privatre+public)</td><td align=\"center\">31,755</td><td align=\"center\">42,504</td><td align=\"center\">46,344</td><td align=\"center\">40,127</td><td align=\"center\">63,059</td><td align=\"center\">45,887</td></tr><tr><td align=\"left\">Private providers</td><td align=\"center\">24,915</td><td align=\"center\">30,004</td><td align=\"center\">34,496</td><td align=\"center\">34,645</td><td align=\"center\">47,882</td><td align=\"center\">35,206</td></tr><tr><td align=\"left\"> Traditional healer</td><td align=\"center\">39,290</td><td align=\"center\">49,659</td><td align=\"center\">52,881</td><td align=\"center\">38,421</td><td align=\"center\">26,224</td><td align=\"center\">38,236</td></tr><tr><td align=\"left\"> Privatehealth care</td><td align=\"center\">23,645</td><td align=\"center\">28,730</td><td align=\"center\">32,432</td><td align=\"center\">33,995</td><td align=\"center\">52,379</td><td align=\"center\">34,803</td></tr><tr><td align=\"left\">Public providers</td><td align=\"center\">56,653</td><td align=\"center\">104,548</td><td align=\"center\">128,806</td><td align=\"center\">60,526</td><td align=\"center\">127,787</td><td align=\"center\">95,795</td></tr><tr><td align=\"left\"> Commune health station</td><td align=\"center\">32,719</td><td align=\"center\">43,950</td><td align=\"center\">82,418</td><td align=\"center\">36,314</td><td align=\"center\">38,808</td><td align=\"center\">48,096</td></tr><tr><td align=\"left\"> District health center</td><td align=\"center\">44,940</td><td align=\"center\">148,116</td><td align=\"center\">187,857</td><td align=\"center\">48,333</td><td align=\"center\">56,813</td><td align=\"center\">80,031</td></tr><tr><td align=\"left\"> Province/central hospital</td><td align=\"center\">158,375</td><td align=\"center\">163,206</td><td align=\"center\">169,700</td><td align=\"center\">110,402</td><td align=\"center\">363,858</td><td align=\"center\">209,144</td></tr><tr><td align=\"left\"> Total</td><td align=\"center\">23,428</td><td align=\"center\">33,997</td><td align=\"center\">38,228</td><td align=\"center\">38,354</td><td align=\"center\">58,179</td><td align=\"center\">39,191</td></tr></tbody></table></table-wrap>" ]
[ "<disp-formula>Where eqsize<sub>h </sub>= hhsize<sub><italic>h</italic></sub><sup><italic>β</italic></sup></disp-formula>" ]
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[ "<table-wrap-foot><p>HH is the abbreviation of household; p-value for the comparison between the lowest and highest quintile groups; <bold>*** </bold>denotes significant at the 1% level; <bold>**</bold>denotes significant at 5% level. Percentage shares are shown within brackets.</p></table-wrap-foot>", "<table-wrap-foot><p>HH is the abbreviation of household; p-value for the comparison between the lowest and highest quintile groups; *** denotes significant at the 1% level; **denotes significant at 5% level. Percentage shares are shown within brackets.</p></table-wrap-foot>", "<table-wrap-foot><p>Adjusted standard errors for correlation between episodes for each person (corrections for clustering). See earlier the method section; bold represents significance.</p></table-wrap-foot>", "<table-wrap-foot><p>HH is the abbreviation of household; p-value for the comparison between the lowest and highest quintile groups; *** denotes significant at the 1% level; **denotes significant at 5% level. Percentage shares are shown within brackets.</p></table-wrap-foot>", "<table-wrap-foot><p>Table 5 shows that the average household expenditure per episode of illness is less for self-treatment (919,616 VND) and for private providers (35,206 VND) than for public providers (95,795 VND). The average household health expenditure for a single illness episode is higher for hospital treatment than for district health centres and commune health stations.</p><p>HH is the abbreviation of household; p-value for the comparison between the lowest and highest quintile groups; *** denotes significant at the 1% level; **denotes significant at 5% level. Percentage shares are shown within brackets.</p></table-wrap-foot>" ]
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[]
[{"surname": ["Phuong", "Hung PM, Harry Minas I, Liu Y, Dahlgren G, Hsio WC"], "given-names": ["DN"], "article-title": ["Issues of equity and effectiveness in health care in Vietnam"], "source": ["Efficient, Equity-oriented strategies for health international perspectives-focus on Vietnam"], "year": ["2000"], "publisher-name": ["CIMH Melbourne, Australia"], "fpage": ["15"], "lpage": ["26"]}, {"collab": ["World Bank"], "article-title": ["Development Indicators"], "source": ["CD-Rom"], "year": ["2003"], "publisher-name": ["The World Bank, Washington, DC"]}, {"collab": ["Ministry of Health"], "source": ["Health Statistics Yearbook"], "year": ["2007"], "publisher-name": ["Hanoi: Ministry of Health"]}, {"collab": ["Ministry of Health"], "source": ["Health Statistics Yearbook"], "year": ["2003"], "publisher-name": ["Hanoi Vietnam: Ministry of Health"]}, {"collab": ["The United Nations Country Team in Viet Nam"], "article-title": ["Health Care Financing for Viet Nam"], "source": ["Discussion paper"], "year": ["2003"], "fpage": ["2"]}, {"article-title": ["Vietnam Health Report 2002"], "source": ["Ministry of Health"], "year": ["2002"], "publisher-name": ["Medical Publishing House, Hanoi Vietnam"]}, {"surname": ["Hung", "Dung", "Dahlgren", "Hung PM, Minas IH, Liu Y"], "given-names": ["PM", "TV", "G"], "article-title": ["Efficient equity-oriented health sector reform: A Vietnamese perspective on some key issues"], "source": ["Efficient, Equity-oriented strategies for health international perspectives-focus on Vietnam"], "year": ["2000"]}, {"collab": ["World Bank", "Sida", "AusAID", "Royal Netherlands Embassy", "Vietnamese Ministry of Health"], "article-title": ["Vietnam \u2013 Growing Healthy: A Review of Vietnam's health sector"], "source": ["Ministry of Health, Vietnam Report No 22210-VN"], "year": ["2001"]}, {"surname": ["Dung"], "given-names": ["PH"], "article-title": ["Health care for the poor and poverty reduction in Vietnam"], "source": ["Health Policy and Medical Sociology Health Strategy and Policy Institute"], "year": ["1999"], "publisher-name": ["MOH, Hanoi, Vietnam"], "fpage": ["17"], "lpage": ["22"]}, {"collab": ["Hanoi Medical University", "Health Strategy and Policy Institute", "Karolinska Institute"], "source": ["FILABAVI, an Epidemiological Field Laboratory- a demographic surveillance site for the study of the health sector reform in Vietnam"], "year": ["2002"], "publisher-name": ["Medical Publishing House, Hanoi, Vietnam"]}, {"surname": ["Chuc", "Diwanm"], "given-names": ["NTK", "VK"], "article-title": ["\"FILABAVI, a demographic surveillance site, an epidemiological field laboratory in Vietnam\""], "source": ["Scandinavian journal of Pubic Health"], "year": ["2003"], "volume": ["31"], "fpage": ["3"], "lpage": ["7"], "pub-id": ["10.1080/14034950310015031"]}, {"surname": ["Xu", "Evans", "Kadama", "Zeramdini", "Klavus", "Murray"], "given-names": ["K", "DB", "P", "R", "J", "CJL"], "article-title": ["Household catastrophic health expenditure: a multicountry analysis"], "source": ["The Lancet"], "year": ["2003"], "volume": ["362"], "fpage": ["111"], "lpage": ["117"], "pub-id": ["10.1016/S0140-6736(03)13861-5"]}, {"surname": ["Xu"], "given-names": ["K"], "article-title": ["Distribution of health payments and catastrophic expenditures Methodology"], "source": ["World Health Organization Geneva; Department \"Health System Financing\" (HSF) Cluster \"Evidence and Information for Policy\" (EIP)"], "year": ["2005"], "fpage": ["2"]}, {"surname": ["Gotstadze", "Bennett", "Ranson", "Gzirshvili1"], "given-names": ["G", "S", "K", "D"], "source": ["Health care-seeking behaviour and out-of-pocket payments in Tbilisi, Georgia"], "year": ["2005"], "publisher-name": ["Oxford University Press in association with The London School of Hygiene and Tropical Medicine"], "comment": ["All rights reserved"], "pub-id": ["10.1093/heapol/czi029"]}, {"collab": ["Ministry of Health of Vietnam"], "article-title": ["Vietnam growing healthy"], "source": ["Hanoi Author"], "year": ["2002"]}, {"surname": ["Toan"], "given-names": ["NV"], "article-title": ["Utilisation of health care service in a transitional society: study in Vietnam 1991\u20131999"], "source": ["(Doctoral thesis) Karolinska Institutet, Stockholm, Sweden"], "year": ["2001"]}, {"surname": ["Swanepoel", "Stuart"], "given-names": ["C", "I"], "article-title": ["Health Care Provider Choice"], "source": ["Bureau For Economic research University of Stellenbosch Private Bad 5050, 7599 Shouth Africa, Stellenbosch Economic Working Papers"], "year": ["2006"], "volume": ["11"]}, {"surname": ["Hjortberg.C"], "given-names": ["Hcuiadc-tcoZ"], "article-title": ["Health care utilisation in a developing country- the case of Zambia"], "source": ["Doctoral thesis"], "year": ["2004"], "publisher-name": ["Lund University KFS AB, Lund"]}, {"collab": ["World Bank"], "article-title": ["Information Document VN PPRAISAL STAGE- Program 135 Phase 2 Support Credit"], "comment": ["P104097(Report No: AB2708)."]}, {"collab": ["Government of Vietnam"], "article-title": ["Decision No. 135/1998/QD-TTg of July 31, 1998 to approve the program on socio-economic development in mountainous, deep-lying and remote commues with special difficulties"], "source": ["Prime Minster of Vietnam"], "year": ["1998"]}, {"collab": ["Ministry of Labour \u2013 Invalids \u2013 Social Affairs"], "article-title": ["Statistics on hunger elimination and poverty eradication 1998\u20132000 and 2001\u20132003"], "source": ["Statistics book"], "year": ["2004"]}]
{ "acronym": [], "definition": [] }
29
CC BY
no
2022-01-12 14:47:26
BMC Health Serv Res. 2008 Jul 30; 8:162
oa_package/f2/0c/PMC2529291.tar.gz
PMC2529292
18717999
[ "<title>Background</title>", "<p>Consistently, studies show that patients with chronic illnesses do not receive optimal treatment [##REF##12399340##1##,##REF##11533422##2##]. Redesigning primary care by separating acute care from planned management of chronic conditions has been proposed to close the quality chasm between current practices and optimal standards [##REF##12365965##3##]. Of all chronic conditions, care for diabetic patients is probably the most manifest and widely spread example of primary care development [##REF##16868301##4##,##REF##11315833##5##]. In the Netherlands, 85% of patients with Diabetes Mellitus type 2 are treated within primary care [##REF##18024508##6##].</p>", "<p>The creation of practice teams with a clear division of labour is an important aspect within this context [##REF##10688568##7##]. Nurses and nurse assistants both are generally involved in management of patients with diabetes. Therefore, key elements of teamwork, such as sharing clear goals, division of labour, training and communication [##REF##15010447##8##] are suspected to potentially improve care for these patients [##REF##10688568##7##,##REF##11145623##9##]. Studies showed positive associations between higher levels of teamwork and such outcomes as clinical performance [##REF##12838294##10##], absence of hospital physicians due to sickness [##REF##11351050##11##], job satisfaction [##REF##17337517##12##], and patient outcomes such as satisfaction of patients with their care [##REF##17337517##12##, ####REF##12897360##13##, ##REF##14619358##14##, ##REF##11588082##15####11588082##15##]. A related construct that is increasingly described in quality improvement research is organizational culture. This interest is based on the increasing recognition that cultural changes are needed alongside the structural changes to secure gains in quality [##REF##12820673##16##]. Some studies showed that organizational cultures that support teamwork and quality improvement may contribute to achieving high quality care [##REF##15083111##17##, ####REF##7782222##18##, ##REF##11926323##19##, ##REF##16946107##20####16946107##20##]. However, it has also been shown that a mix of cultures was associated with higher levels of team effectiveness [##REF##15586830##21##], whereas several other studies failed to find associations between culture and performance [##REF##17591607##22##,##REF##11488265##23##].</p>", "<p>In most countries, primary care practices are small, office-based organizations, usually consisting of no more than a handful of people. Although evidence for the possible relevance of teamwork and culture is growing, most evidence for these-intuitively appealing-concepts is based on studies in hospital settings. In this study we therefore investigate whether higher levels of teamwork and specific types of organizational culture are associated to diabetes care in small office-based general practices.</p>" ]
[ "<title>Methods</title>", "<title>Design and population</title>", "<p>The present cross sectional study was embedded in an intervention study, in which 350 practices in three regions in the middle and south of the Netherlands were invited to participate. Forty general practices agreed to participate (response rate 11.4%), and they were paired on stratification criteria and randomly allocated to intervention or control group [##REF##18024508##6##]. A researcher visited intervention practices at the beginning of the intervention period, in February to April 2003, to discuss the current practice procedures for diabetes care with the staff. Situations in which various staff members shared tasks was a special topic of discussion. Then a diabetes passport was introduced, a patient-held booklet with important personal information that can be used to track results, record treatment targets and give (educational) information. The professionals discussed how the passport could best fit in the practice routines and work processes. The researcher summarized the various responsibilities involved in diabetes care and the use of the passport on a desk-top card. In the first three months, patients received their passport. Three months later, a researcher visited the practice to discuss the progress of the project and to see whether the division of tasks was being maintained as planned. After 6 months, all patients completed a short questionnaire on the use of the diabetes passport, after which each practice received benchmarked feedback on the introduction and use of the passports [##REF##18024508##6##]. At post-intervention, in May to July 2004, all practice members in the 40 practices who indicated to be actively involved in medical care for patients with diabetes type II (general practitioners, nurse practitioners, and practice assistants) were invited to complete questionnaires on team climate and organizational culture. Team and culture measures were combined with data of diabetes mellitus type II patients younger than 80. The study was approved by the ethics committee Arnhem-Nijmegen. Written, informed consent was received from all study participants.</p>", "<title>Measures</title>", "<p>Clinical outcomes were HbA1c level, systolic blood pressure and total cholesterol levels. A fourth outcome was clinical performance which was measured with a sum score of 10 process indicators of diabetes care quality, based on national guidelines on diabetes care [##UREF##0##24##] (see Figure ##FIG##0##1##; measured at the level of the individual patients, Chronbach's alpha 0.86). A patient could be given a score between 0 and 10, because each indicator was scored either done (1) or not done (0). All outcomes were derived by scrutinizing the electronic medical record systems (EMR) by trained research personnel at post-intervention in July 2004.</p>", "<title>Independent factors</title>", "<p>To measure organizational culture, we used the 'Competing Values Framework' (CVF) in which respondents were asked to distributed 100 points across four sets of organizational statements according to the description that best fits their own organization in five questions [##UREF##1##25##]. This approach recognizes that no organization exhibits only one culture or set of values, but that multiple cultures and values coexist simultaneously and compete for attention. The framework distinguishes two dimensions: 'internally oriented' versus 'externally oriented', and 'stability' versus 'flexibility and change', resulting in four ideal types of culture. The <italic>group </italic>culture emphasizes teamwork, cohesiveness, and participation. The <italic>developmental </italic>culture is characterized by the promotion of innovation and risk-taking, and is oriented towards growth. The <italic>rational </italic>culture emphasizes achievement and meeting objectives; people are rewarded to achieve organizational goals and working efficiently. Finally, the <italic>hierarchical </italic>culture emphasizes stability, rules, regulations and coordination. The statements reflect the four culture types. For each question, non blank respondent errors (i.e. the allocation of more or less than 100 points) were corrected by proportionally adjusting the responses to sum up to 100. For each practice, we determined the mean scores on the four types of culture. Internal consistency reliability for the four culture types, using Cronbach's alpha, were 0.64 for group culture, 0.51 for developmental culture, 0.55 for hierarchical culture, and 0.46 for rational culture. In addition, we calculated how well the scores for the different organizational types of culture were in balance, using the Blau index that has been described in previous studies [##REF##15586830##21##,##REF##17591607##22##]. The hypothesis underlying this measure is that it is the relative balance among the four culture types that is associated with team effectiveness. Higher scores on this index indicate a more even distribution of points among the four culture types, so practices that distributed their points in a 25/25/25/25 pattern had the highest score on 'culture balance' (1), whereas practices with more points for one or the other culture type had lower balance scores (&lt; 1).</p>", "<p>Teamwork was measured with the 14 item short version of the 'Team Climate Inventory' (TCI) [##UREF##2##26##,##UREF##3##27##], answered on 5-point Likert scales. The underlying theory argues that group innovations often result from team activities which are characterized by 1) focusing on clear and realistic objectives in which the team members are committed (vision), 2) interaction between team members in a participative and inter-personally non-threatening climate (participative safety), 3) commitment to high standards of performance and, thus, preparedness for basic questions and appraisal of weaknesses (task orientation), and finally, 4) enacted support for innovation attempts including, e.g. cooperation to develop and apply new ideas (support for innovation). For each scale, mean scores were calculated per individual and then averaged to practice-level scores. Chronbach's alphas were 0.81, 0.79, 0.78, and 0.82 respectively, and correlations (r) ranged from 0.49 to 0.53. We finally combined these to one single score [##REF##11588082##15##]. Overall Chronbach's alpha for the 14 questions was 0.91. Correlations between scales and the overall measure ranged from 0.75 to 0.84.</p>", "<p>We translated both the team and culture instruments into Dutch according to guidelines for cross-cultural translation [##REF##11124735##28##]. Analysis of variance tests verified that individual level responses to the culture and team climate instrument could be validly aggregated to the level of the teams for all but one scale. The within-team variability of responses was less than the between-team variability (F values ranging from 2.29 to 3.90 (p &lt; 0.005)). This test was not significant for the hierarchical culture scale (F value 1.3; p = 0.19).</p>", "<p>The following-possibly confounding-factors were included: whether the practice had special diabetes consulting hours, and whether it was an intervention or control practice, measured by a checklist that was completed by a member of each practice personnel at the start of the project. Finally, age and gender of the patients were included, derived from mailed patient questionnaires, and the baseline measures of the four outcomes derived from the EMR.</p>", "<title>Analysis</title>", "<p>We performed multi level regression analyses (mixed models) with patients (level 1) nested within the practices (level 2). We examined bivariate correlations to check for high correlations (Pearson's correlation and cross tabulations with χ<sup>2 </sup>test and studied single relationships between the outcomes and all predictors before adding the control variables. Since we were interested in the effect of each of our variables of interest separately (different types of organizational culture and team climate), we used separate models to study one of these variables at a time. Thus, for each outcome, six models were conducted; four different models examined the four organizational cultures, one examined the balance among these culture types, and one examined team climate. Each model controlled for patient age, sex, and the baseline measure on the particular outcome, whether the practice had special diabetes consulting hours, and whether it was an intervention or control practice. All analyses were performed using SPSS version 12.0.1.</p>" ]
[ "<title>Results</title>", "<title>Practice characteristics</title>", "<p>In total, 146 practice members in 40 practices were invited to complete the questionnaires. We obtained team climate and culture data from 92 respondents, 46 general practitioners (response rate 71%), 8 practice nurses (response rate 73%) and 38 practice assistants (response rate 54%) working in 39 practices (overall response rate: 63%). The analysis on organizational culture and team climate was restricted to the practices in which at least two practice members returned the questionnaires. Therefore, we excluded 9 practice members in 10 practices in which this was not the case. The mean number of appointed members per practice was 3.7 (SD 1.0) and did not differ significantly for excluded practices as compared to included practices (3.4, range 2 to 5 and 3.8, range 2 to 6 respectively, p = 0.2). Also, excluded practices were as often single handed practices as included practices (p = 0.3).</p>", "<p>Table ##TAB##0##1## shows the characteristics of the practices. Single handed practices were underrepresented in our sample as compared to the national mean (40% versus 60%) [##UREF##4##29##]. Among the four types of culture, group culture by far received most of the points (mean across practices = 51.6), followed by hierarchical (19.7), developmental (16.9) and finally rational culture (11.8). The balance among these values of culture was 0.60 on average. We also explored the data for the dominant culture [##REF##15083111##17##] (the culture scoring highest in each practice; data not shown). In only 3 practices, hierarchical culture received the highest amount of points. All the other practices had a dominant group culture. The overall mean score on team climate was 1.94. Scores on the four scales were 1.84 for vision, 1.83 for participative safety, 1.96 for task orientation and 2.16 for support for innovation; data not shown).</p>", "<title>Patient characteristics</title>", "<p>In 40 practices, 2106 patients received questionnaires. Response rates were 68% for the first, and 69% for the second questionnaire, which resulted in data from 993 patients. Since we excluded 10 practices, 241 patients were excluded, leaving 752 patients for this study. Excluded patients did not differ significantly from included patients with respect to age, sex, and our outcomes.</p>", "<p>Inspection of Table ##TAB##1##2## learns that the mean age of the patients was 63 years, and 48.7% was male. Mean systolic blood pressure was 144.2; mean total cholesterol was 81.5 and mean HbA1c was 7.0. Scores on diabetes care quality differed considerably, and varied from 0 to 9, with a mean score of 5.82.</p>", "<p>Table ##TAB##2##3## shows that none of the selected clinical patient outcomes (HbA1c, systolic blood pressure and total cholesterol) showed significant associations with team climate or culture. However, we did find significant relations with clinical performance. A higher score on group culture was associated with lower scores on diabetes care quality (p = 0.04) with a coefficient of -0.04. This means that every 10-unit change on the group culture score (e.g. from 20 to 30 points) resulted in a 0.4 lower score on the diabetes care quality indicator. In theory, if a practice would move from the lowest group culture score to the highest (a difference of 55.6 points in this sample), the score on the quality indicator would decrease by 5.6 * 0.4 = 2.24 points. Since the range in the mean scores for the quality indicator was from 0 to 9 points, 2.24 points therefore represents a maximum decrease of 24.9%. In total, 15.6% of the variation in the quality indicator outcome was determined by our model that included group culture of which 2.7% was accounted for by group culture. On the other hand, maintaining a balance between the different culture types was positively associated with quality (β = 5.97, p = 0.03), representing a maximum 27.6% of the nine point practice range in our quality indicator. A 0.1-unit change in the balance score (e.g. from 0.6 to 0.7) resulted in a 0.6 higher score on the quality indicator. Our model including cultural balance explained 16.2% of the variation in the quality indicator, of which 3.5% was explained by cultural balance.</p>" ]
[ "<title>Discussion</title>", "<p>Overall, we found that high group culture scores were negatively correlated with adherence to diabetes guidelines in primary care practice (β = -0.04), whereas maintaining a balance among the different types of culture on the other hand was positively correlated to managing diabetes care well (β = 5.97). None of our variables of interest showed associations with our clinical patient outcomes.</p>", "<title>Comparison with other studies</title>", "<p>This study confirmed results of recent studies in primary care in the UK, using the CVF, by showing that primary care organizations primarily have group cultures [##REF##17591607##22##,##REF##12171222##30##]. In one of those studies managers of primary care trusts pointed out the possible disadvantages of group cultures, such as a tendency to be 'inward looking'. They expected quality improvement to be hard to achieve unless practices change their culture to one that valued greater collaboration and sharing of expertise, and a willingness to be more flexible in the way that they operated [##REF##12969929##31##]. In our study, high scores on the group culture variable were negatively correlated with indicators for managing care well. This might be explained in light of the suggestion that different culture types are related to those aspects of performance that are valued by that specific dominant culture type [##REF##12820673##16##]. In other words, for example for <italic>changing </italic>routines (in quality improvement projects), a more team-focused and developmental culture type with a focus on flexibility might be helpful in attaining good results, whereas for <italic>performing routine tasks</italic>, such as inspecting feet every 3 months, aspects valued in the more control orientated rational or hierarchical culture types, with a focus on policies, procedures and production might be needed. Therefore, one could also argue that -to reach <italic>and </italic>sustain high quality care for chronic diseases such as diabetes-teams need to find the balance between flexible and control oriented culture types since continuous measuring and improvement, good teamwork, a drive to gain better results, and working according to protocols are equally important. This might be in line with the fact that we found that a high balance between the different types of culture was positively correlated to high quality diabetes care. An earlier study on the role of perceived team effectiveness in improving chronic illness care reasoned that it would be the relative balance among culture values of participation, achievement, openness to innovation and adherence to rules that is most likely to be associated with perceived team effectiveness. Indeed, this study showed an association between a culture balance and team effectiveness, although it was rather marginal [##REF##15586830##21##]. A recent study in primary care hypothesized that a high score on cultural balance would be associated with high levels of team climate, which was not confirmed by the data [##REF##17591607##22##].</p>", "<p>Although previous studies suggested the relevance of teamwork in diabetes care [##REF##11145623##9##,##REF##12897360##13##,##REF##11588082##15##], we failed to find significant associations between team climate and our outcomes, as did a recent UK study [##REF##17591607##22##]. Again, the type of outcome might shed some light on this topic, since studies that did find associations often included outcomes such as work satisfaction [##REF##17337517##12##], absence from work due to sickness [##REF##11351050##11##] and satisfaction by patients with their care [##REF##17337517##12##, ####REF##12897360##13##, ##REF##14619358##14##, ##REF##11588082##15####11588082##15##]. Interestingly, climate scores were also quite low as compared to other studies [##UREF##5##32##,##UREF##6##33##]. This might point to the fact that different practice members involved in diabetes care may not experience their relationships as a 'true' team when it comes to diabetes care [##REF##11488265##23##,##REF##17286724##34##]. The varied nature of clinical problems in primary care practice make team building especially challenging as compared to 'single specialty practices' [##REF##15010447##8##].</p>", "<p>Our study failed to find associations between our organizational factors of interest and intermediate clinical patient outcomes. These findings are consistent with recent findings in studying and reviewing the link between safety-factors and risk-adjusted patient outcomes [##REF##16195571##35##,##REF##18035261##36##]. Although the selection of a clinical outcome is recommended, the selection of such a specific variable may just be too narrow to reflect the complexity of modern patient care [##REF##17164460##37##].</p>", "<title>Strengths and limitations of this study</title>", "<p>To gain better insight on organizational factors influencing health care quality, it has been suggested that studies should preferably focus on factors on different levels (e.g. organizational as well as team), include patient outcomes and use multi level data analyses to correct for clustering effects [##UREF##7##38##]. In the current study, we have taken these suggestions into account. However, some limitations need to be addressed.</p>", "<p>First, the relative small sample size in our study may have limited the power to find associations. Since general practices are generally small office-based organizations, the number of participants who returned our questionnaire on organizational culture and team climate was relatively low (varying from 2 to 4). Previous studies using the TCI excluded practices if less than 30% of respondents completed questionnaires [##REF##17337517##12##,##REF##17591607##22##]. However, the number of GPs and other care providers per practice seems generally somewhat lower in the Netherlands than in -for instance-UK practices [##REF##12897360##13##,##UREF##4##29##,##REF##11954680##39##]. In this study, we also excluded the practices in which only one person returned our questionnaire. The low numbers of respondents could impact the validity of our culture and team climate measures. Low Cronbach's alphas for the culture measures for instance, and the low F-value for the aggregation of the scores on the hierarchical culture scale might point to that. In addition, the fact that primary care practices-both in our study and in the UK [##REF##17591607##22##,##REF##12171222##30##] – tend to have predominantly group cultures raises questions about the sensitivity of the CVF in this setting, especially if culture is analyzed as categorical variable. We have taken this point partly into account by using continuous culture variables in the analyses, however, this cannot fully clear away some concerns about the appropriateness of use of this particular instrument in small practices. Although this instrument has some clear advantages over others, such as the fact that it has been used in several other studies in varying settings, and the fact that it measures 'culture typologies' rather than simple variables [##REF##12820673##16##], the factors measured may have a different meaning in different health care settings.</p>", "<p>Also, and partly related to our previous point, since climate and culture are considered to be shared attributes, individual measures are aggregated to practice level. Yet, this ignores the fact that different subgroups may have different opinions (for instance general practitioners may experience the culture differently from the practice nurses or assistants) [##REF##17337517##12##,##REF##12820673##16##]. Especially in very small practices (for instance with only one general practitioner and two practice assistants), it is debatable whether the aggregated score is a valid measure of the reality. However, for subgroup analysis researchers would need much bigger samples of respondents, which raises questions about the feasibility of survey based methods in measuring these complicated constructs.</p>", "<p>Further, our process measure was assessed by scrutinising the EMR. However, a considerable gap may exist between what the practice members record, and what they actually do in practice. Especially preventive or counselling activities, such as advising physical exercise, have been found to be under recorded [##REF##10856412##40##]. Also, the guideline indicated that smoking behaviour should be discussed with all patients on a yearly basis, even if they are non-smokers. Therefore, we may have underestimated the scores on the quality indicator. However, it is likely that this holds for all practices to the same extent since they all used an EMR. We cannot rule out the possibility though that other confounding factors may have played a role, such as whether or not a physician received feedback or reminders in the EMR, which may have prompted these GPs to perform and register particular preventive activities. At the time of the study, no specific arrangements with insurance companies existed that may have influenced diabetes management. Some practices had a practice nurse who performed tasks related to care for patients with chronic diseases, however, the availability of practice nurses was equal for all regions in the Netherlands. Single handed practices were underrepresented in our study. However, previous research showed no difference in delegation of preventive tasks and treatment of chronic diseases between GPs in single handed practices compared to GPs in group practices [##REF##15640293##41##] so we can assume that our sample is representative for Dutch practices.</p>", "<p>Finally, it is important to note that it is not possible to conclude we showed causal linkages between culture and our outcomes, since the results were based on cross sectional data. We therefore do not know whether high scores on group culture lead to poor diabetes management, or -the other way around-practices in which quality of care is managed in a certain way develop certain types of culture, or culture and performance emerge together in a reciprocal and reinforcing manner [##REF##12820673##16##].</p>" ]
[ "<title>Conclusion</title>", "<p>This study contributes to the discussion around the evidence for intuitively appealing features such as culture and teamwork that have been suggested as a lever for health care improvement. We did find some significant associations between culture and high quality diabetes care, but the relations were rather marginal. On the one hand, one could argue that if organizational culture would have only limited influence on many aspects of care during a long period of time, the resultant of that might still add up to a substantial level. On the other hand, feasibility of current measurements of constructs such as climate and culture is still debatable-especially in primary care settings-, given the fact that response rates are low, and scores are aggregated, which causes power reduction and loss of information. Further, we failed to find any associations with our clinical outcomes, which begs the question if and exactly how these constructs can contribute to evidence based care, and -eventually-healthier patients.</p>", "<p>Future studies in primary care should preferably combine quantitative and qualitative research methods and use more complex designs to get a better insight into these complex constructs and possibly mediating or moderating factors. Also, it would be worth exploring possible associations between culture and climate and changes in health care quality, as well as the use of other measurement instruments and methods that are more sensitive to -for instance-different subcultures that might exist within organizations, especially in primary care practices where people work in very small teams and deal with a big variety of clinical problems.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Redesigning care has been proposed as a lever for improving chronic illness care. Within primary care, diabetes care is the most widespread example of restructured integrated care. Our goal was to assess to what extent important aspects of restructured care such as multidisciplinary teamwork and different types of organizational culture are associated with high quality diabetes care in small office-based general practices.</p>", "<title>Methods</title>", "<p>We conducted cross-sectional analyses of data from 83 health care professionals involved in diabetes care from 30 primary care practices in the Netherlands, with a total of 752 diabetes mellitus type II patients participating in an improvement study. We used self-reported measures of team climate (Team Climate Inventory) and organizational culture (Competing Values Framework), and measures of quality of diabetes care and clinical patient characteristics from medical records and self-report. We conducted multivariate analyses of the relationship between culture, climate and HbA1c, total cholesterol, systolic blood pressure and a sum score on process indicators for the quality of diabetes care, adjusting for potential patient- and practice level confounders and practice-level clustering.</p>", "<title>Results</title>", "<p>A strong group culture was negatively associated to the quality of diabetes care provided to patients (β = -0.04; p = 0.04), whereas a more 'balanced culture' was positively associated to diabetes care quality (β = 5.97; p = 0.03). No associations were found between organizational culture, team climate and clinical patient outcomes.</p>", "<title>Conclusion</title>", "<p>Although some significant associations were found between high quality diabetes care in general practice and different organizational cultures, relations were rather marginal. Variation in clinical patient outcomes could not be attributed to organizational culture or teamwork. This study therefore contributes to the discussion about the legitimacy of the widespread idea that aspects of redesigning care such as teamwork and culture can contribute to higher quality of care. Future research should preferably combine quantitative and qualitative methods, focus on possible mediating or moderating factors and explore the use of instruments more sensitive to measure such complex constructs in small office-based practices.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>MB, RD, MW, TvdW and RG designed the study. MB performed the data collection and data analyses, and all other authors contributed to interpreting the data. MB wrote the first draft, which was critically revised by RD and then by all others. All authors have read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1472-6963/8/180/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>We thank all participating health care personnel and patients.</p>", "<p>Funding Source: European Commission, Fifth Framework, Rebeqi project, contract nr. QLRT-2001-00657</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Clinical performance measure: diabetes guideline recommendations.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Characteristics of practices (N = 30)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">%/Mean (SD)</td></tr></thead><tbody><tr><td align=\"left\">Type of practice (% Single handed)</td><td align=\"left\">40%</td></tr><tr><td align=\"left\">Special diabetes consulting hours</td><td align=\"left\">36.7%</td></tr><tr><td align=\"left\">Group culture (0 – 100)</td><td align=\"left\">51.6 (13.2)</td></tr><tr><td align=\"left\">Developmental culture (0 – 100)</td><td align=\"left\">16.9 (7.4)</td></tr><tr><td align=\"left\">Hierarchical culture (0 – 100)</td><td align=\"left\">19.7 (8.0)</td></tr><tr><td align=\"left\">Rational culture (0 – 100)</td><td align=\"left\">11.8 (5.6)</td></tr><tr><td align=\"left\">Cultural balance (0 – 1)</td><td align=\"left\">0.60 (0.10)</td></tr><tr><td align=\"left\">Team climate (1 – 5)</td><td align=\"left\">1.94 (0.39)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Characteristics of patients (N = 752)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">N</td><td align=\"left\">%/Mean (SD)</td></tr></thead><tbody><tr><td align=\"left\">Gender, % male</td><td align=\"left\">752</td><td align=\"left\">48.7%</td></tr><tr><td align=\"left\">Age, years (SD)</td><td align=\"left\">752</td><td align=\"left\">63.0 (9.7)</td></tr><tr><td align=\"left\">Systolic blood pressure (SD)</td><td align=\"left\">716</td><td align=\"left\">144.2 (19.4)</td></tr><tr><td align=\"left\">Total Cholesterol (SD)</td><td align=\"left\">716</td><td align=\"left\">81.5 (9.6)</td></tr><tr><td align=\"left\">HbA1c</td><td align=\"left\">696</td><td align=\"left\">7.0 (1.2)</td></tr><tr><td align=\"left\">Quality of diabetes care (0 – 10)</td><td align=\"left\">752</td><td align=\"left\">5.82 (2.8)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Associations between team climate, organizational culture and HbA1c, systolic blood pressure, total cholesterol and the aggregated diabetes process quality indicator, measured at patient level (N = 752).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\">HbA1c</td><td align=\"center\" colspan=\"2\">Systolic blood pressure</td><td align=\"center\" colspan=\"2\">Total cholesterol</td><td align=\"center\" colspan=\"2\">Clinical performance</td></tr><tr><td/><td colspan=\"8\"><hr/></td></tr><tr><td/><td align=\"center\">β</td><td align=\"center\">95% CI</td><td align=\"center\">β</td><td align=\"center\">95% CI</td><td align=\"center\">β</td><td align=\"center\">95% CI</td><td align=\"center\">β</td><td align=\"center\">95% CI</td></tr></thead><tbody><tr><td align=\"left\">Group culture</td><td align=\"center\">-0.01</td><td align=\"center\">-0.02, 0.00</td><td align=\"center\">-0.08</td><td align=\"center\">-0.25, 0.10</td><td align=\"center\">0.00</td><td align=\"center\">-0.01, 0.00</td><td align=\"center\">-0.04</td><td align=\"center\">-0.08, 0.00 *</td></tr><tr><td align=\"left\">Developmental culture</td><td align=\"center\">0.00</td><td align=\"center\">-0.02, 0.01</td><td align=\"center\">0.11</td><td align=\"center\">-0.16, 0.39</td><td align=\"center\">0.01</td><td align=\"center\">-0.01, 0.02</td><td align=\"center\">0.04</td><td align=\"center\">-0.03, 0.11</td></tr><tr><td align=\"left\">Hierarchical culture</td><td align=\"center\">0.01</td><td align=\"center\">0.00, 0.02</td><td align=\"center\">0.10</td><td align=\"center\">-0.14, 0.34</td><td align=\"center\">0.00</td><td align=\"center\">-0.01, 0.01</td><td align=\"center\">0.03</td><td align=\"center\">-0.03, 0.09</td></tr><tr><td align=\"left\">Rational culture</td><td align=\"center\">0.02</td><td align=\"center\">0.00, 0.03</td><td align=\"center\">-0.11</td><td align=\"center\">-0.44, 0.23</td><td align=\"center\">0.00</td><td align=\"center\">-0.01, 0.02</td><td align=\"center\">0.04</td><td align=\"center\">-0.05, 0.12</td></tr><tr><td align=\"left\">Cultural balance</td><td align=\"center\">1.35</td><td align=\"center\">-0.03, 2.72</td><td align=\"center\">9.70</td><td align=\"center\">-14.53, 33.93</td><td align=\"center\">0.65</td><td align=\"center\">-0.42, 1.72</td><td align=\"center\">5.97</td><td align=\"center\">0.66, 11.28 *</td></tr><tr><td align=\"left\">Team climate</td><td align=\"center\">-0.22</td><td align=\"center\">-0.50, 0.05</td><td align=\"center\">2.06</td><td align=\"center\">-2.53, 6.64</td><td align=\"center\">0.09</td><td align=\"center\">-0.13, 0.30</td><td align=\"center\">-0.57</td><td align=\"center\">-1.76, 0.76</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><p>* sign &lt; 0.05</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1472-6963-8-180-1\"/>" ]
[]
[{"surname": ["Rutten", "Verhoeven", "Heine", "De Grauw", "Cromme", "Reenders", "van Ballegooie", "Wiersma"], "given-names": ["GEHM", "S", "RJ", "WJC", "PVM", "K", "E", "T"], "article-title": ["NHG-standaard Diabetes Mellitus Type 2 (eerste herziening)."], "source": ["Huisarts Wet"], "year": ["1999"], "volume": ["42"], "fpage": ["67"], "lpage": ["84"]}, {"surname": ["Quinn", "Kimberly", "Kimberly JR and Quinn RE"], "given-names": ["RE", "JR"], "article-title": ["Paradox, Planning, and Perseverance: Guidelines for Managerial Practice"], "source": ["Managing Organization Transitions"], "year": ["1984"], "publisher-name": ["Homewood, Dow Jones-Irwin"], "fpage": ["295"], "lpage": ["313"]}, {"surname": ["Kivim\u00e4ki", "Elovainio"], "given-names": ["M", "M"], "article-title": ["A short version of the Team Climate Inventory: Development and Psychometric properties"], "source": ["J Occup Org Psych"], "year": ["1999"], "volume": ["72"], "fpage": ["241"], "lpage": ["246"], "pub-id": ["10.1348/096317999166644"]}, {"surname": ["Loo", "Loewen"], "given-names": ["R", "P"], "article-title": ["A confirmatory factor-analytic and psychometric examination of the team climate inventory - Full and short versions"], "source": ["Small Group Res"], "year": ["2002"], "volume": ["33"], "fpage": ["254"], "lpage": ["265"], "pub-id": ["10.1177/104649640203300205"]}, {"surname": ["Kenens", "Hingstman"], "given-names": ["R", "L"], "source": ["Cijfers uit de registratie van huisartsen: peiling 2004"], "year": ["2004"], "publisher-name": ["Utrecht, NIVEL"]}, {"surname": ["Loo"], "given-names": ["R"], "article-title": ["Assessing 'team climate' in project teams"], "source": ["International Journal of Project Management"], "year": ["2003"], "volume": ["21"], "fpage": ["511"], "lpage": ["517"], "pub-id": ["10.1016/S0263-7863(02)00058-3"]}, {"surname": ["Dackert", "Brenner"], "given-names": ["I", "S"], "article-title": ["Team climate inventory with merged organization"], "source": ["Psychol Reports"], "year": ["2002"], "volume": ["91"], "fpage": ["651"], "lpage": ["656"], "pub-id": ["10.2466/PR0.91.6.651-656"]}, {"surname": ["Hearld", "Alexander", "Fraser", "Jiang"], "given-names": ["LR", "JA", "I", "HJ"], "article-title": ["How do hospital organizational structure and processes affect quality of care? A critical review of research methods."], "source": ["Med Care Res Rev OnlineFirst"], "year": ["2007"]}]
{ "acronym": [], "definition": [] }
41
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no
2022-01-12 14:47:26
BMC Health Serv Res. 2008 Aug 21; 8:180
oa_package/9f/28/PMC2529292.tar.gz
PMC2529293
18752660
[ "<title>Background</title>", "<p>The quality of health care services, as measured by standardized indicators and stakeholders' satisfaction, constitutes a cornerstone of health care delivery in the current era of continuous health care reform. However, the achievement of quality of care remains a challenge for many western societies, despite a continuously increasing level of health care expenditure [##REF##9749483##1##]. Furthermore, many efforts in the hospital setting have been devoted to limit practice variation, effectively utilize available electronic resources and improve patient satisfaction; whereas in primary care services the importance of financial savings along with significant health gains has not been adequately explored. In our opinion, high health care quality requires universal access, equity in services and cost-effective care [##REF##11072882##2##]. In particular, improving chronic illness management has been attracting increasing interest by health care providers and government agencies, since the average chronic patient entering the primary care system is not receiving an optimal quality of care [##UREF##0##3##]. Furthermore, the ageing population, predominantly in the western societies, constitutes an ever growing economic and health care burden requiring increasing community-based services [##REF##17847182##4##].</p>", "<p>A number of different quality improvement (QI) interventions implemented at various clinical settings have been evaluated and presented in the medical literature [##REF##7585368##5##, ####REF##9703533##6##, ##UREF##1##7##, ##UREF##2##8####2##8##]. Such interventions range from single-component approaches (e.g. electronic reminder systems) to multifaceted complex strategies combining both patient-mediated activities (e.g. educational leaflets) and health care provider services (e.g. consensus building, training, and audit/feedback processes) [##REF##7953212##9##]. In addition, tailoring interventions to the local group practitioners' needs, has also been proposed as a concerted effort to attain successful and sustainable outcomes compared to interventions that are fixed and lack programmatic flexibility [##REF##9690883##10##]. Moreover, interventions for quality improvement involve significant amounts of financial investment, and therefore require careful modeling in order to maximize potential successes [##REF##15640299##11##]. Finally, process and outcome measure evaluation of such attempts is thought to be of paramount importance in order to assist decision makers in developing appropriate policies for structural and long-term planning.</p>", "<p>Many countries with adequate resources, high capacity in primary care research and past experience in quality improvement efforts, have been actively involved in implementing major modifications in primary health care services in order to incorporate quality indicators based on a broad array of methodological strategies [##REF##17625132##12##,##REF##17456241##13##]. The term 'clinical governance' has been introduced to capture a range of activities required to improve the quality of health care services, including the development of processes for continuous monitoring and accountability systems for delivered quality care [##REF##10968820##14##]. However, countries with little experience and limited resources, including Cyprus, face significant challenges in attempting to design QI interventions tailored to country-specific characteristics as described below.</p>", "<p>Cyprus is currently moving towards the introduction of a primary care driven, universal health care coverage system for the entire population, with quality improvement processes as an important incorporated component. However, for the time being, Cyprus operates in a dual system of health care delivery, offering publicly-funded health care services to low and medium-income citizens, while the rest, mostly well-off part of the society, utilize services from the private sector, covering their expenses either from private health insurance schemes or through out of pocket compensation [##REF##15789453##15##]. The majority of public as well as private primary care settings are characterized by under-use of contemporary information technologies, limited monitoring systems, and variable use of clinical standards of care such as chronic disease management guidelines and patient satisfaction surveys [##REF##16456481##16##]. The Cyprus Ministry of Health is being strongly interested in investing significant resources to promote quality improvement strategies in both sectors of health care services. Within an already existing framework of collaboration between the Clinic of Social and Family Medicine of the University of Crete and the Cyprus Ministry of Health, a pilot intervention for quality improvement in two public primary health care centers in Cyprus was developed. In this correspondence, a detailed description is presented of the theories and methodology used to design a country-specific quality improvement intervention in an urban and rural primary health care center in Cyprus, tailored to local practices and primary care professionals' needs.</p>", "<title>The Theoretical Framework</title>", "<p>Our approach was designed to follow the steps of contemporary theories in order to explore different pathways including the use of information technology and the introduction of standard guidelines for chronic disease management. A literature review and an analysis of the existing organizational and operational context led to the identification of three main areas that required strong theoretical background for a successful design of our intervention; namely: a) an organizational change paradigm; b) the introduction of information technology, and c) the adoption of clinical guidelines into everyday practice for chronic disease management.</p>", "<title>Organizational Change Paradigm</title>", "<p>In anticipation of advanced needs for structural changes in the primary care centers during the intervention, a model of organizational change was adopted, which identifies seven stages: sensing of unsatisfied demands on the system, the search for possible responses, evaluation of alternatives, decision to adopt a course of action, initiation of action within the system, and implementation and institutionalization of a change [##UREF##3##17##].</p>", "<title>Information Technology</title>", "<p>The introduction of an electronic medical record (EMR) followed the four constructs of the <italic>Unified Theory of Acceptance and Use of Technology </italic>(UTAUT), as described in the current literature: performance expectancy, effort expectancy, social influence and facilitating conditions [##UREF##4##18##,##UREF##5##19##]. The UTAUT has been validated empirically amongst four businesses from various industries, and was cross-validated using data from another two businesses, enabling researchers to explain up to 70% of technology acceptance behavior [##UREF##6##20##]. Based on the above, a UTAUT model was adopted for the design of our intervention due to its comprehensive character and high explanatory power. According to the UTAUT model, technology acceptance depends on: a) user determinants (e.g. age, gender, experience, and voluntariness of use), b) information technology expectancy (e.g. performance expectancy and effort expectancy), c) implementation setting and user professional environment, including social influence, and d) organizational facilitating conditions. All of the above were taken into consideration during the preparedness phase of the project as well as for the planned daily interactions of the improvement team, in order to enable physicians and nurses to successfully adopt the use of EMR in a previously computer-naïve professional environment.</p>", "<title>Introducing disease management guidelines</title>", "<p>The selection of chronic illnesses that were used in the intervention was based on the most common diseases encountered in public primary care centers of Cyprus, namely hypertension (HTN) and type II diabetes (T2DM) [##REF##15534575##21##]. The introduction of clinical guidelines and continuing medical and nursing education for chronic disease management was based on the <italic>Chronic Care Model </italic>(CCM) [##REF##10157259##22##,##UREF##7##23##], the <italic>Theory of Planned Behavior </italic>(TPB), and the <italic>Theory of Reasoned Action </italic>(TRA) [##REF##15680740##24##,##REF##9638287##25##]. The CCM model synthesizes evidence-based system changes leading to improved outcomes. It emphasizes six main components: a) the organization of health care, b) community linkages, c) self-management support, d) delivery system design, e) decision support and f) information systems.</p>", "<p>Furthermore, our design was influenced by the Chronic Care Model in order to implement disease management guidelines, electronic reminder systems, and e-library resources as part of the decision support systems. Moreover, a chronic care support was organized through the implementation of a referral scheme, an electronic appointment scheduling, and the introduction of an electronic medical record system (EMR). In addition, patient education activities were scheduled to be deployed through training with educational materials and face to face guidance for disease self-management (e.g. blood glucose monitoring and foot examination). The Theory of Planned Behavior and Theory of Reasoned Action provided the necessary theoretical framework in order to assist us in empirically identifying those QI intervention factors on which our efforts should be targeted. Application of TPB and TRA models also helped us identify, through the conduction of in-depth open-ended elicitation interviews, the underlying beliefs that determine health professional's attitudes, subjective norms, and perceived behavioral control. Thereby, such theories may potentially affect the health professional's likelihood of modifying previous behaviors and successfully adopting newly introduced clinical guidelines.</p>", "<title>Methodological Hypotheses</title>", "<p>Based on the above-described theories and an extensive literature review, we developed specific research hypotheses in accordance with our research model. First, with regards to EMR introduction, we hypothesized that (a) performance expectancy, effort expectancy, and social influence would positively affect Primary Care Physicians' (PCPs) and nurses' attitudes toward adopting EMR technology; (b) organizational facilitating conditions should have a direct effect on performance expectancy, effort expectancy, and health professionals' EMR utilization behavior; and (c) behavioral intention will have a significant positive influence on health professionals' practice.</p>", "<p>In addition, with respect to the adoption of guidelines, we expected that (a) PCPs and nurses would accept and effectively implement clinical practice guidelines on chronic disease management; (b) the quality of primary care services for chronic diseases would be improved following the implementation of our intervention; and (c) the use of CCM, TPB and TRA, would help us shape a positive impact on chronic disease management.</p>", "<p>Finally, we hypothesized that the proposed design, according to given resources and other de facto local characteristics, such as limited technological adoption, computer-naïve environment, lack of previous experiences with quality improvement interventions, and scarce incentives for performance, would help us efficiently examine the effectiveness of a QI intervention in Cyprus.</p>" ]
[ "<title>Methods</title>", "<title>A Multifaceted Quality Improvement Intervention in Cyprus</title>", "<p>The translation and implementation of the above described theories and models into a busy day-to-day clinical practice, represents a formidable challenge. Common experience with other quality improvement interventions [##REF##3416682##26##] coupled with the above described insights from industrial examples, suggest that sustained improvements in chronic illness care require a comprehensive, continuous, and systematic change approach following a specific intervention. Our operational model was based on a multifaceted intervention that was facilitated through a multidisciplinary quality improvement team.</p>", "<p>Current literature supports the implementation of multifaceted interventions in the health care sector, since many components may interact and reinforce each other in encouraging the change of professional practice and promoting workplace satisfaction [##REF##16567820##27##]. Our multifaceted intervention was designed to involve several implementation strategies including a combination of educational components (educational materials, workshops, local opinion leaders' presentations, academic detailing), audit and feedback, and an electronic decision support system enabled through e-library and electronic reminder system tailored to the local needs. Particular tailoring characteristics took into consideration the lack of motivators in the public primary care centers, the scarcity of use of clinical guidelines in daily practice, the absence of referral and appointment systems as well as other organizational weaknesses and the existence of a computer naïve environment. Thus, we incorporated the use of physician facilitators, who introduced several non-monetary incentives for the health professionals, provided them with practical tools such as foot examination screening checklist, and developed tailored organizational changes. We also promoted a strong theoretical framework consisting mainly of the UTAUT and CCM supporting the computer naïve environment, and appointed a responsible individual who had to identify specific resources at each center including the support of a new appointment and referral system. Finally, an informed consent form was developed in order to be used during the implementation phase. The study was approved by the National Bioethics Committee.</p>", "<title>Organizational Changes</title>", "<p>Structural and organizational changes were employed, as equally important components in designing a quality improvement intervention for the management of disease co-morbidities, along with the introduction of standard clinical guidelines [##REF##12588208##28##].</p>", "<p>Consensus building meetings were planned in order to identify potential barriers and evaluate alternatives for the introduction of an appointment-based electronic scheduling system, secure continuity of secretarial support during and after the project ended, and introduce an effective specialist referral system. After reaching a consensus, the adopted decision along with necessary organizational changes could take place awaiting the institutionalization of the selected changes. Additionally, a specific plan was applied through a framework of changes that would guide health professionals in their everyday practice. Upper management support from the administrative health services of the Ministry of Health was actively sought.</p>", "<title>Introduction of Electronic Medical Record (EMR)</title>", "<p>The introduction of the EMR system, which was based on the International Classification System for Primary Care (ICPC-2), consisted of the introduction of a windows-based software program (Transhis) described in detail elsewhere [##REF##18237477##29##]. The secretaries, nurses and physicians at the intervention primary care centers were provided with personal computers, printers and a high-speed broadband internet access for all. Primary care physicians (PCPs) and nurses were asked to serve as evaluators of the EMR system performance. In addition, 18 randomly selected patients, half of which were males and half females, were scheduled to undergo personal interviews in order to provide detailed feedback on their experience with the EMR and identify barriers in its daily implementation. Among many other software programs, <italic>Transhis</italic>, a windows-based EMR system, incorporating episode of care and reminder systems was selected to serve as the supporting electronic interface based on defined criteria for appropriateness, efficiency, and feasibility for the general clinical practice [##REF##11501637##30##].</p>", "<title>Chronic Disease Clinical Guidelines</title>", "<p>Chronic disease guidelines represented our decision support tool, one of the main components of the Chronic Care Model. Such a tool was scheduled to provide evidence-based clinical information to the health care professionals that were readily available through electronic databases facilitated by EMR [##REF##14577900##31##]. Type II diabetes mellitus guidelines were based on the American Diabetes Association and St. Vincent Declaration guidelines. Hypertension management guidelines were based on the VII Report of the Joint National Committee, the European guidelines on cardiovascular disease prevention and the European Society of Hypertension – European Society of Cardiology Guidelines, whereas lipid control practice guidelines were based on the National Cholesterol Education Program III.</p>", "<title>Quality Improvement Team</title>", "<p>Setting up a multidisciplinary quality improvement team was thought to be of paramount importance for the successful facilitation and fide implementation of our intervention. The use of a quality improvement team has been described long ago by industrial quality experts [##UREF##8##32##]. Our study team consisted of two family physicians serving as program facilitators, three academic experts in family medicine with previous extensive experience in QI interventions, and one family physician with expertise in EMR.</p>", "<p>Both study facilitators were scheduled to meet with providers in their practice settings during regular time intervals in order to undertake the following actions: (a) record baseline characteristics of participants in the study and present quality indicators for each selected illness; (b) facilitate consensus building for the management of selected illnesses, according to given clinical guidelines; (c) assist in the development and adaptation of tools and strategies for implementing the intervention; (d) facilitate meetings to assess progress and potential barriers in the implementation of the intervention while being able to modify the plan accordingly; (e) conduct interviews of the participating doctors, nurses and patients; and (f) develop and complete electronic chart audits, monthly reports and outreach visit forms.</p>", "<title>Setting and Participants</title>", "<title>Primary Health Care Centers</title>", "<p>The study was scheduled to take place in Nicosia, the capital of Cyprus, in two urban and two rural public primary health care centers (PHCC), which were selected based on population served and employee criteria (age, duration of medical education, number of years in practice). One rural and one urban center were designed to serve as controls being observed to follow regular practice. Each center was designed to have a worksite leader who would leverage resources and be the primary contact person in collaboration with the quality improvement team. All PCPs and nurses from the intervention primary care centers were scheduled to participate in the study as evaluators of the intervention.</p>", "<title>Patients and Eligibility Criteria</title>", "<p>Several reasons supported the patient selection criteria including our objective to include a relatively small, however homogeneous patient population that has also been identified in the medical literature as a frequently neglected group of patients [##REF##17823464##33##]. In addition, HTN and T2DM were found to be the most common diseases in the primary care system of Cyprus [##REF##15534575##21##]. These health problems often occur in a concurrent fashion and lead to serious complications that may not be optimally treated [##REF##8432214##34##,##REF##10938049##35##]. Finally, despite recommendations for more aggressive hypertension therapy in the presence of coexisting diabetes, it is unclear whether there are any differences in how clinicians manage blood pressure in hypertensive patients with or without diabetes [##REF##10840991##36##].</p>", "<title>Study Design</title>", "<p>The proposed model was planned to be evaluated through a community-based open-label intervention control trial comparing regular practice to an EMR-enhanced practice aided by chronic disease management based on standard clinical guidelines. The design included three phases of evaluation including: (a) a baseline assessment, (b) an end of follow-up comparison and (c) an 18-month post intervention evaluation. A window of 3 months run-in-phase was planned for the eligible patients to enter the study. Due to the possibility that deviations from the protocol during the intervention could impact the validity of the trial, we proposed the proper handling and reporting of any non-adherence to the protocol events. In addition, our study design included process and outcome evaluation as depicted in Figure ##FIG##0##1##[##REF##12571344##37##,##REF##11252222##38##], along with a brief economic analysis (accumulated cost of the personnel, equipment and the intervention itself).</p>", "<title>Process Evaluation</title>", "<title>Structured forms</title>", "<p>The facilitators were expected to complete two structured forms: monthly reports (MR) and outreach visits reports (OVR), which were developed based on previous report from the literature [##REF##11276541##39##]. Monthly reports were expected to provide detailed information on the recording of visits to a Primary Care Health Center (PCHC), the activities within each PCHC, the outcomes of those activities, the number of hours spent for both on-site and off-site activities, the implementation of guidelines in clinical practice and the utilization of the EMR including problems and barriers during its implementation.</p>", "<title>Interviews and Audit</title>", "<p>Apart from the above reports, the facilitators were scheduled to meet with the PCPs and the nurses at 6-month and 18-month follow-up visits in order to conduct semi-structured face-to-face interviews as well as focus group sessions at baseline and end of follow-up. The health professionals were expected to provide information on their overall satisfaction with the intervention, the experiences and potential barriers in the implementation of the study as well as their suggestions for improvements. At the end of the 6-month follow-up period, quality indicators audit were scheduled to be conducted in the intervention PCHCs. Additionally, semi-structured interviews of randomly selected patients from the intervention PCHCs were expected to take place at the end of the 18-month study period.</p>", "<title>Outcome Evaluation</title>", "<p>The outcome evaluation of our intervention included the quality indicators for the selected illnesses along with validated instruments measuring patients' opinion. Quality indicators included specific and measurable elements of practice that can be used to assess the quality of care [##UREF##9##40##]. A set of quality indicators was developed by combining experts' opinion with current scientific evidence. Quality indicators for diabetes included fasting blood sugar, levels of HbA1c, blood pressure (BP), body mass index (BMI), lipid profile (TC, HDL-C, LDL-C, TG), microalbuminuria, fundal and feet examination, and prevalence of smoking. Hypertension quality indicators included: blood pressure measurement (SBP, DBP), BMI, lipid profile and prevalence of smoking.</p>", "<p>In addition, we used the EUROPEP questionnaire, distributed to all patients from the intervention and control PCHC, to evaluate patients' opinion before and after the intervention [##REF##10321387##41##]. The EUROPEP instrument is a reliable and internationally validated questionnaire that measures patients' satisfaction with respect to the care received and the interpersonal skills of primary care physicians. The Greek version was planned to be used in our study [##REF##15495288##42##] after the appropriate cultural adaptation of the questionnaire as deemed appropriate for Cyprus.</p>", "<title>Statistical Analyses</title>", "<p>Summary statistics were planned to be generated for baseline characteristics and clinical evaluations for each study arm. T-test and chi-square statistics were planned to be used to assess the homogeneity of study arms with respect to baseline characteristics. The primary outcome measure was the improvement in the quality indicators of patients with hypertension and diabetes assessed by three different statistical methods. The first was based on a comparison of patients found to be at target levels based on guideline recommendations, before and after the intervention, a comparison of quality improvement measurements using General Linear Model of Repeated Measures, and finally, the percentage of patients with more than 10% improvement over baseline in selected indicators at the end of follow up, as being consistent with previous work of Majumdar, et al [##REF##14578240##43##].</p>", "<p>Quantitative analyses of additional quality indicators included the following variables: monthly visits (total number of visits divided by the duration studied in months), and time to response measures (months until patient achieved target levels for specific indicators). Repeated measures were planned to be analyzed using mixed effects models. Correlations among measurements made on the same subject, were planned to be modeled using random effects and random regression coefficients, and through the specification of a covariance structure. All tests were planned to be two-sided and a level of statistical significance was set at 0.05. All study outcomes were planned to be analyzed on the basis of intention to treat.</p>", "<p>Data obtained during the process evaluation would be qualitatively analyzed using audio tapes from face-to-face interviews. Focus groups and personal interview information would be transcribed and a framework approach analysis [##REF##10661708##44##,##UREF##10##45##], was planned to be performed based on the five-step approach: familiarization, identifying a thematic framework, indexing, mapping and interpretation.</p>" ]
[]
[ "<title>Discussion</title>", "<p>Many countries nowadays are facing financial constraints for health care expenditures. The appropriate design of cost-effective, country-specific QI interventions based on translational research [##REF##17084943##46##] is one of the cornerstones of contemporary health care policy. In the current report we have presented the design of a multifaceted, country-specific and tailored to local practices pilot QI intervention in primary health care centers of Cyprus, grounded on a number of theoretical frameworks including the Unified Theory of Acceptance and Use of Technology, the Chronic Care Model, the Theory of Planned Behaviour and the Theory of Reasoned Action.</p>", "<p>A broad array of key initiatives in improving the quality of primary care services such as national systems for inspection and monitoring of performance and pay-for-performance incentive programs have been extensively described in the recent medical literature in countries with a long tradition in quality improvement efforts [##REF##17625132##47##]. Although such paradigms can be extremely useful, countries without past experience in QI interventions, as well as limited resources, may benefit significantly from examples originating from countries with similar experiences and comparable health care system parameters.</p>", "<p>A number of limitations of our study design are worth noting. First, due to limited resources, the intervention was planned to be implemented in a small number of primary care centers with few physicians and nurses serving as evaluators, thus limiting our study's impact. However our findings could be indicative of the directionality of changes and possible improvements that were to be observed. Moreover, our study was not a randomized double-blind community-based controlled trial, since the PCHCs were not randomly selected. In addition, there was a broad diversity among the centers with respect to the population they served. Nevertheless, our before and after specific study design with concurrent controls provides sufficient validity. However, the power calculations of our pilot study are limited by the number of primary care centers participating in the intervention and control groups. Typically group randomized trials like ours, should include about 5 – 15 practices per group taking into account the intra-class correlation due to practice membership. Finally, although our multifaceted intervention may appears to be expensive, ample evidence suggests that the use of EMR and the improved care of patients with chronic conditions have the highest potential for cost savings. [##REF##18385503##48##] Furthermore, examples in the medical literature suggest that the use of physician facilitators have favorable cost-benefit ratios when targeted at costly system issues. [##REF##3083161##49##,##REF##18021507##50##]</p>", "<p>Our study is one of the first attempts to improve the quality of the primary care system of Cyprus based on contemporary methodological approaches and adoption of novel computerized technology. Our investigation was designed to explore issues of feasibility, acceptability from patients and health care professionals, and effectiveness of a pilot quality improvement intervention. In addition, our study was expected to evaluate the potential effect of theoretical frameworks on the implementation of multifaceted intervention programs in the primary care system of Cyprus and to what extend such theoretical frameworks can offer a safe base for the described objectives. Our study design could also provide the necessary theoretical model and applied methodology as well as the practical tools for future efforts towards universal EMR implementation and the management of chronic diseases based on standard guidelines in the primary care setting of Cyprus. However, the desired quality improvement will need to be implemented and evaluated beyond a pilot setting in order to provide firm evidence with respect to its effectiveness.</p>" ]
[ "<title>Conclusion</title>", "<p>The aim of our correspondence was mainly to illustrate the methodological approach in designing a multifaceted quality improvement intervention based on translational research in a country where applied research is limited. We believe that our design may lead to the implementation of a successful quality improvement intervention using relatively limited resources in an environment lacking previous QI attempts. The effectiveness evaluation of the intervention is expected to provide a strong basis for future efforts to craft a standardized approach for continuous quality improvement interventions in the primary care setting of Cyprus.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Quality Improvement Interventions require significant financial investments, and therefore demand careful consideration in their design in order to maximize potential benefits. In this correspondence we present the methodological approach of a multifaceted quality improvement intervention aiming to improve quality of care in primary care, properly tailored for a country such as Cyprus where general practice is currently seeking recognition.</p>", "<title>Methods</title>", "<p>Our methodological approach was focused on the design of an open label, community-based intervention controlled trial using all patients from two urban and two rural public primary care centers diagnosed with hypertension and type II diabetes mellitus. The design of our intervention was grounded on a strong theoretical framework that included the Unified Theory of Acceptance and Use of Technology, and the Chronic Care Model, which synthesize evidence-based system changes in accordance with the Theory of Planned Behavior and the Theory of Reasoned Action. The primary outcome measure was improvement in the quality of care for two chronic diseases evaluated through specific clinical indicators, as well as the patient satisfaction assessed by the EUROPEP questionnaire and additional personal interviews.</p>", "<title>Results</title>", "<p>We designed a multifaceted quality improvement intervention model, supported by a varying degree of scientific evidence, tailored to local needs and specific country characteristics. Overall, the main components of the intervention were the development and adoption of an electronic medical record and the introduction of clinical guidelines for the management of the targeted chronic diseases facilitated by the necessary model of organizational changes.</p>", "<title>Conclusion</title>", "<p>Health planners and policy makers need to be aware of the potential use of certain theoretical models and applied methodology as well as inexpensive tools that may be suitably tailored to the local needs, in order to effectively design quality improvement interventions in primary care settings.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>CL conceived of the idea for the project, and supervised the project. CL, AP, GAS, TZ and HES developed the methodology. GAS supervised the project implementation and developed the data collection tools. All authors reviewed the methodological approach. GAS wrote the first draft of the manuscript, while all authors contributed to the final version of the manuscript. All authors read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1472-6963/8/181/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>The authors would like to thank the Officers at the department of Medical Services of the Ministry of Health in Cyprus and the department of Clinic of Social and Family Medicine for their support of the current project. This work was supported by a grant from the Ministry of Health in Cyprus.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Schematic Representation of the Evaluation Framework.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"1472-6963-8-181-1\"/>" ]
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[{"surname": ["Etzwiller"], "given-names": ["DD"], "collab": ["Chronic care"], "article-title": ["A need in search of system"], "source": ["Diab Educ"], "year": ["1997"], "volume": ["23"], "fpage": ["569"], "lpage": ["573"], "pub-id": ["10.1177/014572179702300509"]}, {"surname": ["Rose", "Miller", "Nemeth", "Jenkins", "Nietert", "Wessell", "Ornstein"], "given-names": ["HL", "PM", "LS", "RG", "PJ", "AM", "S"], "article-title": ["Alcohol screening and brief counseling in a primary care hypertensive population: a quality improvement intervention"], "source": ["Addiction"], "comment": ["2008 Apr 16"]}, {"surname": ["Althabe", "Buekens", "Bergel", "Beliz\u00e1n", "Campbell", "Moss", "Hartwell", "Wright"], "given-names": ["Fernando", "Pierre", "Eduardo", "Jos\u00e9M", "MarciK", "Nancy", "Tyler", "LindaL"], "article-title": ["A Behavioral Intervention to Improve Obstetrical Care"], "source": ["N Eng J Med"], "volume": ["358"], "fpage": ["1929"], "lpage": ["1940"], "comment": ["2008 May 1"], "pub-id": ["10.1056/NEJMsa071456"]}, {"surname": ["Miller", "Beyer"], "given-names": ["Trace Harrison", "JaniceM"], "source": ["Changing Organizational Culture. In classics of organization Theory"], "year": ["2005"], "edition": ["6"], "publisher-name": ["Belmont, CA: Wadsworth"]}, {"surname": ["Venkatesh", "Morris", "Davis", "Davis"], "given-names": ["V", "M", "G", "FD"], "article-title": ["''User acceptance of information technology: toward a unified view\""], "source": ["MIS Quarterly"], "year": ["2003"], "volume": ["27"], "fpage": ["425"], "lpage": ["478"]}, {"surname": ["Schaper", "Pervan"], "given-names": ["LouiseK", "GrahamP"], "article-title": ["An investigation on factors affecting technology acceptance and use decisions by Australian allied health therapists"], "source": ["Proceedings of the 40 th Hawaii International Conference on System Sciences"], "year": ["2007"]}, {"surname": ["Venkatesh", "Morris", "Davis", "Davis"], "given-names": ["M", "G", "FD"], "article-title": ["\"User acceptance of information technology: toward a unified view\""], "source": ["MIS Quarterly"], "year": ["2003"], "volume": ["27"], "fpage": ["425"], "lpage": ["478"]}, {"surname": ["Wagner", "Austin", "Von Korff"], "given-names": ["EH", "BT", "M"], "article-title": ["Organizing care for patients with chronic illness"], "source": ["Milbank"], "year": ["1996"], "volume": ["Q74"], "fpage": ["511"], "lpage": ["544"], "comment": ["Review"], "pub-id": ["10.2307/3350391"]}, {"surname": ["Juran"], "given-names": ["JM"], "source": ["Managerial breakthrough"], "year": ["1964"], "publisher-name": ["New York, NY: McGraw-Hill"]}, {"surname": ["Marshall", "Campbell", "Hacker", "Roland"], "given-names": ["M", "S", "J", "M"], "article-title": ["Quality indicators for general practice. A practical guide for health professionals and managers"], "source": ["The Royal Society of Medicine Press Ltd"], "year": ["2002"]}, {"surname": ["Bryman", "Burgess"], "given-names": ["A", "R"], "source": ["Analyzing qualitative data"], "year": ["1993"], "publisher-name": ["London: Routledge"], "fpage": ["173"], "lpage": ["9"]}]
{ "acronym": [], "definition": [] }
50
CC BY
no
2022-01-12 14:47:26
BMC Health Serv Res. 2008 Aug 27; 8:181
oa_package/e0/6c/PMC2529293.tar.gz
PMC2529294
18710550
[ "<title>Background</title>", "<p>Breast cancer is the most commonly diagnosed cancer, and is the second leading cause of cancer mortality in women in the U.S. [##REF##17237035##1##]. Metastasis of the tumor is the primary cause of morbidity and mortality. In late-stage breast cancer, tumor metastasis can be found in several tissues, including bone, lung, lymph node, and liver [##REF##3420442##2##]. Because metastasis is a major challenge in cancer management, a better understanding of the metastatic progression is required. Tumor progression and metastasis are both regulated by the surrounding microenvironment, i.e. the local stroma. Therefore, studies targeted towards understanding the function of normal breast stroma will facilitate the development of methods for preventing breast cancer metastasis.</p>", "<p>Normal growth, function, and homeostasis of breast epithelial cells depend on intricate interactions between the numerous stromal cells within the mammary gland. The stromal cells are composed of a diverse assortment of cell types including the vasculature, adipocytes, resident immune cells, and fibroblasts. These cells secrete multiple cellular products, such as growth factors and extracellular matrix components, which have profound effects the behavior of the breast epithelial cells. Alterations in the regular communications between these cells can lead to the progression or expansion of malignant growth.</p>", "<p>It is now well documented that stromal cells have a striking effect on the behavior of mammary epithelial cells in culture [##REF##15642169##3##, ####REF##10472777##4##, ##REF##8543388##5##, ##REF##9096664##6##, ##REF##11245428##7####11245428##7##] as well as on the formation, growth, and metastasis of epithelial-derived tumors <italic>in vivo </italic>[##REF##14996910##8##, ####REF##17322389##9##, ##REF##10728684##10##, ##REF##16082203##11####16082203##11##]. Both <italic>in vitro </italic>and <italic>in vivo </italic>studies have shown that epithelial cell contact with tumor-derived or normal fibroblasts can either promote or inhibit tumorigenic cell growth, respectively [##REF##16082203##11##,##REF##15051869##12##]. In agreement with these reports, one study using microarray analyses demonstrated that the gene expression profiles of cancer-derived fibroblasts had a distinctive gene expression pattern that differentiated them from normal breast stroma [##REF##17899370##13##]. Furthermore, breast cancer stroma differs morphologically from the stroma found in normal breast tissue. For example, in ductal carcinomas <italic>in situ </italic>(DCIS), and most invasive breast carcinomas, the stroma exhibits enhanced accumulation of fibroblasts and a modified collagenized extracellular matrix compared to its normal counterpart [##REF##15642169##3##,##REF##10537355##14##, ####REF##8635127##15##, ##REF##10628798##16##, ##REF##11585740##17##, ##REF##11962761##18##, ##REF##11962762##19####11962762##19##]. Understanding the mechanisms of the interactions between cancerous or normal epithelial cells and the stroma might lead to novel methods for cancer therapies that target the function of the resident stromal cells.</p>", "<p>Most models of breast cancer development are studied using mouse <italic>in vivo </italic>models. However, the stroma within the human mammary gland is fundamentally different from that in the mouse [##REF##5539119##20##]. These differences make it difficult to ascertain the tumor/stromal interactions that would occur in the human breast when epithelial cells are implanted into the mouse mammary fat pad. Compared to the human breast, the mouse mammary gland contains large depots of adipose laced with small amounts of interspersed connective tissue. The functional lobular units of the mouse gland are embedded within the fat pad, and have a considerable amount of space between the minimally branched ducts. In contrast, the functional lobular units of the human mammary gland are surrounded by loose intralobular connective tissue, consisting primarily of fibroblasts. This intralobular stroma is subsequently surrounded by a more compact interlobular stroma, which detaches the lobules and intralobular stroma from any substantial direct contact with the adipose tissue [##REF##15369447##21##]. Stemming from the observations that these stroma subtypes differ in their physical location in relation to the functional epithelial lobules, and that epithelial/stromal interactions can promote or inhibit tumorigenesis, we investigated the differences between the two distinct stromas.</p>" ]
[ "<title>Methods</title>", "<title>Sample Collection</title>", "<p>This study was performed in accordance with the guidelines of the National Cancer Institute Review Board, protocol 02-C-0144. All patients provided written informed consent. Fresh human mammary tissue was collected from four (two Caucasian, one African-American, and one Hispanic) female, pre-menopausal, reduction mammoplasty patients, ages ranging from 18 to 40 years old. The tissue was embedded in Tissue-Tek O.C.T. embedding medium (Sankura Finetek Inc., Torrance CA) and frozen on dry ice immediately after surgery. Eight – 10 micron sections of tissue were cut using a Leica 2800 Frigocut-E cryostat (Bannockburn, IL). Every tenth section was subjected to hematoxylin and eosin staining. For each patient sample, sections with distinctive intralobular and interlobular regions were selected for laser capture and microarray analysis.</p>", "<title>Laser capture microdissection and microarrays</title>", "<p>Laser capture microdissection (LCM) and microarrays were performed by Cogenics, Inc. (Morrisville, NC). Briefly, selected intralobular and interlobular stroma sections of frozen tissue were subjected to an AutoPix™ automated LCM system from Arcturus, using static image settings. RNA was isolated from each specimen, pooled, and then evaluated by spectrophotometry and by using an Agilent Bioanalyzer before proceeding to sample amplification. For each sample, 50 ng of total RNA was amplified using Affymetrix Two-Cycle Target Labeling kit (Santa Clara, CA). Ten micrograms of biotinylated cRNA spiked with bioB, bioC, bioD, and cre as a control was hybridized to the Affymetrix Human Genome U133 GeneChip<sup>® </sup>for 16 h at 45°C. Following hybridization, arrays were washed and stained with Affymetrix GeneChip Fluidics Station. Stained arrays were scanned with an Affymetrix GeneChip Scanner 3000. Quality check and preliminary data analysis were carried out using Affymetrix GeneChip Operating Software and Quality Reporter.</p>", "<title>Microarray analysis</title>", "<p>Microarray data were analyzed using the Affy package available at the Bioconductor website <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.bioconductor.org\"/>. The raw data were first background-corrected by the Robust Multichip Average (RMA) method [##REF##12925520##22##] and then normalized by an invariant set method. Unsupervised hierarchical clustering analysis was performed on 1,115 most variable genes. The difference of gene expression between the inter- and intra-stromal samples was analyzed by the Limma package available at the Bioconductor website. P-values obtained from the multiple comparison tests were corrected by false discovery rates. The microarray data has been deposited in the public repository, Gene Expression Omnibus, accession number GSE12306.</p>", "<title>Immunohistochemistry</title>", "<p>All reagents were obtained from Sigma (St. Louis, MO) unless otherwise indicated. Ten-micron-thick sections of frozen tissue were fixed in 1:1 methanol:acetone for 10 min and washed in 1× phosphate buffered saline, pH 7.4 (PBS). Endogenous peroxidase activity was blocked by a 10 min incubation in 3% hydrogen peroxide followed by a 10 min wash in 1× PBS. Immunostaining was carried out using the Vectastain ABC kit (Vector, Burlingame, CA) according to the manufacturer's instruction. Color was developed with diaminobenzidine peroxidase substrate kit (Vector), and sections were counterstained with hematoxylin. Antibodies were obtained from the following sources: Met, Cell Signaling Technologies (Boston, MA); SOS2, Santa Cruz Biotechnology (Santa Cruz, CA); Tenascin-C, Invitrogen (Gaithersburg, MD); CD44, BD Biosciences (San Jose, CA); CD13, Novocastra (Visions Biosystems Bannockburn IL); CD26, Abcam Inc. (Cambridge, MA). Collagen staining with Sirius red was performed as previously described [##REF##14507022##23##]. A positive and negative control was included in each experiment to validate the specificity of each antibody. A breast tissue sample that had been previously determined to express high levels of the protein of interest was used as a positive control. A serial section of each sample that received all staining steps, with the exception of the primary antibody, was used as a negative control.</p>", "<title>Reverse Transcription and PCR</title>", "<p>Reverse transcription (RT) reactions were performed with 0.5 μg of total RNA isolated from LCM using Moloney murine leukemia virus reverse transcriptase (Invitrogen) primed with oligo-dT and random hexamers in a final volume of 25 μL. PCR was performed on 2.5 μL RT product using PCR Master Mix (Roche, Indianapolis, IN) with 0.2–0.4 μM of each primer. Primer sequences can be found in Table ##TAB##0##1##. Conditions for each PCR reaction were as follows: 94°C for 3 min for one cycle, followed by 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, with a final extension at 72°C for 10 min. For a given experiment, PCR was performed using a predetermined number of cycles that spanned the linear range for the samples tested (20–30 cycles). RT-PCR products were resolved by agarose gel electrophoresis, visualized with ethidium bromide, quantified using NIH Image, and normalized to the respective level of GAPDH mRNA. Semi-quantitative RT-PCR analyses were conducted on a minimum of three patient samples. Appropriate negative controls were included for each RT-PCR.</p>" ]
[ "<title>Results and discussion</title>", "<p>Microarray analysis was employed to identify genes that were differentially regulated between the intralobular and interlobular stromal subtypes. Normal human mammary tissue was obtained from healthy, pre-menopausal, reduction mammoplasty patients with no incidence of neoplasia. For each sample collected, tissue sections with distinctive intralobular and interlobular regions were selected for laser capture and microarray analysis (Fig. ##FIG##0##1##). RNA was extracted from the samples, checked for quality, and then hybridized with the Affymetrix Human Genome133A GeneChips, containing over 22,000 oligonucleotide probes. Surprisingly, no significant difference in gene expression was found between the two stroma subtypes. The microarray data demonstrated a wide range of expression values and a 45-degree straight line in each pair of samples, indicating the microarray assay and the normalization procedure were valid. Despite the small sample number, the scatter plots showed limited spread of the off-diagonal lines, suggesting that any differential expression between samples is subtle, and not significant (Fig. ##FIG##1##2A##). A heatmap with dendrograms was also generated from the data (Fig. ##FIG##1##2B##). At first glance, genes from sample numbers 13L, 27L, and 29R appeared to separate as different clusters with respect to interlobular and intralobular samples. However, further in-depth analysis using hierarchical clustering of the samples, based on the 1,115 most variable genes, did not reveal a distinct expression pattern between the intralobular and interlobular stromal tissues, further indicating there was no significant difference in terms of gene expression at the transcriptional level. Six of the genes with the largest difference in expression levels between intralobular in interlobular stroma are listed in Table ##TAB##1##2##, along with the fold-change and p-value. The lowest p-value was found to be 0.4726, which is far from statistically significant. In order to validate the microarray data, RT-PCR was performed on three of the top six genes listed in Table ##TAB##1##2##. The RT-PCR products from three patient samples as well as the quantitation of the products are shown in Figure (##FIG##2##3A##&amp;##FIG##2##3B##). The expression levels of c-Met, SOS2, and CD44 reflect the findings of the microarray data; there was no significant difference between the intralobular and interlobular stroma.</p>", "<title>Examples of protein levels reflecting the gene expression levels between intralobular and interlobular stroma</title>", "<p>While the microarray and supportive RT-PCR analysis revealed no significant difference between gene expression levels, previous reports have documented a distinctive immunohistochemical difference between intralobular and interlobular stroma [##REF##14507022##23##, ####REF##1794609##24##, ##REF##9477307##25##, ##REF##7876358##26##, ##REF##7913070##27####7913070##27##]. Therefore, we investigated whether we could observe a similar phenomenon using the same patients tissue samples utilized in the microarray analysis. We first investigated the protein expression of c-Met, the gene with the smallest p-value (0.4726). Fixed preparations of human mammary tissue from the four patients used in the microarray analysis, as well as additional samples, were immunoassayed using a specific antibody for c-Met. As shown in Figure ##FIG##2##3C##, all of the stroma uniformly stained positive for c-Met protein expression, with no detectable difference between the two stroma subtypes. The c-Met gene encodes the tyrosine kinase receptor for the hepatocyte growth factor/scatter factor (HGF/SF). c-Met/HGF signaling is required for mammalian embryogenesis and is important in cell migration, morphogenic differentiation, cell growth and angiogenesis. In normal breast tissue, c-Met was reported to be associated with ductal cells, and involved in ductal branching [##REF##7768990##28##]. Additionally, the overexpression of c-Met has been shown to contribute to the development and progression of different human malignancies including lung, prostate, colorectal, gastric, and breast cancer [##REF##14685170##29##]. Recently, it was reported that c-Met protein is overexpressed in inflammatory breast cancer compared to non-inflammatory breast cancer, and that an imbalance of c-Met protein expression between tumor and surrounding normal tissue is associated with an aggressive DCIS phenotype [##REF##17593080##30##,##REF##17242702##31##]. In the present study, c-Met protein was easily detectable and uniformly distributed throughout the normal breast.</p>", "<p>We next investigated the protein expression pattern of SOS2 (Son of Sevenless), the gene with the second lowest p-value (0.7845). SOS2 is a Ras-specific nucleotide-exchange factor that is involved in the receptor tyrosine kinase-Ras-ERK cascade [##REF##12177507##32##]. This cascade has been implicated in the control of diverse biological processes including cell proliferation, differentiation, and survival. All tissues immunoassayed for SOS2 showed sparse, weak, staining in the stroma with no detectable difference in the staining pattern between the stroma subtypes (Fig. ##FIG##2##3C##). The only significant positive staining was found in the luminal epithelium of each sample.</p>", "<p>CD44, a protein which has recently gained much attention in breast cancer [##REF##11026027##33##, ####REF##10409443##34##, ##REF##11547898##35##, ##REF##17142857##36##, ##REF##17873349##37####17873349##37##], is a ubiquitously expressed, multifunctional cell surface adhesion molecule involved in cell-cell and cell-matrix interactions, cell trafficking, and transmission of numerous growth signals [##REF##9111868##38##]. The primary ligand for CD44 is hyaluronic acid, which is an important component of the extracellular matrix. However, other CD44 ligands include collagen, fibronectin, laminin, and chondroitin sulfate. Stromal hyaluronic acid levels are a strong, independent, negative predictor for patient survival in breast cancer [##REF##10666382##39##,##REF##16652145##40##]. Additionally, many cancer cells overexpress CD44 or express CD44 variants [##REF##12786890##41##]. Mouse models of breast cancer tumorigenicity have suggested that CD44 expression is a cell surface marker that differentiates tumor initiating from non-tumorigenic breast cancer cells in immuno-compromised mice [##REF##12629218##42##]. Furthermore, injection of reagents interfering with CD44-ligand interaction, such as CD44-specific antibodies, has been shown to inhibit local tumor growth and metastatic spread in mouse models of human cancer [##REF##9111868##38##]. These findings suggest that CD44 may confer a growth advantage on some neoplastic cells and, therefore, could be used as a target for cancer therapy. In the present study, CD44 was one of the top genes with differential regulation between intralobular and interlobular stroma, although the p-value was 0.7845 and not significant. Immunohistochemical analysis reflected the microarray and RT-PCR results. There was uniform staining of the stroma, and the highest immuno-reactivity for CD44 was found in the epithelial cells (Fig. ##FIG##2##3C##).</p>", "<title>Examples of proteins differentially regulated between the intralobular and interlobular stroma, with no significant change in gene expression</title>", "<p>As previously stated, several reports illustrate a difference in protein expression between the intralobular and interlobular stroma. Atherton <italic>et al</italic>. (1998) reported that immuno-localization of type XIV collagen/undulin in the human mammary gland revealed greater deposition in the interlobular stroma than in the intralobular stroma [##REF##9477307##25##]. Fibroblasts isolated from the interlobular stroma synthesized 3- to 5-fold more type XIV collagen/undulin than intralobular fibroblasts, but synthesized type I and type IV collagens in similar amounts. The authors suggest this protein is a way to separate the two types of distinct stroma for analysis. Collagen fibers have also been reported to be more abundant and densely packed throughout interlobular stroma compared to intralobular stroma in the bovine mammary gland [##REF##14507022##23##]. Thus, we examined the collagen fiber deposition in the tissue samples from the patients used in the microarray data. Using Sirius Red, a pan stain for collagen fibers, there was a clear visible difference in the deposition of collagen fibers between the two types of stroma (Fig. ##FIG##3##4A##). In our microarray data, the fold change and p-value for type XIV collagen/undulin were -0.199 and 0.785, respectively. Undulin had the best p-value compared to all other types of collagen, but again, no values for any of the collagen genes were significant. RT-PCR analysis of the patients samples used in the microarray revealed an inconsistent expression of undulin between samples, resulting in no significant change between intralobular and interlobular expression (Fig. ##FIG##3##4B##&amp;##FIG##3##4C##).</p>", "<p>Our current data illustrated that the interlobular stroma has increased stromal collagen compared to the intralobular stromal. Mammographically dense breast tissue is one of the greatest risk factors for developing breast carcinoma, and regions of high breast density are associated with increased stromal collagen [##REF##18226174##43##, ####REF##11389815##44##, ##REF##17229950##45####17229950##45##]. A recent report investigating the effects of collagen density on mammary tumor formation and progression utilized a bi-transgenic tumor model with increased stromal collagen in mouse mammary tissue [##REF##18442412##46##]. This increased stromal collagen significantly increased tumor formation and resulted in a significantly more invasive phenotype, with increased lung metastasis. This study provided the first data causally linking increased stromal collagen to mammary tumor formation and metastasis, and demonstrated that fundamental differences arise and persist in epithelial tumor cells that progressed within collagen-dense microenvironments. It could be hypothesized that a change in the protein expression of the intralobular stroma to mimic the collagen expression of the interlobular stroma would enhance breast cancer progression. Studying the mechanisms, which lead to the differential levels in collagen deposition between these two stromal subtypes, could facilitate in understanding the physiology of breast density and the resultant influences on mammary epithelial cell function.</p>", "<p>Tenascin-C has also been reported to be expressed in the intralobular stroma as well as in the basement and sub-basement membrane zone of normal breast tissue [##REF##1692795##47##]. Tenascin-C is a member of the tenascin family of modular and multifunctional extracellular matrix glycoproteins. These molecules are expressed in the adult during normal processes such as wound healing and tissue involution, and in pathological states including vascular disease, tumorigenesis, and metastasis [##REF##10842355##48##]. In the present study, there was substantially more immuno-staining in the intralobular stroma compared to the interlobular stroma (Fig. ##FIG##3##4D##). Both the sub-basement membrane as well as the stroma had higher immuno-reactivity compared to the interlobular stroma. The microarray data reported a -0.119 fold change and a p-value of 0.852 for tenascin-C. RT-PCR was also performed on the same patient samples, and similar to the microarray data, showed no significant difference in tenascin-C expression (Fig. ##FIG##3##4B##&amp;##FIG##3##4C##). Tenascin-C has been reported to be overexpressed in the extracellular matrix of the stroma in many solid tumors, including breast tumors [##REF##2428505##49##,##REF##1383484##50##]. Additionally, expression of tenascin-C in DCIS has been demonstrated to predict invasion, and high expression has been related to poor prognosis, as well as local and distant reoccurrence in breast cancer patients. [##REF##9816077##51##, ####REF##9893653##52##, ##REF##9836485##53##, ##REF##12036947##54##, ##REF##10945566##55####10945566##55##]. Interestingly, both the distribution and quantity of tenascin-C changes in the breast during the menstrual cycle [##REF##1692795##47##], which may explain the variations in tenascin staining in normal tissue, as well as hormone-dependent and independent tumors. Although intralobular stroma was reported to undergo cyclic changes during the menstrual cycle [##REF##7258295##56##], there was no measurable difference in protein levels of the estrogen and progesterone receptor status within the two types of stroma (<italic>data not shown</italic>). Similar to the results seen with the collagen deposition, this is another example of gene expression levels that do not reflect the abundance of the protein between the two types of stroma.</p>", "<p>Atherton <italic>et al</italic>. (1994) have reported a unique regulation of ectoenzymes between the intralobular and interlobular stroma [##REF##7876358##26##]. In normal breast tissue, aminopeptidase N (CD13) was reported to be uniformly expressed in all stroma, while dipeptidyl peptidase IV (CD26) was absent in the intralobular stroma, but present in the interlobular stroma. The two subpopulations of stromal cells were isolated by enzymatic digestion and cell culture, and then analyzed via flow cytometry and immunohistochemistry. Interestingly, after several passages on tissue plastic culture dishes, the intralobular stroma lost their expression of CD26 and became phenotypically similar to the interlobular fibroblasts. This suggests that growth on tissue culture plastic causes a reversion of the stroma subpopulations to one phenotype. We subjected the patient samples from the microarray data to both immunohistochemistry and RT-PCR for both CD13 and CD26. In contrast to Atherton's report, immunohistochemical analysis of CD13 illustrated a predominately intralobular stroma staining in all patient samples tested (Fig. ##FIG##3##4D##). However, similar to the microarray data, RT-PCR analysis showed inconsistent expression levels between patients (Fig ##FIG##3##4B##&amp;##FIG##3##4C##). Of the four patients used in the microarray analysis, two samples had higher CD13 expression in the intralobular stroma, while the other two had higher expression in the interlobular stroma. A larger sample size is necessary to determine whether the RT-PCR results were significant. Additionally, the intensity of the immunoreactivity may be attributed to the density of the stromal cells between the stroma subtypes, and the overall stromal density of each patient may influence the immunohistochemical analysis and a larger sample size is required for absolute conclusion.</p>", "<p>As with CD13, CD26 demonstrated inconsistent staining between samples, without specific staining to the intralobular or interlobular stroma (Fig. ##FIG##3##4D##). In some patient samples, CD26 demonstrated a slightly greater deposition in interlobular stroma than intralobular stroma, while in other samples CD26 was ubiquitously expressed throughout all stroma. The RT-PCR results reflected the inconsistency of CD26 protein expression, and similar to the microarray data, quantitation of the samples resulted in no significant difference in expression (Fig. ##FIG##3##4B##&amp;##FIG##3##4C##).</p>" ]
[ "<title>Results and discussion</title>", "<p>Microarray analysis was employed to identify genes that were differentially regulated between the intralobular and interlobular stromal subtypes. Normal human mammary tissue was obtained from healthy, pre-menopausal, reduction mammoplasty patients with no incidence of neoplasia. For each sample collected, tissue sections with distinctive intralobular and interlobular regions were selected for laser capture and microarray analysis (Fig. ##FIG##0##1##). RNA was extracted from the samples, checked for quality, and then hybridized with the Affymetrix Human Genome133A GeneChips, containing over 22,000 oligonucleotide probes. Surprisingly, no significant difference in gene expression was found between the two stroma subtypes. The microarray data demonstrated a wide range of expression values and a 45-degree straight line in each pair of samples, indicating the microarray assay and the normalization procedure were valid. Despite the small sample number, the scatter plots showed limited spread of the off-diagonal lines, suggesting that any differential expression between samples is subtle, and not significant (Fig. ##FIG##1##2A##). A heatmap with dendrograms was also generated from the data (Fig. ##FIG##1##2B##). At first glance, genes from sample numbers 13L, 27L, and 29R appeared to separate as different clusters with respect to interlobular and intralobular samples. However, further in-depth analysis using hierarchical clustering of the samples, based on the 1,115 most variable genes, did not reveal a distinct expression pattern between the intralobular and interlobular stromal tissues, further indicating there was no significant difference in terms of gene expression at the transcriptional level. Six of the genes with the largest difference in expression levels between intralobular in interlobular stroma are listed in Table ##TAB##1##2##, along with the fold-change and p-value. The lowest p-value was found to be 0.4726, which is far from statistically significant. In order to validate the microarray data, RT-PCR was performed on three of the top six genes listed in Table ##TAB##1##2##. The RT-PCR products from three patient samples as well as the quantitation of the products are shown in Figure (##FIG##2##3A##&amp;##FIG##2##3B##). The expression levels of c-Met, SOS2, and CD44 reflect the findings of the microarray data; there was no significant difference between the intralobular and interlobular stroma.</p>", "<title>Examples of protein levels reflecting the gene expression levels between intralobular and interlobular stroma</title>", "<p>While the microarray and supportive RT-PCR analysis revealed no significant difference between gene expression levels, previous reports have documented a distinctive immunohistochemical difference between intralobular and interlobular stroma [##REF##14507022##23##, ####REF##1794609##24##, ##REF##9477307##25##, ##REF##7876358##26##, ##REF##7913070##27####7913070##27##]. Therefore, we investigated whether we could observe a similar phenomenon using the same patients tissue samples utilized in the microarray analysis. We first investigated the protein expression of c-Met, the gene with the smallest p-value (0.4726). Fixed preparations of human mammary tissue from the four patients used in the microarray analysis, as well as additional samples, were immunoassayed using a specific antibody for c-Met. As shown in Figure ##FIG##2##3C##, all of the stroma uniformly stained positive for c-Met protein expression, with no detectable difference between the two stroma subtypes. The c-Met gene encodes the tyrosine kinase receptor for the hepatocyte growth factor/scatter factor (HGF/SF). c-Met/HGF signaling is required for mammalian embryogenesis and is important in cell migration, morphogenic differentiation, cell growth and angiogenesis. In normal breast tissue, c-Met was reported to be associated with ductal cells, and involved in ductal branching [##REF##7768990##28##]. Additionally, the overexpression of c-Met has been shown to contribute to the development and progression of different human malignancies including lung, prostate, colorectal, gastric, and breast cancer [##REF##14685170##29##]. Recently, it was reported that c-Met protein is overexpressed in inflammatory breast cancer compared to non-inflammatory breast cancer, and that an imbalance of c-Met protein expression between tumor and surrounding normal tissue is associated with an aggressive DCIS phenotype [##REF##17593080##30##,##REF##17242702##31##]. In the present study, c-Met protein was easily detectable and uniformly distributed throughout the normal breast.</p>", "<p>We next investigated the protein expression pattern of SOS2 (Son of Sevenless), the gene with the second lowest p-value (0.7845). SOS2 is a Ras-specific nucleotide-exchange factor that is involved in the receptor tyrosine kinase-Ras-ERK cascade [##REF##12177507##32##]. This cascade has been implicated in the control of diverse biological processes including cell proliferation, differentiation, and survival. All tissues immunoassayed for SOS2 showed sparse, weak, staining in the stroma with no detectable difference in the staining pattern between the stroma subtypes (Fig. ##FIG##2##3C##). The only significant positive staining was found in the luminal epithelium of each sample.</p>", "<p>CD44, a protein which has recently gained much attention in breast cancer [##REF##11026027##33##, ####REF##10409443##34##, ##REF##11547898##35##, ##REF##17142857##36##, ##REF##17873349##37####17873349##37##], is a ubiquitously expressed, multifunctional cell surface adhesion molecule involved in cell-cell and cell-matrix interactions, cell trafficking, and transmission of numerous growth signals [##REF##9111868##38##]. The primary ligand for CD44 is hyaluronic acid, which is an important component of the extracellular matrix. However, other CD44 ligands include collagen, fibronectin, laminin, and chondroitin sulfate. Stromal hyaluronic acid levels are a strong, independent, negative predictor for patient survival in breast cancer [##REF##10666382##39##,##REF##16652145##40##]. Additionally, many cancer cells overexpress CD44 or express CD44 variants [##REF##12786890##41##]. Mouse models of breast cancer tumorigenicity have suggested that CD44 expression is a cell surface marker that differentiates tumor initiating from non-tumorigenic breast cancer cells in immuno-compromised mice [##REF##12629218##42##]. Furthermore, injection of reagents interfering with CD44-ligand interaction, such as CD44-specific antibodies, has been shown to inhibit local tumor growth and metastatic spread in mouse models of human cancer [##REF##9111868##38##]. These findings suggest that CD44 may confer a growth advantage on some neoplastic cells and, therefore, could be used as a target for cancer therapy. In the present study, CD44 was one of the top genes with differential regulation between intralobular and interlobular stroma, although the p-value was 0.7845 and not significant. Immunohistochemical analysis reflected the microarray and RT-PCR results. There was uniform staining of the stroma, and the highest immuno-reactivity for CD44 was found in the epithelial cells (Fig. ##FIG##2##3C##).</p>", "<title>Examples of proteins differentially regulated between the intralobular and interlobular stroma, with no significant change in gene expression</title>", "<p>As previously stated, several reports illustrate a difference in protein expression between the intralobular and interlobular stroma. Atherton <italic>et al</italic>. (1998) reported that immuno-localization of type XIV collagen/undulin in the human mammary gland revealed greater deposition in the interlobular stroma than in the intralobular stroma [##REF##9477307##25##]. Fibroblasts isolated from the interlobular stroma synthesized 3- to 5-fold more type XIV collagen/undulin than intralobular fibroblasts, but synthesized type I and type IV collagens in similar amounts. The authors suggest this protein is a way to separate the two types of distinct stroma for analysis. Collagen fibers have also been reported to be more abundant and densely packed throughout interlobular stroma compared to intralobular stroma in the bovine mammary gland [##REF##14507022##23##]. Thus, we examined the collagen fiber deposition in the tissue samples from the patients used in the microarray data. Using Sirius Red, a pan stain for collagen fibers, there was a clear visible difference in the deposition of collagen fibers between the two types of stroma (Fig. ##FIG##3##4A##). In our microarray data, the fold change and p-value for type XIV collagen/undulin were -0.199 and 0.785, respectively. Undulin had the best p-value compared to all other types of collagen, but again, no values for any of the collagen genes were significant. RT-PCR analysis of the patients samples used in the microarray revealed an inconsistent expression of undulin between samples, resulting in no significant change between intralobular and interlobular expression (Fig. ##FIG##3##4B##&amp;##FIG##3##4C##).</p>", "<p>Our current data illustrated that the interlobular stroma has increased stromal collagen compared to the intralobular stromal. Mammographically dense breast tissue is one of the greatest risk factors for developing breast carcinoma, and regions of high breast density are associated with increased stromal collagen [##REF##18226174##43##, ####REF##11389815##44##, ##REF##17229950##45####17229950##45##]. A recent report investigating the effects of collagen density on mammary tumor formation and progression utilized a bi-transgenic tumor model with increased stromal collagen in mouse mammary tissue [##REF##18442412##46##]. This increased stromal collagen significantly increased tumor formation and resulted in a significantly more invasive phenotype, with increased lung metastasis. This study provided the first data causally linking increased stromal collagen to mammary tumor formation and metastasis, and demonstrated that fundamental differences arise and persist in epithelial tumor cells that progressed within collagen-dense microenvironments. It could be hypothesized that a change in the protein expression of the intralobular stroma to mimic the collagen expression of the interlobular stroma would enhance breast cancer progression. Studying the mechanisms, which lead to the differential levels in collagen deposition between these two stromal subtypes, could facilitate in understanding the physiology of breast density and the resultant influences on mammary epithelial cell function.</p>", "<p>Tenascin-C has also been reported to be expressed in the intralobular stroma as well as in the basement and sub-basement membrane zone of normal breast tissue [##REF##1692795##47##]. Tenascin-C is a member of the tenascin family of modular and multifunctional extracellular matrix glycoproteins. These molecules are expressed in the adult during normal processes such as wound healing and tissue involution, and in pathological states including vascular disease, tumorigenesis, and metastasis [##REF##10842355##48##]. In the present study, there was substantially more immuno-staining in the intralobular stroma compared to the interlobular stroma (Fig. ##FIG##3##4D##). Both the sub-basement membrane as well as the stroma had higher immuno-reactivity compared to the interlobular stroma. The microarray data reported a -0.119 fold change and a p-value of 0.852 for tenascin-C. RT-PCR was also performed on the same patient samples, and similar to the microarray data, showed no significant difference in tenascin-C expression (Fig. ##FIG##3##4B##&amp;##FIG##3##4C##). Tenascin-C has been reported to be overexpressed in the extracellular matrix of the stroma in many solid tumors, including breast tumors [##REF##2428505##49##,##REF##1383484##50##]. Additionally, expression of tenascin-C in DCIS has been demonstrated to predict invasion, and high expression has been related to poor prognosis, as well as local and distant reoccurrence in breast cancer patients. [##REF##9816077##51##, ####REF##9893653##52##, ##REF##9836485##53##, ##REF##12036947##54##, ##REF##10945566##55####10945566##55##]. Interestingly, both the distribution and quantity of tenascin-C changes in the breast during the menstrual cycle [##REF##1692795##47##], which may explain the variations in tenascin staining in normal tissue, as well as hormone-dependent and independent tumors. Although intralobular stroma was reported to undergo cyclic changes during the menstrual cycle [##REF##7258295##56##], there was no measurable difference in protein levels of the estrogen and progesterone receptor status within the two types of stroma (<italic>data not shown</italic>). Similar to the results seen with the collagen deposition, this is another example of gene expression levels that do not reflect the abundance of the protein between the two types of stroma.</p>", "<p>Atherton <italic>et al</italic>. (1994) have reported a unique regulation of ectoenzymes between the intralobular and interlobular stroma [##REF##7876358##26##]. In normal breast tissue, aminopeptidase N (CD13) was reported to be uniformly expressed in all stroma, while dipeptidyl peptidase IV (CD26) was absent in the intralobular stroma, but present in the interlobular stroma. The two subpopulations of stromal cells were isolated by enzymatic digestion and cell culture, and then analyzed via flow cytometry and immunohistochemistry. Interestingly, after several passages on tissue plastic culture dishes, the intralobular stroma lost their expression of CD26 and became phenotypically similar to the interlobular fibroblasts. This suggests that growth on tissue culture plastic causes a reversion of the stroma subpopulations to one phenotype. We subjected the patient samples from the microarray data to both immunohistochemistry and RT-PCR for both CD13 and CD26. In contrast to Atherton's report, immunohistochemical analysis of CD13 illustrated a predominately intralobular stroma staining in all patient samples tested (Fig. ##FIG##3##4D##). However, similar to the microarray data, RT-PCR analysis showed inconsistent expression levels between patients (Fig ##FIG##3##4B##&amp;##FIG##3##4C##). Of the four patients used in the microarray analysis, two samples had higher CD13 expression in the intralobular stroma, while the other two had higher expression in the interlobular stroma. A larger sample size is necessary to determine whether the RT-PCR results were significant. Additionally, the intensity of the immunoreactivity may be attributed to the density of the stromal cells between the stroma subtypes, and the overall stromal density of each patient may influence the immunohistochemical analysis and a larger sample size is required for absolute conclusion.</p>", "<p>As with CD13, CD26 demonstrated inconsistent staining between samples, without specific staining to the intralobular or interlobular stroma (Fig. ##FIG##3##4D##). In some patient samples, CD26 demonstrated a slightly greater deposition in interlobular stroma than intralobular stroma, while in other samples CD26 was ubiquitously expressed throughout all stroma. The RT-PCR results reflected the inconsistency of CD26 protein expression, and similar to the microarray data, quantitation of the samples resulted in no significant difference in expression (Fig. ##FIG##3##4B##&amp;##FIG##3##4C##).</p>" ]
[ "<title>Conclusion</title>", "<p>Recently it was reported that the gene expression signatures of cancer-adjacent and breast reduction-normal tissues were essentially homogeneous and not distinguishable [##REF##17054791##57##]. The stroma used in this microarray study was exclusively interlobular stroma, and specifically excluded any intralobular stroma. The authors state this was the most complete study to date of gene expression in normal breast tissue, and that normal tissue adjacent to breast carcinomas has not undergone significant gene expression changes. However, the present study highlights the importance of post-transcriptional or post-translational regulation of proteins. Since surgery is a common procedure performed on tissue with potential for tumor progression, the alterations in the adjacent stroma could have important clinical implications. This study emphasizes the importance of using techniques other than gene expression levels to investigate protein regulation within the stroma.</p>", "<p>A recent report utilizing two-dimensional gel electrophoresis supports the present study and shows that carcinoma-associated fibroblasts, tumor-adjacent fibroblasts (cells 2 cm away from the tumor margin), and normal breast fibroblasts have different proteome profiles, with many different proteins differentially expressed among these cells [##REF##18413739##58##]. Interestingly, the carcinoma-associated fibroblasts and tumor-adjacent fibroblasts expressed high levels of the cancer marker survivin and consequently exhibited high resistance to the chemotherapeutic agent cisplatin and UV light. Furthermore, the tumor-adjacent fibroblasts, although histologically normal and not in contact with the tumor cells, contained genetic changes that were distinct from the normal fibroblasts and the carcinoma-associated fibroblasts. It was hypothesized that the carcinoma-associated fibroblasts, as well as their corresponding tumor-adjacent fibroblasts, acquired tumor-like changes that are necessary for tumor growth. The authors further speculated that certain genes are up-regulated early during carcinogenesis and have a promoting role during cancer development. It would be of interest to investigate, based on the data obtained from the present study, whether the carcinoma-associated fibroblasts and tumor-adjacent fibroblasts arise from the intralobular or the interlobular stroma, and what effects tumorigenic changes in either subtype have on each other and the progression of the cancer.</p>", "<p>The failure to grow normal or premalignant human mammary epithelial cells <italic>in vivo </italic>had previously hindered any possibility of a model for human breast cancer progression using human cells. Recently, Kuperwasser <italic>et al</italic>. [##REF##15051869##12##] successfully developed a dynamic <italic>in vivo </italic>model which recapitulates human breast epithelial morphogenesis. In this model, human mammary fibroblasts are injected into the gland and allowed to grown into the gland and \"humanize\" the mouse fat pad, prior to injection of the epithelial cells. This model demonstrated that stroma promoted the normal or premalignant to malignant growth of the epithelial cells, depending on the type of fibroblasts used. It may be informative to observe the differences in the normal outgrowth or tumorigenesis of epithelial cells when either intralobular or interlobular fibroblasts are chosen to humanize the gland. Furthermore, future studies isolating the differences between these two stromal subtypes may bring further insight into the tumor/stroma environment as well as normal mammary development.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The normal growth and function of mammary epithelial cells depend on interactions with the supportive stroma. Alterations in this communication can lead to the progression or expansion of malignant growth. The human mammary gland contains two distinctive types of fibroblasts within the stroma. The epithelial cells are surrounded by loosely connected intralobular fibroblasts, which are subsequently surrounded by the more compacted interlobular fibroblasts. The different proximity of these fibroblasts to the epithelial cells suggests distinctive functions for these two subtypes. In this report, we compared the gene expression profiles between the two stromal subtypes.</p>", "<title>Methods</title>", "<p>Fresh normal breast tissue was collected from reduction mammoplasty patients and immediately placed into embedding medium and frozen on dry ice. Tissue sections were subjected to laser capture microscopy to isolate the interlobular from the intralobular fibroblasts. RNA was prepared and subjected to microarray analysis using the Affymetrix Human Genome U133 GeneChip<sup>®</sup>. Data was analyzed using the Affy and Limma packages available from Bioconductor. Findings from the microarray analysis were validated by RT-PCR and immunohistochemistry.</p>", "<title>Results</title>", "<p>No statistically significant difference was detected between the gene expression profiles of the interlobular and intralobular fibroblasts by microarray analysis and RT-PCR. However, for some of the genes tested, the protein expression patterns between the two subtypes of fibroblasts were significantly different.</p>", "<title>Conclusion</title>", "<p>This study is the first to report the gene expression profiles of the two distinct fibroblast populations within the human mammary gland. While there was no significant difference in the gene expression profiles between the groups, there was an obvious difference in the expression pattern of several proteins tested. This report also highlights the importance of studying gene regulation at both the transcriptional and post-translational level.</p>" ]
[ "<title>Abbreviations</title>", "<p>LCM: Laser capture microscopy; RT-PCR: reverse transcription polymerase chain reaction; HGF/SF: hepatocyte growth factor/scatter factor; SOS2: Son of Sevenless; CD13: aminopeptidase N; CD26: dipeptidyl peptidase IV</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>JF performed immunohistochemistry, RT-PCR, and drafted the manuscript. LL performed the analysis on the microarray data. EG participated in the design of the study, collection and distribution of patient samples, and helped edit the manuscript. DG prepared the samples for laser capture microscopy, PM and BKV designed the experiments and helped interpret the results. All authors read and approved the final manuscript.</p>" ]
[ "<title>Acknowledgements</title>", "<p>This research was supported by the Center for Cancer Research, an Intramural Research Program of the National Cancer Institute, and by Breast Cancer Research Stamp proceeds awarded through competitive peer review.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Identification of intralobular and interlobular stroma in normal human breast tissue</bold>. Hemotoxylin and eosin staining of 8–10 micron sections of normal mammary tissue. The intralobular stroma isolated for laser capture microscopy is outlined in green while the interlobular stroma is outlined in black. Scale bar = 200 μM.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Scattered plots of normalized data and unsupervised hierarchical clustering of the samples and genes</bold>. A. Raw intensity data was background-corrected and normalized as described in Materials and Methods. The normalized data from seven samples were plotted against one sample (SB13L-Intra). B. 1,115 most variable genes were used for hierarchical clustering among samples. The gene expression values were scaled by row and shown in the heat map.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>c-Met, SOS2, and CD44 expression levels in intralobular and interlobular normal human breast stroma</bold>. A. RT-PCR analysis of the indicated genes expression. B. Mean ± SD gained by densitometric examination of RT-PCR product from three independent samples. C. Tissues were subjected to immunohistochemical analysis with the specific antibody indicated (left panels) or corresponding negative controls (right panels). Scale bar = 200 μM.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Localization and expression levels of collagen fibrils, CD13, Tenascin-C, and CD26 in intralobular and interlobular normal human breast stroma</bold>. A. Tissues were stained with Sirius Red alone (top panel) or with Fast Green counterstain (bottom panel). B. RT-PCR analysis of the indicated genes expression. C. Mean ± SD gained by densitometric examination of RT-PCR product from three independent samples. D. Tissues were subjected to immunohistochemical analysis with the specific antibody indicated (left panels) or corresponding negative controls (right panels). Note large quantities of intensely stained interlobular stroma (asterisks) compared to the paler-staining intralobular stroma (arrow). Scale bar = 200 μM.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Primer sequences</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Primer</bold></td><td align=\"left\"><bold>Primer sequences (listed 5' – 3')</bold></td></tr></thead><tbody><tr><td align=\"left\">GAPDH</td><td align=\"left\">Forward CATGTGGGCCATGAGGTCCACCAC<break/>Reverse TGAAGGTCGGTGTGAACGGATTTGGC</td></tr><tr><td align=\"left\">c-Met</td><td align=\"left\">Forward ACCTGCTGAAATTGAACAGCGAGC<break/>Reverse ACACTTCGGGCACTTACAAGCCTA</td></tr><tr><td align=\"left\">SOS2</td><td align=\"left\">Forward TAGAGAAAGGCGAGCAGCCAATCA<break/>Reverse AGGGTGAGATTTGTGGTATGGCGA</td></tr><tr><td align=\"left\">CD44</td><td align=\"left\">Forward GCCTGGCGCAGATCGATTTGAATA<break/>Reverse CCCTGTGTTGTTTGCTGCACAGAT</td></tr><tr><td align=\"left\">Tenascin-C</td><td align=\"left\">Forward AGATGTCACAGACACCACTGCCTT<break/>Reverse TGTGGCTTGTTGGCTCTTTGGAAC</td></tr><tr><td align=\"left\">CD13</td><td align=\"left\">Forward TCCACACCTTTGCCTACCAGAACA<break/>Reverse TGCCTGATGTGCTGAAGAGATCGT</td></tr><tr><td align=\"left\">CD26</td><td align=\"left\">Forward TGGAGGCATTCCTACACAGCTTCA<break/>Reverse ACAGCTCCTGCCTTTGGATATGGA</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Top six genes with the highest p-value</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Gene name</bold></td><td align=\"center\"><bold>Accession number</bold></td><td align=\"center\"><bold>Log fold change</bold></td><td align=\"center\"><bold>P-value</bold></td></tr></thead><tbody><tr><td align=\"left\">C-Met</td><td align=\"center\">AA005141</td><td align=\"center\">-0.4891</td><td align=\"center\">0.4726</td></tr><tr><td align=\"left\">SOS2</td><td align=\"center\">AI276593</td><td align=\"center\">-0.4345</td><td align=\"center\">0.7845</td></tr><tr><td align=\"left\">CPT1A</td><td align=\"center\">BC000185</td><td align=\"center\">-0.4465</td><td align=\"center\">0.7845</td></tr><tr><td align=\"left\">PDLIM7</td><td align=\"center\">AW206786</td><td align=\"center\">-0.3722</td><td align=\"center\">0.7845</td></tr><tr><td align=\"left\">TSC22D2</td><td align=\"center\">AF201292</td><td align=\"center\">-0.4455</td><td align=\"center\">0.7845</td></tr><tr><td align=\"left\">CD44</td><td align=\"center\">AW851559</td><td align=\"center\">-0.4704</td><td align=\"center\">0.7845</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
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[ "<graphic xlink:href=\"1471-2121-9-46-1\"/>", "<graphic xlink:href=\"1471-2121-9-46-2\"/>", "<graphic xlink:href=\"1471-2121-9-46-3\"/>", "<graphic xlink:href=\"1471-2121-9-46-4\"/>" ]
[]
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{ "acronym": [], "definition": [] }
58
CC BY
no
2022-01-12 14:47:26
BMC Cell Biol. 2008 Aug 18; 9:46
oa_package/f1/e3/PMC2529294.tar.gz
PMC2529295
18673568
[ "<title>Background</title>", "<p>RNAi has become a widely used method for conducting gene perturbation studies [##UREF##0##1##,##UREF##1##2##]. Studies using RNAi to investigate gene function can be highly specific as well as scalable, including whole-genome screens [##REF##14527409##3##, ####REF##15042092##4##, ##REF##15517019##5##, ##REF##15517021##6##, ##REF##15782175##7##, ##REF##15791247##8##, ##REF##17429401##9##, ##REF##17560332##10####17560332##10##]. While RNAi can be robust, there are challenges inherent to any RNAi experiment [##REF##12054897##11##,##REF##15629712##12##]. These challenges arise from problems in predicting the specificity of an individual siRNA <italic>a priori</italic>, as well as directly linking the reduced target protein levels with the observed effects [##REF##16159017##13##,##REF##15475108##14##]. Despite these challenges, RNAi is the most versatile and robust method for broadly testing gene function in most eukaryotes [##REF##16607398##15##].</p>", "<p>High content screening (HCS), or automated quantitative immunofluorescence, is being used to an increasing extent in the target validation stage of drug development, as well as in basic science [##REF##16320133##16##,##UREF##2##17##]. Image analysis is used to identify, quantitate and track multiple measures of individual cells [##REF##16011909##18##, ####REF##17076895##19##, ##REF##15478183##20####15478183##20##]. Usually, these data are averaged, which is analogous to whole-well assays such as caspase activity or reporter gene expression. The advantage of HCS even in analyses at the whole-well level is that cells can be individually screened for inclusion in the well average according to parameters such as the health of the cell, stage in the cell cycle or activation state of a signaling pathway.</p>", "<p>Single cell cytometry (or single cell analysis) has been used historically to analyze complex populations of cells, such as the study of differentiating immune cells by flow cytometry [##REF##15286731##21##,##REF##16582878##22##]. Recently, the use of flow cytometry and single cell analysis has been applied to signaling pathways within cancer cell lines [##REF##15047199##23##, ####REF##15845847##24##, ##REF##15260991##25##, ##REF##15731332##26####15731332##26##]. These studies highlight two advantages to flow cytometry-based single cell analysis. First, the ability to integrate the study of more than one cell-signaling pathway into an assay allows the classification of cancer cells according to perturbation responses, rather than static pathway activation levels. This better recapitulates the complex stimuli cancer cells encounter <italic>in vivo</italic>. Furthermore, advanced solid-tumor cancers are comprised of multiple subpopulations of cells, based on their genetic fluctuations and their interactions with host cells and tissues. Single cell analysis is capable of measuring changes within each of these subpopulations [##REF##15260991##25##,##REF##15475475##27##, ####REF##17401369##28##, ##REF##15539606##29####15539606##29##]. The methods developed to analyze interrelationships between thousands of data points in each of multiple samples are advancing biological and pharmaceutical research beyond the study of single pathways, and towards the study of outcomes that arise from complex interactions between multiple pathways [##REF##15845847##24##,##REF##12522051##30##,##REF##16339439##31##]. Such approaches are gaining favor because single-pathway studies show only limited correlations across cell lines or clinical samples, whereas the integration of multiple pathways and over complex sets of stimuli, enable more accurate understandings of cell signaling by addressing direct signaling as well as cross-pathway regulation [##REF##16699502##32##].</p>", "<p>We have used HCS to characterize the effects of genetic and chemical perturbations on cells by single cell analysis. We find that the wide range of protein expression levels in unperturbed cells is a significant complication for RNAi experiments, but that this complication can be addressed directly by analyzing such experiments at the single cell level. These methods allow the study of protein function by measuring the response in distinct subpopulations of cells in culture that result from stochastic variability of a target protein in a culture of cells.</p>" ]
[ "<title>Methods</title>", "<title>Cell lines, cell culture and reagents</title>", "<p>Immortalized breast cell lines 184A1, and 184B5 were generously provided by Martha Stampfer (LBNL, Berkeley, CA). The C19 derivative of T/C-28a2 was developed and generously provided by Manas Majumdar (Wyeth Research, Cambridge, MA). MCF-7, T47D, MDA-MB-235, DLD-1, RWPE-1 were obtained from ATCC (Mannasas, VA). RWPE-1-GFP was developed by transduction of a lentivirus that encodes the GFP gene under the control of the CMV promoter. Media used for each cell line were according to instructions from the source.</p>", "<p>Antibodies against γ-phosphorylated histone H2A-x, were obtained from Upstate Biotechnologies (Lake Placid, NY); antibodies against caspase-cleaved PARP and p53 were obtained from Cell Signaling Technologies (Beverly, MA). Fluorescent probes, including DAPI, and antibodies conjugated to Alexa dyes, were obtained from Molecular Probes/Invitrogen (Carlesbad, CA). Adriamycin, 16% paraformaldehyde, and Tween-20 were obtained from Sigma, Inc. (St. Louis, MO). siRNAs targeting p53 were obtained from Ambion, Inc (Austin, TX). Custom synthesized and unmodified siRNAs targeting GFP were obtained from Qiagen (Valencia, CA).</p>", "<title>siRNA transfections</title>", "<p>siRNAs were transfected as complexes with cationic liposomes from one of several manufacturers. For each experiment 3–5 commercially-available lipids were tested in a series of concentrations and siRNA:lipid ratios, according to manufacturers instructions. Transfections were four hours long and terminated by a change in media. For each cell line used in each experiment, the optimal lipid and siRNA:lipid ratios were determined using a test siRNA that targets GAPDH and GAPDH enzyme activity was measured for each condition, using the KD Alert kit from Ambion (Austin, TX). Optimal conditions were chosen as those that gave the greatest reduction in GAPDH activity when treated with the GAPDH-targeting siRNA, but minimal toxicity as identified by the NTC siRNA. Optimal conditions for each experiment are listed in Additional File ##SUPPL##1##2##.</p>", "<title>Quantitative immunofluorescence</title>", "<p>Cells labeled as described in the figures were fixed with 4% paraformaldehyde, washed, permeabilized with 0.2% Triton X-100 and stained with 300 nM DAPI, primary and secondary antibodies and washed again. Antibodies were titrated for optimal imaging, and the lowest concentration that gave a highly-specific labeling of the antigen was used. Sources, dilution levels and fluorescence conditions are listed in Additional File ##SUPPL##1##2##.</p>", "<p>Antigen intensities and localizations within cells following fixation and staining were imaged using an ArrayScan V<sup>TI </sup>(Cellomics, Pittsburgh, PA), using a 20 × 0.63 NA objective. Images were analyzed using the Target Activation and Compartmental Analysis image analysis applications from Cellomics. Cellular imaging was accomplished by first locating cell nuclei using DAPI-chromatin fluorescence and expanding the diameter of the nuclei to encompass the cytoplasmic region. Specific adjustments are required for each cell line. Cytoplasmic regions of neighboring cells were optimized in an iterative cycle of algorithm modifications and testing. Fluorescence intensity was captured and interpreted by one of several methods, typically mean fluorescence intensity per cell. Fluorescence measurements were well within the linear range of the image capture system (illumination, light filtering and detection using a cooled-CCD camera), so relative changes in protein levels could be made using relative changes in fluorescence between cells and samples. Non-specific detection is low, as shown in Additional File ##SUPPL##2##3##, and this enabled relative changes in protein levels to be determined from the fluorescence intensities.</p>", "<p>Nuclear morphology was used as an indicator of cell health. Specifically, changes in nuclear area are indicative of severe cell stresses that result in necrosis or apoptosis. The identification of cells lethally treated with etoposide using nuclear area as an indicator of imminent cell death has been used by several laboratories in both classical apoptosis studies without the use of automation and in cytological profiling approaches. The change in nuclear area following treatment with an inducer of apoptosis is shown in Additional File ##SUPPL##0##1##. SW480 cells were treated with 5 μM etoposide for 24 hr, fixed and stained as described above. Cells treated with 10 μM and 20 μM etoposide showed similar distributions of nuclear area.</p>", "<title>Quantitation and statistical analyses</title>", "<p>We have used HCS to examine protein levels within cells, and how these levels are manipulated by RNAi, at the single cell level. Data extraction and processing were performed using the statistical programming language R <ext-link ext-link-type=\"uri\" xlink:href=\"http://cran.r-project.org\"/>. Data from individual cells were extracted directly from the Cellomics' STORE database using a custom R function getCellData(), which uses a SQL query provided by Cellomics. The getCellData() function allows single cell data to be queried by well, row, column, or plate, one feature at a time, and is described in Additional File ##SUPPL##3##4##.</p>", "<p>R scripts utilizing the getCellData() function are executed on a LINUX cluster. An auxiliary text file lists the plates and wells to be extracted, as well as the annotation associated with each well. The R script reads the auxiliary file 1nd replicates and merges the annotation with the single cell data as it is extracted from the database. Averaging, normalizations, and transformations are performed in R prior to export as a flat text file. Data is visualized either directly in R or imported into Spotfire for interactive analysis.</p>" ]
[ "<title>Results</title>", "<title>Analysis of RNAi-mediated knockdown of GFP at the whole-well and single cell levels</title>", "<p>The reduction of GFP levels in cells by the transfection of siRNAs targeting the GFP mRNA sequence is a common and robust system for the study of RNAi biology and mechanism [##REF##10903441##33##]. Its intrinsic robustness notwithstanding, a high degree of variability is frequently observed in experiments modulating GFP expression. We have used this system to understand the extent of variability on experimental results by analyzing the knockdown of GFP levels at the whole well and single cell level. A prostate epithelial cell line (RWPE-1) that constitutively expressed GFP was treated with an siRNA that targets GFP. Despite carefully optimizing transfection efficiency, an appreciable level of heterogeneity was evident in the cells transfected with the GFP-targeting siRNA, the samples treated with an non-targeting control siRNA (NTC) and even in untreated samples. In all cases, a high range of GFP expression can be observed, despite clear overall differences in the samples treated with an siRNA that targets GFP. This heterogeneity is evident in the case of cells transfected with a rhodamine-labeled siRNA that targets GFP, shown in Figure ##FIG##0##1A##. As can be observed in the figure, siRNAs effectively transfected localize near the nucleus in P-bodies [##REF##15937477##34##, ####REF##15908945##35##, ##REF##17403906##36####17403906##36##]. In these studies, the siRNA is labeled with rhodamine on the sense strand, which allows uptake to be monitored, but the label itself does not interfere with silencing, at least in part because the label is on the passenger, or non-targeting, strand. Instead, it allows uptake to be quantitated on a per-cell basis. Perinuclear accumulation of the sense strand is frequently observed in cationic liposome-mediated siRNA transfections [##REF##15324818##37##], and its accumulation enables limiting the evaluation of GFP levels to only those cells that had been transfected effectively. Box plots were generated using eight independent transfections for each siRNA concentration, as shown in Figure ##FIG##0##1B##. More GFP expression remains in this experiment than in GFP knockdown experiments reported by others (which can report greater than 90% reduction in GFP levels, [##REF##12054897##11##,##REF##16793020##38##]), however these studies evaluated the effectiveness of targeting sequences in co-transfection experiments, which limits GFP expression to only those cells transfected with the RNAi reagents. Studies that examine RNAi knockdown in cell lines stably expressing GFP show knockdown levels consistent with the data in Figure ##FIG##0##1B##[##REF##15158602##39##, ####REF##16095743##40##, ##REF##17194936##41####17194936##41##]. Some of the difficulties of working with RNAi can be observed in Figure ##FIG##0##1B##, where average effects of siRNA treatment are subject to limitations in transfection reagent concentrations. In particular, in the specific conditions as set up in the experiment, the higher concentrations produce a small reduction in functional knockdown. We have observed this in specific combinations of cell type, transfection reagent and conditions. Overall, transfection reagents have limited ranges of optimal effectiveness, but the exact ranges are highly dependent on the configuration of the experiment, including source of the cell line used. As such, each experiment needs to be individually optimized, as factors that limit the effective range can be either toxicity or siRNA:lipid and complex:cell number ratios that result in suboptimal introduction of the siRNA (Lapan, P. Zhang, J., Pan, J. and Haney, S.A., manuscript in preparation). In the results shown here, the higher siRNA levels are changing the siRNA:lipid ratio, which is the most likely source of diminished efficacy at the higher siRNA levels.</p>", "<p>To investigate the extent to which transfection and other sources of variability play a role in the analysis of GFP knockdown by an siRNA, we analyzed the same data at the single cell level. The data for one well where the siRNA was transfected at 3.13 nM are presented in Figure ##FIG##0##1C##. These data are reported as single cell values that correlate the expression of GFP with the amount of siRNA taken up on a per-cell basis for the GFP siRNA, which was labeled with Rhodamine. The siRNA shows a clear ability to reduce GFP levels. It can also be readily observed that the sample treated with the NTC siRNA includes a significant numbers of cells that intrinsically express low levels of GFP. The number of cells that express low levels of GFP in the control sample affects the mean level of GFP for the pool of untreated cells, and therefore, the extent of knockdown of the treated sample. While the effectiveness of the siRNA in reducing GFP levels is scored as roughly 60% using a whole-well analysis, gating on data within GFP-positive regions (analogous to the gating of cell populations in flow cytometry), the experimental effect is 10-fold, or a 90% reduction in high GFP-expressing cells, with 457 GFP expressing cells in the NTC siRNA treated sample, and 48 in the GFP siRNA treated sample. Heterogeneity of GFP expression is observed by other investigators. In particular, it has been noted that a variety of factors contribute to the perception of stochastic effects on protein expression levels when individual cells are examined. These effects contribute to the observed variability in lines developed from clonally expanded isolates [##REF##17828790##42##], and from constitutive promoters [##REF##17615263##43##].</p>", "<title>Intrinsic target protein levels are highly variable on a per cell basis</title>", "<p>Prior to extending the results we observed using siRNA-mediated knockdown of GFP to endogenous proteins, we characterized protein abundance in cell culture populations at the single cell level. This analysis provides a context for understanding how changes in protein levels are measured at the single cell level, and how changes in protein levels affect cellular functions. Eight examples of frequently studied proteins are shown for two breast cell lines in Figure ##FIG##1##2##. A broad distribution is observed for the proteins indicated in the figure, as well as in other cell lines, including cell lines derived from human tumors (including MCF-7, MDA-MB-235, LnCaP, DU-145, and DLD-1), and epithelial cells that have been immortalized (including the prostate line RWPE-1, the chondrocyte line T/C-28a2 and the breast line 184B5). Proteins we have characterized include transcription factors (STAT1, STAT3, p53, Rb), protein kinases (CDC2, AKT1, ribosomal protein S6 kinase), and other signaling proteins (p16<sup>INK4A</sup>, PTEN). The inherent variability of these proteins is greater than what can be linked to changes resulting from changes in proliferation rates or the cell cycle (as determined by DNA content per cell). Average protein intensities for the indicated proteins are shown in Figure ##FIG##1##2A##. These data are reported in a manner similar to common methods for describing protein levels in cell lines (e.g. Western blotting and ELISA assays). The data shows that for many of the proteins, levels are higher in the cancer line T47D than they are for the immortalized breast line 184A1, particularly CDC2, STAT3 and p53. Such differences are frequently used to distinguish immortalized breast lines as being \"normal,\" although numerous studies have shown that such immortalized lines bear significant similarities to breast cancer cell lines at the phenotypic and transcriptional levels [##REF##9398663##44##,##REF##11214324##45##]. As such, increased similar levels of BIRC5, BRCA1 and c-Myc between the two lines are consistent with previous studies from this [##REF##17233903##46##] and other laboratories [##REF##9674704##47##,##REF##9817205##48##] that these proteins are significantly affected by immortalization in breast cell lines. Of relevance to the current discussion, different protein levels in the immortalized and cancer cell lines do not exist as discrete examples of cells with high and low levels of a particular protein, but as broad and overlapping ranges of protein levels on a per cell basis (Figure ##FIG##1##2B##). The increased average levels of such proteins are reflected in these distributions, creating a significant \"weighting\" of the cells with higher abundances, as shown in Figure ##FIG##1##2C## (e.g. HDAC3 and p53), while at the same time including a portion of the sample with lower levels. Such broad distributions bear an impact on drug development, as these \"side populations\" for proteins involved in the cell cycle or DNA damage response may represent cells that are particularly important to disease progression. Subpopulations of cells may be more resistant to chemotherapeutics at the low end of antigen intensity, and may have little contribution to disease progression due to excessive stress and an increased proportion of dying cells at the high end. In such cases, focusing on the disease-relevant populations will have an important benefit to drug development.</p>", "<p>We have examined the variability in intrinsic protein levels in cells, including a potential role for bias during the fixation and staining process, by dual-color staining (Figure ##FIG##1##2D–2F##). We observe that for many pairs, the extent of covariation is low, as observed for p53 and BRCA1 (r = 0.379) and Rb and HDAC3 levels (r = 0.353) in T47D cells. These data indicate that fixation and permeabilization do not play dominant roles in the distribution of antigen intensity. We do observe a higher correlation between c-MYC and BRCA1 levels (r = 0.814), in this particular case, the co-variation may reflect a biological correlation. In addition to the analytical comparison of co-staining patterns, we have examined several pairs of antigen staining to determine whether the staining patterns themselves are independent in cases where abundances are independent, by high-resolution confocal microscopy (results not shown). We find that in cases where two antigens are characterized in the same cells, the patterns are consistent for each antigen, regardless the level of staining for the second antigen. For example, the extent of nuclear staining and the degree of punctate staining observed were independent for the pairs examined (pairwise combinations of HDAC3, Rb and p53), further indicating that artifactual factors, such as uneven permeabilization or fixation, are not the cause of the wide range in antigen levels observed for these cells.</p>", "<title>RNAi-mediated knockdown of PTEN affects phosho-S6 levels</title>", "<p>The regulation of the AKT/mTor pathway represents several important and clinically relevant targets, particularly the inhibition of mTor through rapamycin-related compounds such as temsirolimus [##REF##15998902##49##,##REF##15983389##50##]. The relationship between sensitivity to temsirolimus, PTEN status and phospho-S6 levels have been studied closely for both pharmacogenomic indicators that can be used in patient selection, and in the case of phospho-S6 levels, as a phamacodynamic marker that can be used in drug dosing [##REF##14585353##51##,##REF##15254063##52##]. However, PTEN is only one contributor to activation of the AKT/mTor pathway. This is true in cell culture systems as well as in human tumor samples. We were interested in whether the analysis of RNAi knockdown of PTEN at the single cell level could elaborate on the relationship between its levels and activation of the AKT/mTor pathway. Phosphorylation of S6 is highly sensitive to the activation state of the pathway, both in cellular systems and clinically, where it is a validated biomarker of increased PI3K activity and is correlated with PTEN status. Knockdown results for PTEN are shown at the single cell level in Figure ##FIG##2##3A##. Testing a range of transfection conditions for PTEN knockdown (similar to Figure ##FIG##0##1A##) shows that this system is more robust to higher lipid concentrations that is observed for the immortalized chondrocyte line used in the GFP expression studies. The effect of PTEN depletion on pS6 phosphorylation is shown in Figure ##FIG##2##3B##, where the population of cells treated with the PTEN siRNA shows higher levels of pS6 phosphorylation. In Figure ##FIG##2##3C##, the levels of PTEN and phospho-S6 are compared for the same samples. The reduction of PTEN level and increase in phospho-S6 levels observed above can be seen as a shift in the PTEN siRNA treated sample.</p>", "<p>Figure ##FIG##2##3C## also shows the complexity of the AKT/mTor pathway when each sample is examined at the single cell level. That is to say, the effect observed in the whole well analyses, a decrease in PTEN results in an increase in phospho-S6 levels, would be expected to cause a negative correlation between these two proteins at the single cell level. Instead, a moderate positive correlation is observed, similar to the correlation observed in the unperturbed endogenous protein levels studied in Figure ##FIG##1##2##. Although often depicted as a linear pathway that leads to the activation of transcription, translation and metabolic activity, this pathway is under multiple levels of positive and negative feedback regulation of PI-3 kinase, AKT, mTor and ribosomal protein S6 kinase [##REF##15718470##53##, ####REF##15533996##54##, ##REF##9603962##55####9603962##55##], which complicates strict correlations between any two points that are separated by one or more of these additional regulatory channels (discussed below). The extensive number of interactions between the AKT/mTor pathway and other regulatory pathways means that cells in culture are in a large number of discrete states. This has been observed elsewhere by our laboratory [##UREF##3##56##], and has been noted as a complicating factor in therapies that target this pathway, including those that target Her-2<sup><sc>NEU</sc></sup>, PI3K and ERK [##REF##16939811##57##,##REF##17885672##58##]. The use of single cell analysis to track multiple signaling states presents a valuable advance in the study of current and novel theapeutics.</p>", "<title>Defining the role of STAT3 in colon carcinoma growth and survival by single cell analysis of RNAi-mediated reduction in STAT3 levels</title>", "<p>To further investigate the contribution of single cell analysis to cellular signaling studies, we turned to a less complex signaling pathway, the role of STAT3 in cancer cell proliferation and apoptosis suppression. Two examples are shown in Figure ##FIG##3##4##. In Figure ##FIG##3##4A##, knockdown of STAT3 in SW480 colon carcinoma cells are shown at the single cell level. Knockdown of STAT3 at the protein level is about 30%, based on average values for replicate wells (3 for each condition, data not shown). Although weakly separated when analyzed at the whole well level, the single cell distributions show a clear effect of treating with the STAT3 siRNA; a K-S test (the Kolmogorov-Smirnov statistic, [##REF##15539606##29##,##REF##15475475##59##]) shows a difference of 0.349 (p &lt; 2.2e-16). Such reductions are typically too small to produce robust phenotypic differences in most whole-well assay formats. There are likely to be many cases where this is correct, but Figure ##FIG##3##4A## provides a different perspective that more accurately states the situation. It is clear that distribution of STAT3 levels in SW480 cells is too wide for an average reduction of 30% to effectively demonstrate a phenotype associated with STAT3 levels at the whole well level. The overall reduction can be observed in the shift of the distributions, but residual overlap is greater than 50%. If a 30% reduction in STAT3 level does in fact have an effect on these cells, an average change of 30% of STAT3 levels in these samples may not show such an effect because of the wide range in each sample.</p>", "<p>While strong changes in average protein levels are required for experiments at the whole well level, analysis at the single cell level shows that STAT3 levels vary over a broad range under both control and STAT3 siRNA treatments. As such, comparisons between low and high STAT3 levels can be made by single cell analysis in cases where whole well differences are less dramatic. As an example, the effect of reducing STAT3 levels by RNAi can be analyzed in the experiment shown in Figure ##FIG##3##4##. Specifically, STAT3 is constitutively activated in many cancer cell lines, and reduction in STAT3 levels or activity (through RNAi or inhibitors of the JAK/STAT pathways) have been shown to result in growth arrest and apoptosis [##REF##13678425##60##, ####REF##15264241##61##, ##REF##15805244##62####15805244##62##].</p>", "<p>Proliferation inhibition is the result of the essential role of the protein in growth, but the induction of apoptosis or other forms of cell death has been ascribed to more complex interactions, such as oncogene dependency [##REF##16894390##63##] or oncogenic shock [##REF##17097564##64##]. In these models, cancer cell death results from a release in apoptosis suppression mediated by the signal transduction pathway. The data in Figure ##FIG##3##4A## can be used to determine whether reducing STAT3 levels through RNAi results in a change in cell health that is distinct from cells with equivalent levels of STAT3 as a result of expression adjustments made during growth in standard culture conditions. This was done through comparing the distribution of cells through the cell cycle in the entire dataset versus a subset of cells where STAT3 levels were low in the STAT3 siRNA-treated sample. For the cells treated with the STAT3 siRNA, 22034 cells were analyzed in the complete dataset and 5471 cells were analyzed in the low-STAT3 population, as indicated in the annotation of Figure ##FIG##3##4A##. Samples were initially compared for DNA content, as a measure of cell cycle distributions. The data for the entire STAT3 siRNA-treated sample is shown in Figure ##FIG##3##4B##, and that for the low-STAT3 subset are shown in Figure ##FIG##3##4C##.</p>", "<p>The data in Figure ##FIG##3##4B## shows that the cells are proliferating, with a significant number of cells in the G2/M phases of the cell cycle. For the low STAT3-containing cells (Figure ##FIG##3##4C##), the distribution shows a reduction in cells in these phases of the cell cycle, and a majority of the cells in G1. The cell cycle distribution is similar for the low-STAT3 cells of the NTC treated samples, but there are fewer cells and the histogram is not as smooth (not shown). Looking at subgroups with higher levels of STAT3, the proportion of cells in G2 increases somewhat.</p>", "<p>In addition to measuring the effect on the cell cycle, the effect of lowering STAT3 levels through RNAi on cell stress and cell death can be determined as well. In this case, such effects would indicate a dependence on high STAT3 levels for survival, either through oncogene addiction or oncogenic shock, two models derived from observations that reduction in oncogene activity can induce cell death. Severe cell stress and cell death are manifest in several ways, including changes to the chromatin and nuclei [##REF##17401369##28##,##REF##16945015##65##, ####REF##17588932##66##, ##REF##12844433##67####12844433##67##], which can be quantitated in image-based assays. In the present example, an effect of lowering STAT3 levels on viability would manifest itself as a change in nuclear size in the STAT3 siRNA-treated cells as compared to the NTC siRNA-treated cells. This has been noted in cytometry-based profiling studies [##REF##17401369##28##,##REF##15539606##68##,##UREF##4##69##], and is shown for SW480 colon carcinoma cells as a function of etoposide treatment in Additional File ##SUPPL##0##1## (details are provided in the Methods section). Nuclear size as a function of DNA content is shown in Figure ##FIG##3##4D## and Figure ##FIG##3##4E## for the entire dataset and for the low STAT3-expressing fraction of cells, respectively. Nuclear size increases as a function of DNA content through the cell cycle, as shown for both panels, with increasing nuclear size as cells progress into S phase and again in late G2, immediately prior to anaphase. For the data shown in Figure ##FIG##3##4##, the relationship between DNA content and nuclear size is essentially identical for the NTC siRNA-treated sample (in blue) and the STAT3 siRNA-treated sample (in red) in both analyses, indicating that cells that have had STAT3 levels reduced through RNAi treatment are not undergoing cell death to a greater extent than control cells. If STAT3 levels were critical to the suppression of apoptosis or necrosis, the nuclear diameter of the cells with low STAT3 abundance would change, relative to the control cells. They would increase in size as a general function of cell stress [##REF##15475475##27##, ####REF##17401369##28##, ##REF##15539606##29####15539606##29##], but would typically shrink and become more variegated in classical apoptosis [##REF##16945015##65##,##REF##15547975##70##]. None of these changes are observed in any of the subsets. Taken together, these results suggest that STAT3 is playing an important role in the proliferation of SW480 cells, but is not acting as an essential oncogene through the suppression of apoptosis or necrosis, as would be evident if the nuclei were significantly different.</p>", "<title>p53 dependence on adriamycin sensitivity can be observed following p53 knockdown at the whole well level, and in naturally-occurring low p53-expressing cells at the single cell level</title>", "<p>As a final example of the value of single cell analysis, we characterized the effect of p53 levels on apoptosis and activation of the DNA damage response. The DNA damaging agent adriamycin is toxic to all cells, but the toxicity is more pronounced when p53 is either not expressed or non-functional [##REF##15805289##71##]. We have looked at the dependence of p53 levels in DLD-1 colon carcinoma cells on adriamycin sensitivity at the single cell level. The sensitivity of p53-depleted cells to adriamycin is shown in Figure ##FIG##4##5A##, where the number of cells per well is reported as a function of adriamycin concentration and treatment with either an siRNA that targets p53 or a non-targeting control (NTC). Control cells shown as not treated with adriamycin were treated with DMSO at the same concentration as the cells treated with the highest concentration of adriamycin. Confidence limits for the data were 0.021 (standard error of 0.0044) for the NTC treated cells and 0.0053 (standard error of 0.0007) for the p53 siRNA treated cells. The levels of p53 for each sample are shown in Figure ##FIG##4##5B##. This data shows that transfection of an siRNA targeting p53 reduces p53 levels in DLD-1 cells prior to adriamycin treatment, as well as limiting the ability of these cells to fully recover p53 levels as a function of increasing adriamycin concentrations, despite the fact that the increase in p53 levels following DNA damage occurs through post-translational stabilization of p53 protein.</p>", "<p>For the sample treated with the NTC siRNA, the amount of p53 per cell was used to divide the cells into groups, and the fraction of cells for each group as a function of adriamycin concentration is shown in Figure ##FIG##4##5C##. Cells with high levels of p53 are compared to cells with low levels of p53 for each dose of adriamycin. The data shows that cells expressing low levels of p53 are sharply reduced as adriamycin concentrations increase, and to an extent comparable to the reduction of the total cell numbers. This suggests that cells with low levels of p53 are particularly sensitive to adriamycin treatment. Since p53 levels can rise as a direct result of DNA damage, it is also possible that cells with low levels of p53 initially are actually stabilizing p53 and levels are increasing. Therefore, we sought to resolve these two factors in p53-mediated cell survival mechanisms.</p>", "<p>We have addressed the question of whether adriamycin sensitivity is affected by p53 levels at the time of DNA damage by looking at how cells respond to treatment prior to when cell death and increased p53 levels are observed. In Figure ##FIG##4##5D##, the level of p53 in cells treated with siRNAs targeting p53 and the NTC control are shown for cells treated with increasing concentrations of adriamycin for 6 hours. At this time, we do not observe cell death (as reported by the number of cells per well), or a significant increase in average p53 levels (as shown in the figure). However, DNA damage can be observed in these cells in a dose-dependent manner, as determined by changes in DNA and nuclear morphology (data not shown). We have binned these cells by p53 level for each concentration of adriamycin treatment, and measured the levels of γ-H2A-x phosphorylation for each group, as shown in Figure ##FIG##4##5E##. Phosphorylation of this variant histone occurs in cells following DNA damage [##REF##11571274##72##] independently of changes in p53 level or modification [##REF##11571274##72##,##REF##15632067##73##]. The data shows that cells with higher levels of p53 show stronger DNA damage responses, as evidenced by increased γ-phosphorylated histone-H2A-x levels. Since these are independent responses to DNA damage, it suggests that cells with higher p53 levels may result from a stronger (or more activated) DNA damage response pathway prior to the onset of DNA damage itself, up until a point where the damage is beyond the ability of the cells to respond effectively (1.2 μM and higher concentrations). At high concentrations, significant cell death is observed for all cells (&gt;85% cell killing), and no differential is observed between untreated cells and those treated with an siRNA targeting p53. At concentrations where the dependence of p53 status on adriamycin sensitivity can be observed, single cell analysis has been able to correlate the extent of the DNA damage response induction with p53 levels in cells where p53 levels have not been altered prior to DNA damage. The same general response can be observed in separate experiments using DLD-1 cells that have not been treated with any siRNA prior to that with adriamycin (i.e. no mock or control siRNA transfection at all), shown in Figure ##FIG##4##5F##. The cells are somewhat more resistant to adriamycin in general, possibly a result of no treatment with liposomes in a transfection, but the pattern of higher p53 levels correlating with higher DNA damage response is still evident.</p>" ]
[ "<title>Discussion and conclusion</title>", "<p>We have applied the general concept of multiparametric single cell analysis to the use of RNAi, and to the relationship between protein levels and chemotherapeutic response. High Content Screening is becoming an important and general approach to biological and therapeutic studies. In addition to increasing the options available for cell-based assays in general, it is opening up new approaches to biological processes and drug development, such as cytological profiling [##REF##17401369##28##,##REF##15539606##29##,##REF##17588932##66##]. Inherent in the latter approaches is the use of single cell cytometry to analyze complex patterns in cellular responses [##REF##15475475##27##]. We have generalized the use of single cell cytometry in several experimental systems and have found that it generally improves experimental analysis, and in some cases, enables challenging questions to be addressed directly. We have used single cell cytometry to address four biological problems: identifying the relevant cells in a knockdown of GFP, correlating the knockdown of PTEN with the increase in activity of pS6 kinase, the effect of knockdown of STAT3 on proliferation and death of colon carcinoma cells and the relationship between p53 levels and responsiveness to DNA damage (both as manipulated by RNAi and as occur intrinsically through standard cell culture conditions).</p>", "<p>For RNAi screening in general, there are two applications of single cell cytometry that are potentially valuable. First is a general analysis of knockdown phenotypes by number of cells showing an altered phenotype, rather than average phenotypic change for the two samples. This approach is more in line with other distribution-based methods such as sectoring samples in flow cytometry, and can present data in more biologically-relevant way than reporting as percent-of-control (discussed below). Rigorous analysis of RNAi screening data is currently challenging [##REF##16607398##15##,##REF##17716236##74##], and would benefit from clearer definitions of what constitutes a hit [##REF##17429401##9##,##REF##17435171##75##]. The second benefit of single cell cytometry is the capacity to score cells as a function of the amount of siRNA effectively introduced in cells, as evidenced by the accumulation of the (non-functional) sense strand in P-bodies following efficient transfection. Transfection of siRNAs are frequently associated with off-target effects [##REF##16373489##76##, ####REF##12754523##77##, ##REF##16489337##78####16489337##78##], particularly at concentrations typically used for library-based screening (&gt;20 nM) [##REF##15864305##79##,##REF##14769947##80##]. Off-target effects result in many false positive hits in RNAi screens, and impose a significant burden on the post-screening confirmation phase of a project [##REF##16990807##81##]. Transfection at low concentrations (&lt; 10 nM) has been shown to reduce such artifacts, however library screening is performed with many siRNAs that have not been well-validated, particularly for off-target effects. Library screening typically involves higher concentrations because a productive screen requires that cells be reliably transfected, and some balance between the efficiency of transfection and a lack of specificity can be tolerated in the initial screen [##REF##16607398##15##], as long as an effective strategy exists for demonstrating authentic gene-phenotype connections [##REF##16990807##81##,##REF##17716236##82##]. Therefore, off-target effects resulting from high concentrations of siRNA transfections are a common and perhaps unavoidable complication of running siRNA screens. Reduced off-target effects have been associated with pooling or multiplexing siRNAs, particularly in highly complex pools such as are generated by enzymatic preparation of gene-specific siRNA pools (esiRNAs, [##REF##15616564##83##]), at least in part because the concentration of any single siRNA is low.</p>", "<p>Reverse-transfection, including the live cell array [##REF##15782175##7##,##REF##14525932##84##,##REF##16628209##85##], is frequently used in functional screens. This format spots the siRNA (or dsRNA for screens in <italic>Drosophila </italic>cells) onto a surface prior to use with cultured cells, and therefore cells are not transfected at a specific concentration, strictly speaking. Single cell analysis can be readily performed on assays following reverse transfection, since these explicitly require image-based readouts. Selecting a subpopulation with consistent siRNA uptake for each siRNA is computationally intensive, and therefore would be difficult to use directly in the primary screen endpoint, but could be used to analyze data from a primary screen that uses a high content (image-based) assay. The siRNAs need to be labeled directly or co-transfected with a labeled siRNA, in order for siRNA levels to be quantitated. However, the benefit of this is that knockdown phenotypes can be scored for cells within specific thresholds of siRNA accumulation, and these thresholds can be adjusted as the data is reviewed, rather than during image analysis. </p>", "<p>Scoring perturbations by fraction of responding cells (in the case of GFP knockdown at the single cell level) and by response magnitude as a function of target level (such as in the example of DNA damage response as a function of p53 levels) highlight important characteristics of biological samples, particularly in the development of human diseases such as cancer. Clinically important roles are played by minor populations within cell types, such as the growth of solid tumors through tumor-initiating cells (cancer stem cells) and the importance of regions within tumors that control angiogenesis and chemoresistance (the hypoxic core of cells within solid tumors). These properties can be observed in cell culture models, but this differentiation is lost in whole-well methods. Tracking effects of candidate therapeutics among rare cells or cells that have reduced proliferation rates can focus decisions on how well promising a strategy may be by limiting analysis to the cells that play the biggest role in disease progression.</p>", "<p>A similar situation occurs with pathway analyses. An assay that measures a change in a complex pathway, such as the PI3K/AKT/mTor pathway, cannot help but exclude important factors that contribute to a diverse set of outputs. This heterogeneity may be as much a part of the discordance between target inhibition and clinical response as widely cited factors, such as tumor heterogeneity as a result of genetic instability. In both cases, variability in the cells that constitute a tumor enable a significant number of cells to escape death. The difference between these two scenarios is that genetic instability suggests a somatic evolutionary process, whereas signaling heterogeneity suggests that insufficient control of the pathway results in escape from a therapeutic. In such cases, single cell analysis could improve the search for combination therapeutic strategies. mTor activity is subject to multiple levels of feedback regulation [##REF##16915295##86##,##REF##16226444##87##] and to cross-talk with other pathways, particularly the influence of amino acid and cellular energy levels on mTor activity [##REF##9603962##55##]. As such these influences would need to be measured in a multiparametric assay system, to track changes between two points in such a complex pathway. Taken together, the results presented here suggest that pathways that are quiescent (such p53 during periods of low DNA damage) or truly linear (such as activation of STAT signaling by JAK kinases) should show correlations between two points at the single cell level. This correlation could be used to validate results from RNAi experiments by providing a separate method of linking protein levels to pathway function.</p>", "<p>Studies that integrate complex signaling interactions, as opposed to linear events within single pathways, are at the root of systems biology [##REF##16339439##31##,##REF##16699502##32##], and are better able to characterize pathway states in their biological contexts. Such approaches are being shown to be of direct relevance to signaling in disease biology [##REF##15260991##25##,##REF##16564013##88##]. HCS is a strong complement to flow cytometry as a method of single cell analysis because signaling pathway responses can be integrated with cytological dynamics, and as such will extend systems biology into areas such as cancer cell motility and invasion [##REF##15475475##27##,##REF##15539606##29##,##REF##16426740##89##]. These approaches will lead to more innovative approaches to treating disease [##REF##16453012##90##], including complex molecular studies which can be integrated with genetic and epidemiological studies that show subtle but important interactions between common disease loci.</p>" ]
[ "<title>Discussion and conclusion</title>", "<p>We have applied the general concept of multiparametric single cell analysis to the use of RNAi, and to the relationship between protein levels and chemotherapeutic response. High Content Screening is becoming an important and general approach to biological and therapeutic studies. In addition to increasing the options available for cell-based assays in general, it is opening up new approaches to biological processes and drug development, such as cytological profiling [##REF##17401369##28##,##REF##15539606##29##,##REF##17588932##66##]. Inherent in the latter approaches is the use of single cell cytometry to analyze complex patterns in cellular responses [##REF##15475475##27##]. We have generalized the use of single cell cytometry in several experimental systems and have found that it generally improves experimental analysis, and in some cases, enables challenging questions to be addressed directly. We have used single cell cytometry to address four biological problems: identifying the relevant cells in a knockdown of GFP, correlating the knockdown of PTEN with the increase in activity of pS6 kinase, the effect of knockdown of STAT3 on proliferation and death of colon carcinoma cells and the relationship between p53 levels and responsiveness to DNA damage (both as manipulated by RNAi and as occur intrinsically through standard cell culture conditions).</p>", "<p>For RNAi screening in general, there are two applications of single cell cytometry that are potentially valuable. First is a general analysis of knockdown phenotypes by number of cells showing an altered phenotype, rather than average phenotypic change for the two samples. This approach is more in line with other distribution-based methods such as sectoring samples in flow cytometry, and can present data in more biologically-relevant way than reporting as percent-of-control (discussed below). Rigorous analysis of RNAi screening data is currently challenging [##REF##16607398##15##,##REF##17716236##74##], and would benefit from clearer definitions of what constitutes a hit [##REF##17429401##9##,##REF##17435171##75##]. The second benefit of single cell cytometry is the capacity to score cells as a function of the amount of siRNA effectively introduced in cells, as evidenced by the accumulation of the (non-functional) sense strand in P-bodies following efficient transfection. Transfection of siRNAs are frequently associated with off-target effects [##REF##16373489##76##, ####REF##12754523##77##, ##REF##16489337##78####16489337##78##], particularly at concentrations typically used for library-based screening (&gt;20 nM) [##REF##15864305##79##,##REF##14769947##80##]. Off-target effects result in many false positive hits in RNAi screens, and impose a significant burden on the post-screening confirmation phase of a project [##REF##16990807##81##]. Transfection at low concentrations (&lt; 10 nM) has been shown to reduce such artifacts, however library screening is performed with many siRNAs that have not been well-validated, particularly for off-target effects. Library screening typically involves higher concentrations because a productive screen requires that cells be reliably transfected, and some balance between the efficiency of transfection and a lack of specificity can be tolerated in the initial screen [##REF##16607398##15##], as long as an effective strategy exists for demonstrating authentic gene-phenotype connections [##REF##16990807##81##,##REF##17716236##82##]. Therefore, off-target effects resulting from high concentrations of siRNA transfections are a common and perhaps unavoidable complication of running siRNA screens. Reduced off-target effects have been associated with pooling or multiplexing siRNAs, particularly in highly complex pools such as are generated by enzymatic preparation of gene-specific siRNA pools (esiRNAs, [##REF##15616564##83##]), at least in part because the concentration of any single siRNA is low.</p>", "<p>Reverse-transfection, including the live cell array [##REF##15782175##7##,##REF##14525932##84##,##REF##16628209##85##], is frequently used in functional screens. This format spots the siRNA (or dsRNA for screens in <italic>Drosophila </italic>cells) onto a surface prior to use with cultured cells, and therefore cells are not transfected at a specific concentration, strictly speaking. Single cell analysis can be readily performed on assays following reverse transfection, since these explicitly require image-based readouts. Selecting a subpopulation with consistent siRNA uptake for each siRNA is computationally intensive, and therefore would be difficult to use directly in the primary screen endpoint, but could be used to analyze data from a primary screen that uses a high content (image-based) assay. The siRNAs need to be labeled directly or co-transfected with a labeled siRNA, in order for siRNA levels to be quantitated. However, the benefit of this is that knockdown phenotypes can be scored for cells within specific thresholds of siRNA accumulation, and these thresholds can be adjusted as the data is reviewed, rather than during image analysis. </p>", "<p>Scoring perturbations by fraction of responding cells (in the case of GFP knockdown at the single cell level) and by response magnitude as a function of target level (such as in the example of DNA damage response as a function of p53 levels) highlight important characteristics of biological samples, particularly in the development of human diseases such as cancer. Clinically important roles are played by minor populations within cell types, such as the growth of solid tumors through tumor-initiating cells (cancer stem cells) and the importance of regions within tumors that control angiogenesis and chemoresistance (the hypoxic core of cells within solid tumors). These properties can be observed in cell culture models, but this differentiation is lost in whole-well methods. Tracking effects of candidate therapeutics among rare cells or cells that have reduced proliferation rates can focus decisions on how well promising a strategy may be by limiting analysis to the cells that play the biggest role in disease progression.</p>", "<p>A similar situation occurs with pathway analyses. An assay that measures a change in a complex pathway, such as the PI3K/AKT/mTor pathway, cannot help but exclude important factors that contribute to a diverse set of outputs. This heterogeneity may be as much a part of the discordance between target inhibition and clinical response as widely cited factors, such as tumor heterogeneity as a result of genetic instability. In both cases, variability in the cells that constitute a tumor enable a significant number of cells to escape death. The difference between these two scenarios is that genetic instability suggests a somatic evolutionary process, whereas signaling heterogeneity suggests that insufficient control of the pathway results in escape from a therapeutic. In such cases, single cell analysis could improve the search for combination therapeutic strategies. mTor activity is subject to multiple levels of feedback regulation [##REF##16915295##86##,##REF##16226444##87##] and to cross-talk with other pathways, particularly the influence of amino acid and cellular energy levels on mTor activity [##REF##9603962##55##]. As such these influences would need to be measured in a multiparametric assay system, to track changes between two points in such a complex pathway. Taken together, the results presented here suggest that pathways that are quiescent (such p53 during periods of low DNA damage) or truly linear (such as activation of STAT signaling by JAK kinases) should show correlations between two points at the single cell level. This correlation could be used to validate results from RNAi experiments by providing a separate method of linking protein levels to pathway function.</p>", "<p>Studies that integrate complex signaling interactions, as opposed to linear events within single pathways, are at the root of systems biology [##REF##16339439##31##,##REF##16699502##32##], and are better able to characterize pathway states in their biological contexts. Such approaches are being shown to be of direct relevance to signaling in disease biology [##REF##15260991##25##,##REF##16564013##88##]. HCS is a strong complement to flow cytometry as a method of single cell analysis because signaling pathway responses can be integrated with cytological dynamics, and as such will extend systems biology into areas such as cancer cell motility and invasion [##REF##15475475##27##,##REF##15539606##29##,##REF##16426740##89##]. These approaches will lead to more innovative approaches to treating disease [##REF##16453012##90##], including complex molecular studies which can be integrated with genetic and epidemiological studies that show subtle but important interactions between common disease loci.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>High Content Screening has been shown to improve results of RNAi and other perturbations, however significant intra-sample heterogeneity is common and can complicate some analyses. Single cell cytometry can extract important information from subpopulations within these samples. Such approaches are important for immune cells analyzed by flow cytometry, but have not been broadly available for adherent cells that are critical to the study of solid-tumor cancers and other disease models.</p>", "<title>Results</title>", "<p>We have directly quantitated the effect of resolving RNAi treatments at the single cell level in experimental systems for both exogenous and endogenous targets. Analyzing the effect of an siRNA that targets GFP at the single cell level permits a stronger measure of the absolute function of the siRNA by gating to eliminate background levels of GFP intensities. Extending these methods to endogenous proteins, we have shown that well-level results of the knockdown of PTEN results in an increase in phospho-S6 levels, but at the single cell level, the correlation reveals the role of other inputs into the pathway. In a third example, reduction of STAT3 levels by siRNA causes an accumulation of cells in the G1 phase of the cell cycle, but does not induce apoptosis or necrosis when compared to control cells that express the same levels of STAT3. In a final example, the effect of reduced p53 levels on increased adriamycin sensitivity for colon carcinoma cells was demonstrated at the whole-well level using siRNA knockdown and in control and untreated cells at the single cell level.</p>", "<title>Conclusion</title>", "<p>We find that single cell analysis methods are generally applicable to a wide range of experiments in adherent cells using technology that is becoming increasingly available to most laboratories. It is well-suited to emerging models of signaling dysfunction, such as oncogene addition and oncogenic shock. Single cell cytometry can demonstrate effects on cell function for protein levels that differ by as little as 20%. Biological differences that result from changes in protein level or pathway activation state can be modulated directly by RNAi treatment or extracted from the natural variability intrinsic to cells grown under normal culture conditions.</p>" ]
[ "<title>Authors' contributions</title>", "<p>PL codeveloped the data extraction and single cell analysis pipeline, and conducted the GFP knockdown experiments, analyzed data, and developed the manuscript; JZ conducted and analyzed the p53 knockdown experiments; JP conducted the antigen distribution, PTEN and STAT3 experiments; AH codeveloped the data extraction and single cell analysis pipeline; SH analyzed data and wrote the draft. All authors contributed to interpretation of data and manuscript revisions.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>We thank Spyro Mousses, Kim Arndt and Robert T. Abraham for helpful comments on the manuscript. We thank John Morris, Bill Hussey, Tom Cannon and Ming Cui for information management support.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Single cell analysis of siRNA knockdown of GFP</bold>. siRNAs transfected at increasing doses into RWPE-1 cells stably transduced to constitutively express GFP, are correlated with the reduction of GFP expression, as determined by fluorescence intensity. <bold>A</bold>. GFP-siRNA accumulation and correlation with GFP levels observed by fluorescence microscopy. <bold>B</bold>. Average GFP fluorescence levels of wells treated with a GFP-specific siRNA or a non-targeting control siRNA, as indicated. Each box plot displays the median and intrerquartile range of 8 wells. <bold>C</bold>. For the transfection of siRNAs at a concentration of 3.13 nM, the cells of one well are plotted individually for both GFP and rhodamine fluorescence intensities.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Wide distributions are observed in endogenous protein levels for cultured cell lines</bold>. Antigen intensity was determined by high content screening following fixation and staining with antibodies that were specific for the indicated protein. Quantitation was achieved by a whole cell mask, which was dilated out from the nuclear region identified by DAPI staining. <bold>A</bold>. Endogenous expression levels of proteins are shown for two breast cell lines, the immortalized line 184B5 and the estrogen-sensitive breast cancer cell line T47D, shown as well or sample mean values of all of the cells. Proteins examined in this study, as indicated in the figure, were quantitated by indirect immunofluorescence and image analysis, and values for each cell are plotted as individual points. All graphs are to the same scale, indicated in the lower left. Approximately 7000 cells were quantitated per sample (antigen/cell line). <bold>B</bold>. Display of sample distributions for the proteins indicated in (A). Data is presented as histograms of cells with increasing levels (total fluorescence intensity, or the sum of all pixel intensities, per cell) of the indicated proteins. <bold>C</bold>. Contribution of cells stratified by antigen intensities on overall abundance measurements. Data is as in panel B, but calculated as the product of the number of cells per bin times the average antigen intensity for that bin. As such, the contribution of each bin to the total well mean response is represented. <bold>D-F</bold>. Correlations between two proteins in cell populations. Protein levels per cell are shown for T47D cells, as indicated in the graphs. Protein levels are mean average fluorescence intensities per cell, as indicated in the axes labels.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>siRNA-mediated knockdown of PTEN and its effect on phosphorylation of ribosomal protein S6</bold>. The breast carcinoma cell line MDA-MB-231 was treated with siRNAs to characterize the correlation between PTEN levels and ribosomal protein S6 phosphorylation levels. ~15,000 cells are presented. <bold>A</bold>. PTEN levels displayed as a histogram for samples treated with an siRNA targeting PTEN or a non-targeting control, as indicated. <bold>B</bold>. Ribosomal protein S6 phosphorylation for the same cells as in A, shown as a histogram of phosphorylation levels as reported by immunofluorescence intensities. <bold>C</bold>. Pairwise correlation for PTEN and phospho-S6 levels at the single cell level for data presented in A and B.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>siRNA mediated knockdown of STAT3</bold>. <bold>A</bold>. Histogram of STAT3 levels in SW480 colon carcinoma cells treated with STAT3 and NTC (non-targeting) siRNAs. Red bars denote STAT3 siRNA treated cells and blue bars represent NTC treated cells. Data presents ~22,000 cells for samples treated with STAT3 and NTC siRNAs each. A region of low-STAT3 expressing cells examined in panels (C) and (E) is indicated in the panel (top left corner). <bold>B</bold>. DNA histogram of cells treated with the STAT3 siRNA. <bold>C</bold>. DNA histogram of low-STAT3 expressing cells (cells are highlighted in panel A). <bold>D</bold>. Nuclear size as a function of DNA content for the entire dataset. <bold>E</bold>. Nuclear size as a function of DNA content for the low-STAT3 cells highlighted in part A, for both STAT3 and NTC treated cells. The measure of DNA content for panels B-E are identical, and therefore the comparison of nuclear size as a function of DNA content may be made directly to the fraction of cells in each phase of the cell cycle (panels C and D, respectively). Color schemes for panels D and E are as in A.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Dependence of p53 on the response to adriamycin is observed in both p53 siRNA treated cells and in untreated cells with low levels of p53</bold>. <bold>A</bold>. DLD-1 colon carcinoma cells treated with increasing doses of adriamycin as indicated in the figure. Cells were treated with an siRNA targeting GFP (blue) or an NTC (red). Cells not treated with adriamycin were treated with DMSO. <bold>B</bold>. p53 levels following siRNA treatment. An siRNA that targets p53 (blue) or a non-targeting control (red), are shown. siRNA treatments as described in A. <bold>C</bold>. The fraction of cells within each concentration of adriamycin for the NTC treated sample is shown. The fractions of cells with the highest and lowest 20% of the range of p53 levels (burgundy and teal, respectively) in the untreated sample are shown at each concentration of adriamycin. The range of p53 levels in each bin is 0–200 FU for the lowest bin and 800–1000 FU for the highest bin. <bold>D</bold>. p53 levels following p53 or NTC siRNA treatment for 48 hours, and adriamycin treatment for 6 hours. siRNA treatments as described in A <bold>E</bold>. Levels of γ-phosphorylated histone H2A-x levels as a function of p53 levels per cell and adriamycin treatment for 6 hours. Adriamycin doses are as shown in other panels, in a color range from yellow (no adriamycin) to orange (1.222 μM adriamycin). Each data point represents 200–400 cells. <bold>F</bold>. Same as (E), except cells were not treated with an NTC siRNA. Adriamycin concentrations are indicated in the panel.</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Supplementary Figure 1 Effect of etoposide treatment on nuclear area of SW 480 colon carcinoma cells. SW480 cells were plated in a 96-well microtiter plate and cultured for 24 hr, at which time they were treated with 5 mM etoposide (shown in red) or a vehicle control (shown in blue). Nuclear size for each cell is shown as a histogram of the entire dataset after binning as shown in the figure.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional file 2</title><p>Immunofluorescence and siRNA transfection conditions. Specific catalog and treatment conditions for siRNAs, transfection reagents and immunofluorescence microscopy.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional file 3</title><p>Supplementary Figure 2 Representative specific and non-specific staining intensities for the primary and secondary antibodies. Box plots of antigen levels as detected by high content screening. Specific antigens were detected using antibodies as indicated in the panel and non-specific background staining was detected using specific isotypes, is indicated as well. IgG is from rabbit, IgG1 and IgG2a are from mouse.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S4\"><caption><title>Additional file 4</title><p>Extracting high content single cell data for analysis. SWEAVE document describing the routine used to retrieve single cell data from the database.</p></caption></supplementary-material>" ]
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[{"surname": ["Hannon"], "given-names": ["GJ"], "source": ["RNAi: A Guide to Gene Silencing"], "year": ["2003"], "edition": ["1"], "publisher-name": ["Cold Spring Harbor, NY, USA , Cold Spring Harbor Press"], "fpage": ["436"]}, {"surname": ["Appasani", "Appasani K"], "given-names": ["K"], "source": ["RNA Interference Technology: From Basic Science to Drug Development"], "year": ["2005"], "edition": ["1st"], "publisher-name": ["Cambridge, UK , Cambridge University Press"], "fpage": ["510"]}, {"surname": ["Comley"], "given-names": ["J"], "article-title": ["High content screening-Emerging importance of novel reagents/probes and pathway analysis"], "source": ["Drug Discovery World"], "year": ["2005"], "volume": ["6"], "fpage": ["31"], "lpage": ["54"]}, {"surname": ["Haney", "Zhang", "Pan", "LaPan", "Haney SA"], "given-names": ["SA", "J", "J", "P"], "article-title": ["HCS in cellular oncology and tumor biology."], "source": ["High Content Screening: Science, Techniques and Applications"], "year": ["2008"], "publisher-name": ["Hoboken, NJ , John Wiley Press"], "fpage": ["113"], "lpage": ["143"]}, {"surname": ["Hill", "Li", "LaPan", "Haney", "Haney S"], "given-names": ["A", "Y", "P", "S"], "article-title": ["Analysis of multiparametric high-content data."], "source": ["High Content Screening: Science, Techinques and Applications"], "year": ["2008"], "publisher-name": ["Hoboken, NJ , Wiley Interscience"], "fpage": ["329"], "lpage": ["353"]}]
{ "acronym": [], "definition": [] }
90
CC BY
no
2022-01-12 14:47:26
BMC Cell Biol. 2008 Aug 1; 9:43
oa_package/db/94/PMC2529295.tar.gz
PMC2529296
18681978
[ "<title>Background</title>", "<p>China's population of 1.3 billion accounts for 20% of the world population, making it the most populated country in the world. With rapid economic growth and globalization, Chinese people's lifestyle has been changing dramatically towards being more physically inactive, eating more fast food, and being overall more stressed compared their old lifestyle. Because of the rapid change in lifestyle, population health is quickly shifting from a high mortality rate due to infectious and domestic diseases, related to 'poverty' before implementation of 'open door policy' in 1980, to a currently greater life expectancy and higher prevalence of chronic and non-communicable diseases brought on by 'affluence' [##REF##15036820##1##].</p>", "<p>In the last five decades, the Chinese population has become healthier as measured by decreased mortality rate and increased life expectancy. For example, infant mortality has decreased from 80.8/1000 live births in 1958 to 21.5/1000 live births in 2004 [##UREF##0##2##], maternal mortality has declined from 88.9/100000 live births in 1990 to 48.3/100000 live births in 2004, and life expectancy has increased from 57.0 years in 1957 to 71.8 years in 2004 [##UREF##1##3##]. The most recent data showed that heart disease, cancer and stroke are the major causes of death, accounting for 65% of all deaths and infectious disease has become the least important cause of death, only accounting for 3% of all deaths in Chinese population aged 40 or above [##REF##16162883##4##].</p>", "<p>Life expectancy is an internationally used health measure because many countries, including China, periodically calculated it using vital data. However, such measure mirrors mortality, and particularly infant mortality, but is insensitive to nonfatal and psychosocial conditions that contribute indirectly to death. Hence, it is imperative to assess population health using indicators that reflect contemporary health issues. This study aimed to describe the male and female adult Chinese population health status in multiple dimensions, including well-being, morbidity, quality of life, and health behaviors of smoking, alcohol consumption and physical activity, using data from the most recent National Health Services Survey from the Chinese government. Our descriptive study on Chinese population health status has application for international organizations because China's large population will influence global health status. Our study provided important information for generating research questions for future studies.</p>" ]
[ "<title>Methods</title>", "<title>Study Population</title>", "<p>We derived data from the China Third National Health Services Survey, which collected data through face-to-face interviews from September 18 to October 20, 2003. Of the 193,689 respondents surveyed, we included individuals who were 18 years of age or older and excluded 3,677 respondents with missing values, resulting in a total of 139,831 (69,748 males and 70,083 females) respondents in our analysis.</p>", "<p>The national survey employed a multiple stage cluster sampling method to select the sample randomly. The mainland of China was clustered according to the government administrative geographic system (i.e., county, town and village in rural areas, and city, community, and neighbourhood in urban areas). Firstly, 95 counties and cities were randomly selected from rural and urban areas. Secondly, 5 towns and 5 communities were randomly selected in each county and city, respectively. Thirdly, 2 villages in each town and 2 neighbourhoods in each community were randomly selected. Fourthly, 60 households were randomly selected in each village and neighbourhood, respectively, resulting in about 57,000 households. All family members aged 15 years or older were invited to participate in the face-to-face interview.</p>", "<title>Data collection</title>", "<p>Medical doctors and nurses conducted the survey. Before the survey, interviewers were trained and practiced interviewing; their understanding and knowledge about the survey method and content were examined through testing. During the survey, interviewers visited each household up to three times on different days and times. Interviewers explained the purposes and confidentiality of the survey, and then invited family members to participate. Respondents could choose not to participate and their participation in the survey was accepted as oral consent. The completeness of questionnaires was checked by a district survey manager at the end of every day. If there was missing information on the survey, individuals would be re-surveyed if possible. After the survey, 5% of households were randomly selected and re-surveyed on 14 questions to examine survey quality; the agreement was 95%. The survey response rate for adults was 77.8% [##UREF##2##5##].</p>", "<title>Demographic Characteristics</title>", "<p>Demographic variables included age, sex, marital status, education; rural/urban residence, and geographic region. Educational level was categorized into five categories, illiterate (it was defined as people who could not read newspaper or magazines, or write a short note), elementary school (i.e. those who attended up to 6 years of schooling or were not illiterate for those without schooling), junior high school (i.e. schooling 7 – 9 years), senior high school (i.e. schooling 10–12 years), and college or university or higher (i.e. complete or incomplete of post-secondary school). Residence was divided based on rural and urban area and then economic development. Rural area included towns and villages. Based on economic development, Eastern China, the most developed region, included 11 provinces and metropolitans such as Beijing, Shanghai, and Liaoning. Middle China included 8 provinces, such as provinces of Heilongjiang, Shanxi, and Hunan. Western China, the least developed region, included 12 provinces such as Yunnan, Tibet, In-Mongolia, and Ningxia.</p>", "<title>Health Status Indicators</title>", "<p>Self-perceived overall wellbeing was assessed using a five -point Likert-type scale of being excellent, good, fair, poor or very poor. Presence of illness in the last two weeks and physician-diagnosed chronic disease in the last six months was recorded. The two-week illness was surveyed by asking: \"Have you had any physical and mental discomforts during the last two weeks?\" Chronic disease referred to disease diagnosed by medical doctors and occurring in the last 6 months prior to the survey, or chronic disease that was diagnosed more than 6 months prior to the survey but reoccurred within the last 6 months and received treatment. Non-physician diagnosed chronic disease was not included because the validity of self-diagnosed medical conditions depends on the level of the respondent's knowledge and their perceptions on the definition of 'disease' and 'health'. Physician diagnosed chronic disease was further confirmed by asking diagnosis location including community clinics, county hospital, city hospital, provincial hospital, military hospital, and others. Respondent reported up to three specific chronic diseases. The reported diseases were coded and classified using the disease classification scheme designed by China Ministry of Health for the survey.</p>", "<p>Quality of life was measured using a seven-item instrument. Respondents were asked about presence and level of severity of their dysfunction and disability in the last 30 days in 1) ability about washing or dressing themselves, 2) ability to do job work or housework, 3) feeling of pain or physical discomfort, 4) ability of concentration on work or study and memory, 5) ability of recognizing familiar people from 20 meters away (with glasses for those wearing glasses), 6) emotional discomfort due to restlessness, and 7) anxiety or depression. Under each item, five itemized answers about presence and severity were provided, including: none, mild, moderate, severe, and extremely severe.</p>", "<title>Health determinant</title>", "<p>Information about smoking, alcohol consumption and physical exercise was collected. For smoking, the survey asked: \"Are you currently smoking?\" (with answer: Yes, No) Under the survey question of \"Do you drink alcohol?\", the three answers were provided: \"No or rarely\", \"Sometimes\" (defined drinking &lt; 3 times per week), and \"Frequently\" (defined drinking ≥ 3 times per week). For exercise, a question of \"What is the sport or exercise that you have been regularly doing in the last 6 months?\" was asked with providing a list of recreational physical activities, such as running, Tai Chi, Wushu, dancing, and playing balls. Regularity of exercise was not defined in the survey and determined by respondent's perception.</p>", "<title>Statistical Analysis</title>", "<p>Proportion was employed to describe respondents in demographic characteristics, health status and health determinants. Because of the large sample size and multiple categories in some variables, the P-value for sex difference was not reported. Frequencies of variables in the survey were not weighted because sampling weight was not available. The same sampling method had been used in the previous two National Health Services Surveys in China. Analyses of previous surveys suggest that this sampling method is adequate to generate a nationally representative sample [##UREF##2##5##]. The survey respondent age and sex composition was comparable with the 2000 census. Finally, multiple logistic regressions were used to generate risk adjusted P-value for gender difference in health indicators after adjustment for demographic characteristics and correction of clustering of individuals within family using the repeated measure [##REF##12746247##6##,##REF##15987728##7##].</p>", "<p>The data were analyzed at the health information centre of the Ministry of Health in Beijing. Confidentiality of the survey was protected through storing the data on password protected computers at the Ministry, and removing personal identifiable information (such as name and address) from the database available for researchers and examining analysis outputs for release of aggregated data by the centre staff.</p>" ]
[ "<title>Results</title>", "<title>Demographic Characteristics</title>", "<p>Demographic characteristics for respondents are presented in Table ##TAB##0##1##. A majority of the respondents were married (80.3%) and resided in rural areas (71.2%). There was a similar proportion of male and female (49.9%. versus 50.1%). More males than females were unmarried (14.8% versus 9.2%) but had higher education (illiterate rate: 12.2% for males and 27.7% for females). The composition by age, rural/urban and region was similar between males and females.</p>", "<title>Health Status and Determinant</title>", "<p>Of the respondents, 5.5% rated their overall wellbeing as being poor or very poor, 15.8% reported illness in the last 2 weeks and 16.3% reported presence of chronic disease (see Table ##TAB##1##2##). Compared to males, more females rated their overall wellbeing as being poor or poorer (4.8% versus 6.2%, risk adjusted P &lt; 0.001), and reported presence of illness in the last 2 weeks (14.1% versus 17.4%, risk adjusted P &lt; 0.001) and chronic disease (15.0% versus 17.7%, risk adjusted P &lt; 0.001). Males had lower prevalence of heart disease (1.4% versus 2.4%), hypertension (3.2% versus 4.0%) and rheumatologic arthritis (0.8% versus 1.5%) than females. However, prevalence of chronic pulmonary disease was slightly higher for males than that for females (1.7% versus 1.1%).</p>", "<p>In all seven items of the quality of life, 29.9% reported at least one problem (see Table ##TAB##2##3##). The rate was significantly lower for males than that for females (26.9% versus 32.8%, risk adjusted P &lt; 0.001). Males were more likely than females to report no problems on all seven items (such as 86.6% versus 81.8% for pain, 88.1% versus 84.3% for concentration or memory, 90.6% versus 87.5% for vision, 88.2% versus 84.3% for emotional discomfort and 89.3% versus 86.1% for anxiety/depression).</p>", "<p>Respondents aged 65 years or older had much poorer health status than those aged less than 65 years old among males and females (see Table ##TAB##3##4##). A similar proportion of rural and urban respondents rated their health status as being poor or very poor (4.8% versus 4.7% for males and 6.3% versus 6.1% for females), and reported the presence of illness in the last 2 weeks (14.2% versus 14.0% for males and 17.3% versus 17.6% for females). However, fewer rural respondents reported chronic disease than urban respondents (13.0% versus 19.9% for males and 15.5% versus 22.8% for females). In all seven items of the quality of life measured, rural respondents reported fewer problems than urban respondents (26.2% versus 28.7% for males and 32.0% versus 34.7% for females).</p>", "<p>Of the respondents, 27.9% were smoking, 8.8% drank alcohol frequently and 13.6% exercised regularly (see Table ##TAB##4##5##). Compared to males, females were significantly less likely to smoke (52.4% versus 3.4%, risk adjusted P &lt; 0.001), drink alcohol (frequent alcohol consumption 16.5% versus 1.1%, risk adjusted P &lt; 0.001) but less likely to do regular exercise (14.2% versus 13.0%, risk adjusted P &lt; 0.001). Smoking and frequent alcohol consumption rate were particularly high among males aged 35 to 64 years and regular exercise rate was especially high among male and female seniors (age ≥ 65 years) and among urban respondents (see Table ##TAB##5##6##).</p>" ]
[ "<title>Discussion</title>", "<p>This study highlighted the Chinese adult population health status as the following: 1) only a small proportion of Chinese adults perceived their health as being poor; 2) chronic diseases were high, particularly hypertension, heart disease, chronic pulmonary disease and diabetes; 3) one third of Chinese had a functional problem; 4) prevalence of emotional and/or mental health problems surpassed prevalence of physical functional problems; 5) smoking and alcohol abuse was very common in men; 6) most of the Chinese surveyed were physical inactive and 7) male health status was better than female health status.</p>", "<p>Non-communicable, rather than communicable, diseases are the major burden in China and the burden has dramatically increased in the last decade as that in some developing countries [##REF##16150869##8##, ####REF##16731270##9##, ##REF##17229946##10####17229946##10##]. Compared to the self-reported health conditions in 1993 [##UREF##0##2##], our analysis of 2003 national survey data showed that prevalence of hypertension and stroke had doubled and prevalence of diabetes had tripled while the prevalence of pulmonary disease and infectious disease had declined by half. In reality, the prevalence of chronic diseases should be higher than our reports because of unawareness of their presence. For example, we reported prevalence of 3.6% for hypertension and 0.8% for diabetes. Based on previous report of unawareness rate of 55.3% for hypertension [##REF##12468580##11##] and 66.6% for diabetes in China [##UREF##3##12##], the prevalence should be 8.1% for hypertension and 2.4% for diabetes.</p>", "<p>In contrast with the social-economic gradient in health commonly found in the literature [##UREF##4##13##, ####REF##17287304##14##, ##REF##17478442##15##, ##REF##12522017##16####12522017##16##], the wealthier urban population in China is not found to be healthier than the rural population in terms of physician diagnosed chronic disease. Our findings are consistent with a previous report in China [##UREF##5##17##]. That study measured glucose tolerance among 42,751 residents who were randomly selected from 11 provinces in China, and reported diabetes prevalence rates of 5.8% in municipal areas, 2.9% in high income rural areas and 1.8% in low income rural areas [##UREF##5##17##].</p>", "<p>There are several possible explanations for the above findings. First, the rural population had lower incidence of chronic disease compared with the urban population. The urban population, who has benefited most from China's economic development, has experienced a dramatic lifestyle change in the past two decades. Compared with before, they are becoming more physically inactive (commuting by cars rather than bicycles), and eating more fast food and high protein/fat food. Prior to the implementation of the \"open door policy\" in the 1980s, China's population health was characterized with a high prevalence of infectious diseases as a result of poverty. It has now shifted to a high prevalence of chronic and non-communicable diseases, brought on by 'affluence.' Such change is much more dominant in urban than rural populations. Of all daily sources of energy, cereals accounted for 61.4% and meat 10.8% for rural residents, compared with 48.5% and 17.6% respectively for urban residents [##UREF##6##18##].</p>", "<p>The second possible explanation is that compared with the urban population, the rural population had a higher mortality rate (6.1/1000 versus 5.6/1000) [##UREF##7##19##], with a shorter duration from disease occurrence to death, and thus a lower life expectancy (69.5 versus 75.2 years) [##UREF##8##20##,##UREF##9##21##]. It was reported that the rural population had higher rates of heart disease and stroke specific mortality than the urban population (330.7 versus 279.5/100,000 person-years for heart disease and 304.1 versus 256.1/100,000 person-years for stroke) [##REF##16162883##4##]. The higher mortality rate is related to lower insurance coverage and lower ability to afford treatment among rural population compared with their counterparts in urban areas [##REF##15452014##22##,##REF##16162889##23##]. China's current healthcare system relies heavily on a non-regulated market to reduce government health expenditure and allows public hospitals to determine the price of services within a certain range [##UREF##10##24##]. The rural population utilized physicians more than the urban population (52.0% versus 43.0%), but utilized hospitals far less (7.6% versus 11.1%, p &lt; 0.001) when they were ill [##REF##17667311##25##]. More people in rural than in urban areas opted for no treatment when suffering from an illness and were more likely to die earlier.</p>", "<p>The third possible explanation is that rural populations were more likely than the urban population to be unaware of the presence of chronic disease. China's national physical measurement study [##UREF##3##12##] reported that the unawareness of diabetes was 71% and 62% for rural and urban populations, respectively.</p>", "<p>Quality of life has been less studied in China although it is an important parameter of population health status. The reason may be due to unawareness of its importance and unavailability of well-developed and validated instruments in the Chinese language. A few previous reports on quality of life either in English or Chinese [##REF##16341052##26##, ####REF##16294368##27##, ##REF##16162120##28####16162120##28##] focused on general populations in small geographic areas or patients receiving certain healthcare services. Our study findings of about 30% population with functional problems could not be compared to previous Chinese studies. Compared to a Canadian report [##REF##17571009##29##], our study indicated that Chinese in mainland China had 5% more physical problems but 5% fewer emotional functional problems than Chinese in Canada. However, we have noticed that differences between these two studies in social demographic characteristics and cultural influence were not adjusted.</p>", "<p>Health promotion for better nutrition, tobacco and alcohol reduction, increase in exercise, and hypertension control is critical for avoiding population health declining and promote quality of life. Smoking and alcohol abuse was very common in male population, particularly those in the middle age group, but was rare among females. This is related to the Chinese culture, which accepts male smoking and drinking but not female [##UREF##11##30##]. China's recent health promotion activities have achieved a decline in the male smoking rate from 70% in 1996 to 52% in 2003 [##UREF##12##31##]. However, many people are still unaware of the dangers in smoking; the proportion who were unaware of smoking's dangers was over 60% in some provinces and higher in rural than urban area [##UREF##12##31##]. Physical inactivity was common for both men and women. Interestingly, Chinese seniors were more active than the younger populations.</p>", "<p>Hypertension is an important risk factor for many chronic diseases, particularly for stroke, heart disease and chronic renal disease. He and colleagues [##REF##16162883##4##] found that hypertension contributed 11.7% to total mortality, smoking 7.9% and physical inactivity 6.8%, resulting in a total of 28.4% (when combined) to mortality in the Chinese adult population. However, these factors were very poorly controlled. About 30% of hypertensive patients took antihypertensive medication with 8.1% achieved blood pressure control [##REF##12468580##11##], and 27.2% of diabetics took medication with 9.7% controlled diabetes [##UREF##3##12##]. The huge gap between presence, awareness, treatment and control of hypertension strongly indicates imperative needs for a national education program that targets the public, clinicians and decision makers to eliminate the gap. Reforming the healthcare system towards the universal insurance coverage is also essential to remove financial barrier to access the system.</p>", "<p>There are four major limitations in the study. First limitation was that validity of self-report health condition was suboptimal. Our prevalence of chronic disease was likely underestimated as stated above. Second limitation was that we did not conduct risk factorial analysis for health status due to the nature of the cross sectional survey. Third limitation is that we did not assess child health status. The reason for that is the survey did not include children under age 15. Fourth limitation is that we only analyzed three major risk factors but were unable to assess other important risk factors of diet and obesity.</p>" ]
[ "<title>Conclusion</title>", "<p>Our analysis demonstrated that males had better health status than females in terms of presence of self-perceived poor wellbeing, illness, chronic disease, and poor quality of life. However, smoking and frequent drinking of alcohol was more prevalent among males than that among females. Our results also indicated that prevalence of chronic illnesses was higher among the urban residents, as compared with rural residents among males and females. Along with reduction of the risk factors to chronic disease, promotion of emotional and mental health should be considered to increase quality of life. Further research on measuring mental health is imperative. Without intervening preventable risk factors for chronic diseases (i.e. reducing hypertension, smoking, alcohol abuse, and physical inactivity), the Chinese population health status will deteriorate even faster as the population ages rapidly due to one-child per family policy in the last thirty years.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>With rapid economic growth and globalization, lifestyle in China has been changing dramatically. This study aimed to describe the male and female adult Chinese population health status.</p>", "<title>Methods</title>", "<p>The Chinese Third National Health Services Survey was conducted in 2003 to collect information about health status and quality of life from randomly selected residents. Of the 193,689 respondents to the survey (response rate 77.8%), 139,831 (69,748 male and 70,083 female) respondents who were 18 years of age or older were analyzed.</p>", "<title>Results</title>", "<p>Among the respondents, fewer males than females rated their overall wellbeing as being poor or very poor (4.8% versus 6.2%), reported illness in the last 2 weeks (14.1% versus 17.4%), presence of physician diagnosed chronic disease (15.0% versus 17.7%) and at least one functional problem in seven items of the quality of life (26.9% versus 32.8%). More males than females were currently smoking (52.4% versus 3.4%) and drank alcohol more than three times per week (16.5% versus 1.1%). Physically inactive rate was similar between males and females (85.8% versus 87.0%). Fewer rural respondents reported chronic disease than urban respondents (13.0% versus 19.9% for males and 15.5% versus 22.8% for females). In all seven items of the quality of life measured, rural respondents reported less problems than urban respondents (26.2% versus 28.7% for males and 32.0% versus 34.7% for females).</p>", "<title>Conclusion</title>", "<p>Males had better health status than females in terms of self-perceived wellbeing, presence of illness, chronic disease, and quality of life. However, smoking and frequent alcohol drinking was more prevalent among males than that among females. In contrast with the social-economic gradient in health commonly found in the literature, the wealthier urban population in China was not found to be healthier than the rural population in terms of physician diagnosed chronic disease.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>JS designed the study and drafted the manuscript. ML performed the statistical analysis, interpreted the results and participated in coordination. QZ performed the statistical analysis and interpreted the results. MLu participated in the study design and interpretation of the results. HQ conceived the study, participated in its design and drafted the manuscript. All authors read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2458/8/277/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>Dr. Hude Quan is supported by a Population Health Investigator Award from the Alberta Heritage Foundation for Medical Research, and a New Investigator Award from the Canadian Institutes of Health Research, Canada. Dr. Mingshan Lu acknowledges the financial support from the Institute of Health Economics in Alberta. The authors thank the China Ministry of Health for providing the data for analysis.</p>" ]
[]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Characteristics of the survey respondents aged 18 years or older in China</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Variables</td><td align=\"center\">Total<break/>N (% of 139831)</td><td align=\"center\">Male<break/>N (% of 69748)</td><td align=\"center\">Female<break/>N (% of 70083)</td></tr></thead><tbody><tr><td align=\"left\">Age</td><td/><td/><td/></tr><tr><td align=\"left\"> 18–34</td><td align=\"center\">43055 (30.8)</td><td align=\"center\">21475 (30.8)</td><td align=\"center\">21580 (30.8)</td></tr><tr><td align=\"left\"> 35–44</td><td align=\"center\">31770 (22.7)</td><td align=\"center\">15646 (22.5)</td><td align=\"center\">16124 (23.0)</td></tr><tr><td align=\"left\"> 45–54</td><td align=\"center\">30023 (21.5)</td><td align=\"center\">15088 (21.6)</td><td align=\"center\">14935 (21.3)</td></tr><tr><td align=\"left\"> 55–64</td><td align=\"center\">16942 (12.1)</td><td align=\"center\">8746 (12.5)</td><td align=\"center\">8196 (11.7)</td></tr><tr><td align=\"left\"> ≥ 65</td><td align=\"center\">18041 (12.9)</td><td align=\"center\">8793 (12.6)</td><td align=\"center\">9248 (13.2)</td></tr><tr><td align=\"left\">Marital status</td><td/><td/><td/></tr><tr><td align=\"left\"> Married</td><td align=\"center\">112274 (80.3)</td><td align=\"center\">55622 (79.8)</td><td align=\"center\">56652 (80.8)</td></tr><tr><td align=\"left\"> Unmarried</td><td align=\"center\">16736 (12.0)</td><td align=\"center\">10324 (14.8)</td><td align=\"center\">6412 (9.2)</td></tr><tr><td align=\"left\"> Divorce</td><td align=\"center\">1584 (1.1)</td><td align=\"center\">938 (1.3)</td><td align=\"center\">646 (0.9)</td></tr><tr><td align=\"left\"> Widow</td><td align=\"center\">9237 (6.6)</td><td align=\"center\">2864 (4.1)</td><td align=\"center\">6373 (9.1)</td></tr><tr><td align=\"left\">Education</td><td/><td/><td/></tr><tr><td align=\"left\"> Illiterate</td><td align=\"center\">27905 (20.0)</td><td align=\"center\">8494 (12.2)</td><td align=\"center\">19411 (27.7)</td></tr><tr><td align=\"left\"> Elementary school</td><td align=\"center\">38332 (27.4)</td><td align=\"center\">19374 (27.7)</td><td align=\"center\">18958 (27.1)</td></tr><tr><td align=\"left\"> Junior high school</td><td align=\"center\">45654 (32.7)</td><td align=\"center\">25788 (37.0)</td><td align=\"center\">19866 (28.3)</td></tr><tr><td align=\"left\"> Senior high school</td><td align=\"center\">15024 (10.7)</td><td align=\"center\">8787 (12.6)</td><td align=\"center\">6237 (8.9)</td></tr><tr><td align=\"left\"> College or university</td><td align=\"center\">12916 (9.2)</td><td align=\"center\">7305 (10.5)</td><td align=\"center\">5611 (8.0)</td></tr><tr><td align=\"left\">Residence area</td><td/><td/><td/></tr><tr><td align=\"left\"> Urban</td><td align=\"center\">40244 (28.8)</td><td align=\"center\">19516 (28.0)</td><td align=\"center\">20728 (29.6)</td></tr><tr><td align=\"left\"> Rural</td><td align=\"center\">99587 (71.2)</td><td align=\"center\">50232 (72.0)</td><td align=\"center\">49355 (70.4)</td></tr><tr><td align=\"left\">Region of China</td><td/><td/><td/></tr><tr><td align=\"left\"> East of China</td><td align=\"center\">48554 (34.7)</td><td align=\"center\">23957 (34.4)</td><td align=\"center\">24597 (35.1)</td></tr><tr><td align=\"left\"> Middle of China</td><td align=\"center\">39056 (27.9)</td><td align=\"center\">19541 (28.0)</td><td align=\"center\">19515 (27.8)</td></tr><tr><td align=\"left\"> West of China</td><td align=\"center\">52221 (37.4)</td><td align=\"center\">26250 (37.6)</td><td align=\"center\">25971 (37.1)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Self-perceived overall physical and emotional wellbeing, illness, and morbidity in the respondents aged 18 years or older in China</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"right\">Total<break/>N (% of 139831)</td><td align=\"right\">Male<break/>N (% of 69748)</td><td align=\"right\">Female<break/>N (% of 70083)</td></tr></thead><tbody><tr><td align=\"left\"><italic>Physical and emotional wellbeing</italic></td><td/><td/><td/></tr><tr><td align=\"left\"> Excellent</td><td align=\"right\">49088 (35.1)</td><td align=\"right\">25816 (37.0)</td><td align=\"right\">23272 (33.2)</td></tr><tr><td align=\"left\"> Good</td><td align=\"right\">50996 (36.5)</td><td align=\"right\">26156 (37.5)</td><td align=\"right\">24840 (35.5)</td></tr><tr><td align=\"left\"> Fair</td><td align=\"right\">32055 (22.9)</td><td align=\"right\">14447 (20.7)</td><td align=\"right\">17608 (25.1)</td></tr><tr><td align=\"left\"> Poor</td><td align=\"right\">6724 (4.8)</td><td align=\"right\">2873 (4.1)</td><td align=\"right\">3851 (5.5)</td></tr><tr><td align=\"left\"> Very poor</td><td align=\"right\">968 (0.7)</td><td align=\"right\">456 (0.7)</td><td align=\"right\">512 (0.7)</td></tr><tr><td align=\"left\">Combination of poor and very poor*</td><td align=\"right\">7692 (5.5)</td><td align=\"right\">3329 (4.8)</td><td align=\"right\">4363 (6.2)</td></tr><tr><td align=\"left\"><italic>Morbidity</italic></td><td/><td/><td/></tr><tr><td align=\"left\">Presence of illness in the last 2 weeks before the survey*</td><td align=\"right\">22050 (15.8)</td><td align=\"right\">9865 (14.1)</td><td align=\"right\">12185 (17.4)</td></tr><tr><td align=\"left\">Presence of physician diagnosed chronic disease in the last 6 months before the survey*</td><td align=\"right\">22808 (16.3)</td><td align=\"right\">10432 (15.0)</td><td align=\"right\">12376 (17.7)</td></tr><tr><td align=\"left\"> Infectious and parasitic disease</td><td align=\"right\">485 (0.4)</td><td align=\"right\">303 (0.4)</td><td align=\"right\">182 (0.3)</td></tr><tr><td align=\"left\"> Cancer</td><td align=\"right\">231 (0.2)</td><td align=\"right\">122 (0.2)</td><td align=\"right\">109 (0.2)</td></tr><tr><td align=\"left\"> Diabetes</td><td align=\"right\">1062 (0.8)</td><td align=\"right\">472 (0.7)</td><td align=\"right\">590 (0.8)</td></tr><tr><td align=\"left\"> Heart disease*</td><td align=\"right\">2644 (1.9)</td><td align=\"right\">997 (1.4)</td><td align=\"right\">1647 (2.4)</td></tr><tr><td align=\"left\"> Stroke</td><td align=\"right\">1257 (0.9)</td><td align=\"right\">693 (1.0)</td><td align=\"right\">564 (0.8)</td></tr><tr><td align=\"left\"> Chronic pulmonary disease*</td><td align=\"right\">1911 (1.4)</td><td align=\"right\">1153 (1.7)</td><td align=\"right\">758 (1.1)</td></tr><tr><td align=\"left\"> Hypertension*</td><td align=\"right\">4989 (3.6)</td><td align=\"right\">2203 (3.2)</td><td align=\"right\">2786 (4.0)</td></tr><tr><td align=\"left\"> Peptic ulcer</td><td align=\"right\">707 (0.5)</td><td align=\"right\">446 (0.6)</td><td align=\"right\">261 (0.4)</td></tr><tr><td align=\"left\"> Chronic liver disease</td><td align=\"right\">192 (0.1)</td><td align=\"right\">123 (0.2)</td><td align=\"right\">69 (0.1)</td></tr><tr><td align=\"left\"> Chronic renal disease</td><td align=\"right\">258 (0.2)</td><td align=\"right\">83 (0.1)</td><td align=\"right\">175 (0.3)</td></tr><tr><td align=\"left\"> Rheumatologic arthritis*</td><td align=\"right\">1598 (1.1)</td><td align=\"right\">534 (0.8)</td><td align=\"right\">1064 (1.5)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Quality of life in the respondents aged 18 years or older in China</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Quality of life</td><td align=\"right\">Total<break/>N (% of 139831)</td><td align=\"right\">Male<break/>N (% of 69748)</td><td align=\"right\">Female<break/>N (% of 70083)</td></tr></thead><tbody><tr><td align=\"left\"><italic>Problem about washing or dressing yourself in the last 30 days</italic></td><td/><td/><td/></tr><tr><td align=\"left\"> No problem</td><td align=\"right\">131927 (94.4)</td><td align=\"right\">66277 (95.0)</td><td align=\"right\">65650 (93.7)</td></tr><tr><td align=\"left\"> Mild problem</td><td align=\"right\">5193 (3.6)</td><td align=\"right\">2216 (3.1)</td><td align=\"right\">2977 (4.2)</td></tr><tr><td align=\"left\"> Moderate problem</td><td align=\"right\">1666 (1.2)</td><td align=\"right\">734 (1.1)</td><td align=\"right\">932 (1.3)</td></tr><tr><td align=\"left\"> Severe problem</td><td align=\"right\">777 (0.6)</td><td align=\"right\">389 (0.6)</td><td align=\"right\">388 (0.6)</td></tr><tr><td align=\"left\"> Extremely severe problem</td><td align=\"right\">268 (0.2)</td><td align=\"right\">132 (0.2)</td><td align=\"right\">136 (0.2)</td></tr><tr><td align=\"left\"><italic>Problem about usual activities such as work, or housework in the last 30 days</italic></td><td/><td/><td/></tr><tr><td align=\"left\"> No problem</td><td align=\"right\">126403 (90.4)</td><td align=\"right\">64061 (91.9)</td><td align=\"right\">62342 (89.0)</td></tr><tr><td align=\"left\"> Mild problem</td><td align=\"right\">8876 (6.3)</td><td align=\"right\">3621 (5.2)</td><td align=\"right\">5255 (7.5)</td></tr><tr><td align=\"left\"> Moderate problem</td><td align=\"right\">2723 (2.0)</td><td align=\"right\">1186 (1.7)</td><td align=\"right\">1537 (2.2)</td></tr><tr><td align=\"left\"> Severe problem</td><td align=\"right\">1362 (1.0)</td><td align=\"right\">644 (0.9)</td><td align=\"right\">718 (1.0)</td></tr><tr><td align=\"left\"> Extremely severe problem</td><td align=\"right\">467 (0. 3)</td><td align=\"right\">236 (0.3)</td><td align=\"right\">231 (0.3)</td></tr><tr><td align=\"left\"><italic>Level of pain and physical discomfort in the last 30 days</italic></td><td/><td/><td/></tr><tr><td align=\"left\"> No pain or physical discomfort</td><td align=\"right\">117701 (84.2)</td><td align=\"right\">60364 (86.6)</td><td align=\"right\">57337 (81.8)</td></tr><tr><td align=\"left\"> Mild pain or physical discomfort</td><td align=\"right\">16228 (11.6)</td><td align=\"right\">6835 (9.7)</td><td align=\"right\">9393 (13.4)</td></tr><tr><td align=\"left\"> Moderate pain or physical discomfort</td><td align=\"right\">4373 (3.1)</td><td align=\"right\">1847 (2.7)</td><td align=\"right\">2526 (3.6)</td></tr><tr><td align=\"left\"> Severe pain or physical discomfort</td><td align=\"right\">1274 (0.9)</td><td align=\"right\">580 (0.8)</td><td align=\"right\">694 (1.0)</td></tr><tr><td align=\"left\"> Extremely severe pain or physical discomfort</td><td align=\"right\">255 (0.2)</td><td align=\"right\">122 (0.2)</td><td align=\"right\">133 (0.2)</td></tr><tr><td align=\"left\"><italic>Problem about concentration or memory in the last 30 days</italic></td><td/><td/><td/></tr><tr><td align=\"left\"> No problem</td><td align=\"right\">120534 (86.2)</td><td align=\"right\">61433 (88.1)</td><td align=\"right\">59101 (84.3)</td></tr><tr><td align=\"left\"> Mild problem</td><td align=\"right\">14232 (10.2)</td><td align=\"right\">6173 (8.8)</td><td align=\"right\">8059 (11.5)</td></tr><tr><td align=\"left\"> Moderate problem</td><td align=\"right\">3765 (2.7)</td><td align=\"right\">1553 (2.2)</td><td align=\"right\">2212 (3.2)</td></tr><tr><td align=\"left\"> Severe problem</td><td align=\"right\">1027 (0.7)</td><td align=\"right\">467 (0.7)</td><td align=\"right\">560 (0.8)</td></tr><tr><td align=\"left\"> Extremely severe problem</td><td align=\"right\">273 (0.2)</td><td align=\"right\">122 (0.2)</td><td align=\"right\">151 (0.2)</td></tr><tr><td align=\"left\"><italic>Problem of recognizing a familiar person in 20 meter or more away (with glasses for people wearing glasses)</italic></td><td/><td/><td/></tr><tr><td align=\"left\"> No problem</td><td align=\"right\">124449 (89.0)</td><td align=\"right\">63157 (90.6)</td><td align=\"right\">61292 (87.5)</td></tr><tr><td align=\"left\"> Mild problem</td><td align=\"right\">9895 (7.1)</td><td align=\"right\">4354 (6.1)</td><td align=\"right\">5541 (7.9)</td></tr><tr><td align=\"left\"> Moderate problem</td><td align=\"right\">3772 (2.7)</td><td align=\"right\">1503 (2.2)</td><td align=\"right\">2269 (3.2)</td></tr><tr><td align=\"left\"> Severe problem</td><td align=\"right\">1316 (0.9)</td><td align=\"right\">558 (0.8)</td><td align=\"right\">758 (1.1)</td></tr><tr><td align=\"left\"> Extreme problem</td><td align=\"right\">399 (0.3)</td><td align=\"right\">176 (0.3)</td><td align=\"right\">223 (0.3)</td></tr><tr><td align=\"left\"><italic>Problem about emotional discomfort due to restlessness in the last 30 days</italic></td><td/><td/><td/></tr><tr><td align=\"left\"> No problem</td><td align=\"right\">120632 (86.3)</td><td align=\"right\">61542 (88.2)</td><td align=\"right\">59090 (84.3)</td></tr><tr><td align=\"left\"> Mild problem</td><td align=\"right\">15504 (11.1)</td><td align=\"right\">6680 (9.6)</td><td align=\"right\">8824 (12.6)</td></tr><tr><td align=\"left\"> Moderate problem</td><td align=\"right\">2938 (2.1)</td><td align=\"right\">1185 (1.7)</td><td align=\"right\">1753 (2.5)</td></tr><tr><td align=\"left\"> Severe problem</td><td align=\"right\">586 (0.4)</td><td align=\"right\">258 (0.4)</td><td align=\"right\">328 (0.5)</td></tr><tr><td align=\"left\"> Extremely severe problem</td><td align=\"right\">171 (0.1)</td><td align=\"right\">83 (0.1)</td><td align=\"right\">88 (0.1)</td></tr><tr><td align=\"left\"><italic>Anxiety or depression in the last 30 days</italic></td><td/><td/><td/></tr><tr><td align=\"left\"> No anxiety or depression</td><td align=\"right\">122612 (87.7)</td><td align=\"right\">62263 (89.3)</td><td align=\"right\">60349 (86.1)</td></tr><tr><td align=\"left\"> Mild anxiety or depression</td><td align=\"right\">13230 (9.4)</td><td align=\"right\">5786 (8.3)</td><td align=\"right\">7444 (10.6)</td></tr><tr><td align=\"left\"> Moderate anxiety or depression</td><td align=\"right\">3040 (2.2)</td><td align=\"right\">1271 (1.8)</td><td align=\"right\">1769 (2.5)</td></tr><tr><td align=\"left\"> Severe anxiety or depression</td><td align=\"right\">764 (0.6)</td><td align=\"right\">349 (0.5)</td><td align=\"right\">415 (0.6)</td></tr><tr><td align=\"left\"> Extremely severe anxiety or depression</td><td align=\"right\">185 (0.1)</td><td align=\"right\">79 (0.1)</td><td align=\"right\">106 (0.2)</td></tr><tr><td align=\"left\"><italic>Presence of mild, moderate, severe or extremely severe problem on any one of seven items above*</italic></td><td align=\"right\">41756 (29.9)</td><td align=\"right\">18753 (26.9)</td><td align=\"right\">23003 (32.8)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Self-perceived overall physical and emotional wellbeing, illness, morbidity and quality of life by age and region in the male and female respondents aged 18 years or older in China</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\">Male Age</td><td align=\"center\" colspan=\"2\">Female Age</td><td align=\"center\" colspan=\"2\">Male Residence</td><td align=\"center\" colspan=\"2\">Female Residence</td></tr><tr><td/><td colspan=\"2\"><hr/></td><td colspan=\"2\"><hr/></td><td colspan=\"2\"><hr/></td><td colspan=\"2\"><hr/></td></tr><tr><td align=\"left\">Quality of life</td><td align=\"center\">&lt; 65<break/>N (%)</td><td align=\"center\">≥ 65<break/>N (%)</td><td align=\"center\">&lt; 65<break/>N (%)</td><td align=\"center\">≥ 65<break/>N (%)</td><td align=\"center\">Rural<break/>N (%)</td><td align=\"center\">Urban<break/>N (%)</td><td align=\"right\">Rural<break/>N (%)</td><td align=\"right\">Urban<break/>N (%)</td></tr></thead><tbody><tr><td align=\"left\">Total N (denominator)</td><td align=\"center\">60955</td><td align=\"center\">8793</td><td align=\"center\">60835</td><td align=\"center\">9248</td><td align=\"center\">50232</td><td align=\"center\">19516</td><td align=\"right\">49355</td><td align=\"right\">20728</td></tr><tr><td align=\"left\">Perceived poor or very poor physical and emotional wellbeing</td><td align=\"center\">1905 (3.1)</td><td align=\"center\">1424 (16.2)</td><td align=\"center\">2589 (4.3)</td><td align=\"center\">1774 (19.2)</td><td align=\"center\">2419 (4.8)</td><td align=\"center\">910 (4.7)</td><td align=\"right\">3106 (6.3)</td><td align=\"right\">1257 (6.1)</td></tr><tr><td align=\"left\">Presence of illness in the last 2 weeks before the survey</td><td align=\"center\">7198 (11.8)</td><td align=\"center\">2667 (30.3)</td><td align=\"center\">9194 (15.1)</td><td align=\"center\">2991 (32.3)</td><td align=\"center\">7142 (14.2)</td><td align=\"center\">2723 (14.0)</td><td align=\"right\">8533 (17.3)</td><td align=\"right\">3652 (17.6)</td></tr><tr><td align=\"left\">Presence of physician diagnosed chronic disease in the last 6 months before the survey</td><td align=\"center\">6899 (11.3)</td><td align=\"center\">3533 (40.2)</td><td align=\"center\">8575 (14.1)</td><td align=\"center\">3801 (41.1)</td><td align=\"center\">6541 (13.0)</td><td align=\"center\">3891 (19.9)</td><td align=\"right\">7659 (15.5)</td><td align=\"right\">4717 (22.8)</td></tr><tr><td align=\"left\">Presence of mild to extremely severe problem in the last 30 days on any one of seven items below</td><td align=\"center\">13173 (21.6)</td><td align=\"center\">5580 (63.5)</td><td align=\"center\">16376 (26.9)</td><td align=\"center\">6627 (71.7)</td><td align=\"center\">13144 (26.2)</td><td align=\"center\">5609 (28.7)</td><td align=\"right\">15814 (32.0)</td><td align=\"right\">7189 (34.7)</td></tr><tr><td align=\"left\"> Washing or dressing yourself</td><td align=\"center\">1815 (3.0)</td><td align=\"center\">1656 (18.8)</td><td align=\"center\">2322 (3.8)</td><td align=\"center\">2111 (22.8)</td><td align=\"center\">2559 (5.1)</td><td align=\"center\">912 (4.7)</td><td align=\"right\">3291 (6.7)</td><td align=\"right\">1142 (5.5)</td></tr><tr><td align=\"left\"> Usual activities such as work, or housework</td><td align=\"center\">3113 (5.1)</td><td align=\"center\">2574 (29.3)</td><td align=\"center\">4320 (7.1)</td><td align=\"center\">3421 (37.0)</td><td align=\"center\">4179 (8.3)</td><td align=\"center\">1508 (7.7)</td><td align=\"right\">5584 (11.3)</td><td align=\"right\">2157 (10.4)</td></tr><tr><td align=\"left\"> Pain and physical discomfort</td><td align=\"center\">6219 (10.2)</td><td align=\"center\">3165 (36.0)</td><td align=\"center\">8688 (14.3)</td><td align=\"center\">4058 (43.9)</td><td align=\"center\">6689 (13.3)</td><td align=\"center\">2695 (13.8)</td><td align=\"right\">8880 (18.0)</td><td align=\"right\">3866 (18.7)</td></tr><tr><td align=\"left\"> Concentration or memory</td><td align=\"center\">4720 (7.7)</td><td align=\"center\">3595 (40.9)</td><td align=\"center\">6525 (10.7)</td><td align=\"center\">4457 (48.2)</td><td align=\"center\">5723 (11.4)</td><td align=\"center\">2592 (13.3)</td><td align=\"right\">7360 (14.9)</td><td align=\"right\">3622 (17.5)</td></tr><tr><td align=\"left\"> Recognizing a familiar person in 20 meter or more away (with glasses for people wearing glasses)</td><td align=\"center\">3248 (5.3)</td><td align=\"center\">3343 (38.0)</td><td align=\"center\">4352 (7.2)</td><td align=\"center\">4439 (48.0)</td><td align=\"center\">4634 (9.2)</td><td align=\"center\">1957 (10.0)</td><td align=\"right\">5973 (12.1)</td><td align=\"right\">2818 (13.6)</td></tr><tr><td align=\"left\"> Emotional discomfort due to restlessness</td><td align=\"center\">5531 (9.1)</td><td align=\"center\">2675 (30.4)</td><td align=\"center\">7525 (12.4)</td><td align=\"center\">3468 (37.5)</td><td align=\"center\">5668 (11.3)</td><td align=\"center\">2538 (13.0)</td><td align=\"right\">7479 (15.2)</td><td align=\"right\">3514 (17.0)</td></tr><tr><td align=\"left\"> Anxiety or depression</td><td align=\"center\">5476 (9.0)</td><td align=\"center\">2009 (22.9)</td><td align=\"center\">7171 (11.8)</td><td align=\"center\">2563 (27.7)</td><td align=\"center\">5260 (10.5)</td><td align=\"center\">2225 (11.4)</td><td align=\"right\">6845 (13.9)</td><td align=\"right\">2889 (13.9)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Prevalence of smoking, alcohol consumption and physical activity in the respondents aged 18 years or older in China</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><italic>Factors</italic></td><td align=\"center\"><italic>Total</italic><break/><italic>N (% of 139831)</italic></td><td align=\"center\"><italic>Male</italic><break/><italic>N (% of 69748)</italic></td><td align=\"center\"><italic>Female</italic><break/><italic>N (% of 70083)</italic></td></tr></thead><tbody><tr><td align=\"left\">Currently smoking*</td><td align=\"center\">38943 (27.9)</td><td align=\"center\">36544 (52.4)</td><td align=\"center\">2399 (3.4)</td></tr><tr><td align=\"left\">Frequency of alcohol consumption* <sup>#</sup></td><td/><td/><td/></tr><tr><td align=\"left\"> No or rarely</td><td align=\"center\">109283 (78.1)</td><td align=\"center\">42749 (61.3)</td><td align=\"center\">66534 (95.0)</td></tr><tr><td align=\"left\"> Sometimes (&lt; 3 times per week)</td><td align=\"center\">18258 (13.1)</td><td align=\"center\">15507 (22.2)</td><td align=\"center\">2751 (3.9)</td></tr><tr><td align=\"left\"> Frequently (≥ 3 times per week)</td><td align=\"center\">12290 (8.8)</td><td align=\"center\">11492 (16.5)</td><td align=\"center\">798 (1.1)</td></tr><tr><td align=\"left\">Regular exercise in the last 6 months*<sup>&amp;</sup></td><td align=\"center\">19057 (13.6)</td><td align=\"center\">9932 (14.2)</td><td align=\"center\">9125 (13.0)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T6\"><label>Table 6</label><caption><p>Prevalence of smoking, frequent alcohol consumption and physical activity by age and gender in the respondents aged 18 years or older in China</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"3\">Male</td><td align=\"center\" colspan=\"3\">Female</td></tr><tr><td/><td colspan=\"3\"><hr/></td><td colspan=\"3\"><hr/></td></tr><tr><td/><td align=\"center\">Smoking<break/>N (%)</td><td align=\"center\">Frequently drinking alcohol*<break/>N (%)</td><td align=\"center\">Regular exercise<sup>#</sup><break/>N (%)</td><td align=\"center\">Smoking<break/>N (%)</td><td align=\"center\">Frequently drinking alcohol*<break/>N (%)</td><td align=\"center\">Regular exercise<sup>#</sup><break/>N (%)</td></tr></thead><tbody><tr><td align=\"left\">Age</td><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> 18–34</td><td align=\"center\">8966 (41.8)</td><td align=\"center\">1656 (7.7)</td><td align=\"center\">2478 (11.5)</td><td align=\"center\">218 (1.0)</td><td align=\"center\">62 (0.3)</td><td align=\"center\">1961 (9.1)</td></tr><tr><td align=\"left\"> 35–44</td><td align=\"center\">9504 (60.7)</td><td align=\"center\">2902 (18.6)</td><td align=\"center\">1525 (9.8)</td><td align=\"center\">409 (2.5)</td><td align=\"center\">183 (1.1)</td><td align=\"center\">1405 (8.7)</td></tr><tr><td align=\"left\"> 45–54</td><td align=\"center\">9425 (62.4)</td><td align=\"center\">3517 (23.3)</td><td align=\"center\">1807 (12.0)</td><td align=\"center\">580 (3.9)</td><td align=\"center\">230 (1.5)</td><td align=\"center\">2015 (13.5)</td></tr><tr><td align=\"left\"> 55–64</td><td align=\"center\">4842 (55.4)</td><td align=\"center\">1914 (21.9)</td><td align=\"center\">1723 (19.7)</td><td align=\"center\">464 (5.7)</td><td align=\"center\">147 (1.8)</td><td align=\"center\">1763 (21.5)</td></tr><tr><td align=\"left\"> ≥ 65</td><td align=\"center\">3807 (43.3)</td><td align=\"center\">1503 (17.1)</td><td align=\"center\">2399 (27.3)</td><td align=\"center\">728 (7.9)</td><td align=\"center\">176 (1.9)</td><td align=\"center\">1981 (21.4)</td></tr><tr><td align=\"left\">Residence</td><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Rural</td><td align=\"center\">27245 (54.2)</td><td align=\"center\">8736 (17.4)</td><td align=\"center\">2789 (5.6)</td><td align=\"center\">1615 (3.3)</td><td align=\"center\">572 (1.2)</td><td align=\"center\">1759 (3.6)</td></tr><tr><td align=\"left\"> Urban</td><td align=\"center\">9299 (47.7)</td><td align=\"center\">2756 (14.1)</td><td align=\"center\">7143 (36.6)</td><td align=\"center\">784 (3.8)</td><td align=\"center\">226 (1.1)</td><td align=\"center\">7366 (35.5)</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><p>* Note: P value &lt; 0.001 for males versus females after adjustment for age, marital status, education, urban/rural residence and geographic region.</p></table-wrap-foot>", "<table-wrap-foot><p>* Note: P value &lt; 0.001 for males versus females after adjustment for age, marital status, education, urban/rural residence and geographic region.</p></table-wrap-foot>", "<table-wrap-foot><p>* Note: P value &lt; 0.001 for males versus females after adjustment for age, marital status, education, urban/rural residence and geographic region.</p><p><sup>#</sup>P-value &lt; 0.001 for frequent alcohol drinkers versus none, rare or sometimes drinkers.</p><p><sup>&amp;</sup>Regularity of exercise was determined based on respondent's perception.</p></table-wrap-foot>", "<table-wrap-foot><p>* Note: Frequent drinker was defined as drinking ≥ 3 times per week.</p><p><sup># </sup>Regularity of exercise was determined based on respondent's perception.</p></table-wrap-foot>" ]
[]
[]
[{"collab": ["China Ministry of Health"], "source": ["Health Statistics Yearbook 2006"], "year": ["2006"], "publisher-name": ["Beijing: China Ministry of Health"], "comment": ["[in Chinese]"]}, {"collab": ["China Ministry of Health"], "source": ["Life expectancy"], "comment": ["[in Chinese]. Accessed on August 14, 2008"]}, {"collab": ["China Ministry of Health"], "source": ["The Third National Health Services Survey Design"], "year": ["2003"], "publisher-name": ["Beijing, China Ministry of Health"], "comment": ["[in Chinese]"]}, {"surname": ["Wu", "Xie", "Li", "Zhao", "Zhou"], "given-names": ["YF", "GQ", "Y", "LC", "BF"], "article-title": ["The current status on the prevalence, awareness, treatment and control of diabetes mellitus in several Chinese subpopulations"], "source": ["Chinese J of Epidemioplogy"], "year": ["2005"], "volume": ["26"], "fpage": ["564"], "lpage": ["568"], "comment": ["[in Chinese]"]}, {"surname": ["Deaton"], "given-names": ["A"], "article-title": ["Policy implications of the gradient of health and wealth"], "source": ["Health Aff"], "year": ["2002"], "volume": ["21"], "fpage": ["13"], "lpage": ["30"], "pub-id": ["10.1377/hlthaff.21.2.13"]}, {"surname": ["Wang", "Li", "Xiang", "Liu", "Bai", "Feng", "Fu", "Ma", "Chen", "Jin", "Li", "Qian", "Chen", "Sun", "Man"], "given-names": ["KA", "TL", "DH", "ZY", "J", "JG", "ZY", "LM", "JS", "SX", "YQ", "RL", "H", "TJ", "QQ"], "article-title": ["Study on the epidemiological characteristics of diabetes mellitus and IGT in China"], "source": ["Chinese J of Epidemiology"], "year": ["1998"], "volume": ["19"], "fpage": ["282"], "lpage": ["85"], "comment": ["[in Chinese]"]}, {"surname": ["Li", "Rao", "Kong", "Yao", "Xiang", "Zhai", "Ma", "Yang"], "given-names": ["LM", "KQ", "LZ", "CH", "HD", "FY", "GS", "XG"], "article-title": ["A description on the Chinese national nutrition and health survey in 2002"], "source": ["Chinese J of Epidemiology"], "year": ["2005"], "volume": ["26"], "fpage": ["478"], "lpage": ["84"], "comment": ["[in Chinese]"]}, {"collab": ["Ministry of Health, China"], "source": ["China Health Statistics Yearbook"], "year": ["2004"], "comment": ["[in Chinese]. Accessed on July 31, 2008"]}, {"collab": ["China Statistics Department"], "source": ["Statistics of 1% national sample, 2005"], "year": ["2006"], "publisher-name": ["Beijing, China Statistics Department"], "comment": ["[in Chinese]"]}, {"collab": ["Information Centre, Helath Ministry of China"], "source": ["China health development report, 1997\u20132001"], "year": ["2003"], "publisher-name": ["Beijing, Health Ministry of China"], "comment": ["[in Chinese]"]}, {"surname": ["Ma", "Lu", "Quan"], "given-names": ["J", "M", "H"], "article-title": ["Healthcare system from central planning to market-based: lessons from China"], "source": ["Health Aff"], "year": ["2008"], "volume": ["27"], "fpage": ["937"], "lpage": ["48"], "pub-id": ["10.1377/hlthaff.27.4.937"]}, {"surname": ["Ma", "Sun", "Luan", "Li", "Hu", "Wang", "Yang"], "given-names": ["GS", "LZ", "DC", "YP", "XQ", "JZ", "XG"], "article-title": ["The description analysis of the smoking pattern of people in China"], "source": ["Chinese J of Prevention and Control for Chronic Disease"], "year": ["2005"], "volume": ["13"], "fpage": ["195"], "lpage": ["99"], "comment": ["[in Chinese]"]}, {"surname": ["Yang", "Ma", "Liu", "Zhou"], "given-names": ["GH", "JM", "N", "LN"], "article-title": ["Smoking and passive smoking in Chinese, 2002"], "source": ["Chinese J of Epidemiology"], "year": ["2005"], "volume": ["26"], "fpage": ["77"], "lpage": ["83"], "comment": ["[in Chinese]"]}]
{ "acronym": [], "definition": [] }
31
CC BY
no
2022-01-12 14:47:26
BMC Public Health. 2008 Aug 5; 8:277
oa_package/3a/1d/PMC2529296.tar.gz
PMC2529297
18664301
[ "<title>Background</title>", "<p>In Uganda, HIV/AIDS remains a major public health problem, mainly affecting people in the productive and reproductive age group of 15 to 49 years. About 1.2 million people are living with HIV while 1.8 million people have died of HIV/AIDS [##UREF##0##1##]. However, Uganda has reversed the HIV sero-prevalence from 30% in 1990 to 6.5% in 2003 [##UREF##1##2##]. This remarkable success has been achieved through promotion of the <italic>ABC </italic>strategy (<italic>abstinence, be faithful, condom use</italic>), effective treatment of opportunistic infections, prevention of mother-to-child transmission of HIV (PMTCT), use of antiretroviral therapy (ART) and HIV voluntary counselling and testing (VCT) [##UREF##1##2##]. During 2005, the national ART programme was operational in hospitals but had not been rolled out to the health sub-districts.</p>", "<p>HIV voluntary counselling and testing (VCT) is now widely accepted as the cornerstone of HIV prevention programmes in many countries because of its multiple benefits [##REF##16327323##3##, ####REF##12511218##4##, ##REF##10963247##5##, ##REF##16091240##6##, ##REF##15305474##7##, ##REF##10963246##8####10963246##8##]. Furthermore, VCT is the gateway to comprehensive HIV care and support including access to antiretroviral therapy [##UREF##2##9##]. Newer approaches of VCT delivery including routine HIV counselling and testing[##REF##17667332##10##], home-based VCT[##REF##16885775##11##,##REF##15746219##12##], use of community-based lay counsellors[##REF##17207292##13##], couple counselling and testing[##REF##16176896##14##], and same-day mobile VCT[##REF##16394855##15##,##REF##16249708##16##], have been added to the traditional VCT delivery systems of free standing, health unit based, and outreach VCT services [##UREF##3##17##].</p>", "<p>Despite the array of delivery approaches and the advantages of VCT, uptake in sub-Saharan Africa is disappointingly low with reports of 12% to 56% among couples or the general public [##UREF##2##9##,##REF##16261266##18##, ####REF##12959817##19##, ##REF##16777636##20##, ##REF##14663117##21##, ##REF##15764856##22####15764856##22##]. Furthermore, there is very little information on VCT uptake among men, and also on the factors that influence it [##UREF##1##2##,##UREF##4##23##]. It is not known whether factors that influence VCT uptake in the general population are also operational in VCT uptake by men only [##REF##17667332##10##, ####REF##16885775##11##, ##REF##15746219##12##, ##REF##17207292##13##, ##REF##16176896##14##, ##REF##16394855##15####16394855##15##,##REF##14663117##21##,##REF##17949401##24##,##REF##17415040##25##]. Men's utilization of VCT is important because in many societies including those in Uganda, men are the heads of households and control decisions and resources that are essential for HIV prevention and care. As Uganda gears to consolidate gains in HIV prevention, it is vitally important that men are fully involved in the HIV prevention and control strategies. It is against this background that we conducted a study to determine the prevalence and factors associated with VCT utilization among men in Kasese district, Uganda.</p>" ]
[ "<title>Methods</title>", "<title>Study site</title>", "<p>Kasese District is situated in Uganda's western region and is divided into two counties namely Bukonzo and Busongora. It had a population of 530,000 people in 2005, of which 80% were rural. The population is predominantly <italic>Bakonzo </italic>and the main language is <italic>Lhukonzo</italic>. The literacy rate was 37% for men and 33% for women, and the main economic activity was subsistence farming [##UREF##5##26##]. In 2005, the HIV prevalence was reported to be 13.3% [##UREF##1##2##]. The study site was Bukonzo West health sub-district (HSD). Bukonzo West HSD has two static VCT sites namely Bwera hospital and Kasanga health centre III that provide preventive and curative outpatient services. It also has a few outreach VCT sites in some of the parishes. The HSD consists of 5 sub-counties, 32 parishes and 170 villages and is predominantly a rural setting. It has a total population of about 123,967 persons of which 48% are males. The mean household size is 5.1, and on average, 3 in 4 households are headed by males [##UREF##6##27##].</p>", "<title>Study design, population and sampling</title>", "<p>The study was a population-based cross-sectional study employing quantitative and qualitative methods of data collection and conducted between January and April 2005. Triangulation of the quantitative and qualitative research methods and data were used for validation and completeness of data[##REF##8807387##28##]. We used a conceptual framework consisting of independent predictors of VCT including socio-demographic, socio-cultural, and health service factors as well as knowledge and perceptions on VCT to design the instruments and to guide the analysis (Figure ##FIG##0##1##).</p>", "<title>Quantitative study</title>", "<p>The study participants were adult men aged 18 years and above, residing in Bukonzo West HSD during the study period, and who consented to participate in the study. We excluded men who were very sick, deaf or mentally ill. Cluster sampling and simple random sampling were used to obtain the sample. A cluster was defined as a village. A list of villages (Local Council I) in Bukonzo West health sub-district was obtained from the District Planning unit. A total of 170 villages were numbered according to the order in which they appeared on the list to form a sampling frame.</p>", "<p>For each village, the cumulative population was calculated. A sampling interval was calculated by dividing the total population by the number of clusters required in the sample [##REF##8970692##38##]. A random number not exceeding the sampling interval was then chosen using a table of random numbers. The first cluster selected was the village in whose cumulative population the random number fitted. Subsequent clusters were selected by adding the sampling interval to the previous population. Thus a sample of 38 clusters (villages) was selected by using probability proportional to size (PPS).</p>", "<p>In each village, the households were randomly selected using a list of heads of households provided by Local Council chairpersons of the respective villages. The principal investigator and research assistants, with the help of a guide walked around the selected villages. Eligible household members who consented to participate in the study were interviewed. A maximum of 25 households were randomly selected for each village. If in a household there was more than one eligible person, one of them was randomly selected for the interview. Households where eligible men could not be found were skipped.</p>", "<title>Sample size estimation</title>", "<p>The sample size was estimated using the modified Kish and Leslie (1965) formula. The prevalence of VCT use among men was estimated at 50% to give the highest power possible and hence the lowest sample size. A design effect of two was incorporated in the formula to cater for the design effect due to the cluster sampling method. The number of clusters C, was calculated using the formula C = [p (1 - P) D]/S<sup>2</sup>b, where b = number of responses per cluster (village) = 21 (on average), and S = standard error = √[P (1-P) D/n] [##REF##1949887##29##]. The required number of clusters was 38 and the sample size was estimated at 780 participants.</p>", "<title>Variables</title>", "<p>The independent variables included socio-demographic characteristics (age, marital status, occupation, education level, type of marriage, religion and income), knowledge and perceptions (knowledge of VCT, source of information about VCT, perceived risk of HIV, perceived benefit of VCT, discordance and importance of couple testing), socio-cultural factors (decision-making, sexual behaviour, stigma, fear of rejection by sexual partner, couple testing, disclosure of test results and social beliefs), and health service factors (willingness to pay, distance to VCT site, travel times, availability of VCT service, confidentiality, and quality of service). The outcome variable for the study was VCT use and was measured at three levels: pre-test counseling, HIV testing, and post-test counseling and receiving HIV test results. VCT use was considered complete if the participant received all three.</p>", "<p>The principal investigator and research assistants administered a pre-tested semi-structured questionnaire to eligible men. The instruments were translated into <italic>Lhukonzo </italic>and back translated into English. Twelve research assistants were trained and commissioned. These were mainly men who had attained Senior Four education (11 years), were fluent in <italic>Lhukonzo </italic>and English, and were residents of Bukonzo West HSD. Throughout data collection, regular meetings were held with the research assistants in order to edit the completed questionnaires and to review field experiences and performance so as to improve subsequent interviews. Data collected were kept under lock and key by the principal investigator.</p>", "<title>Data analysis</title>", "<p>Quantitative data was entered into EPINFO version 6.04 and then exported to Stata version 8.0 for analysis that adjusted for the design effect. Unadjusted analysis was performed between VCT use by the men as the outcome variable and each independent variable. Variables that were significant (p-value &lt; 0.05) were then entered into a model for logistic regression using the backward elimination method so as to control for confounding. The criteria used for the backward elimination model was the default of a p-value &gt; 0.10. Associations between the outcome and independent variables were assessed using odds ratios, 95% confidence limits and p-values. A p-value of 0.05 or less was taken to be statistically significant.</p>", "<title>Qualitative study</title>", "<p>Four focus group discussions consisting of 10–12 participants per group were conducted in <italic>Lhukonzo </italic>by the Principal Investigator as the moderator assisted by a note taker. We used a focus group discussion guide composed of predetermined topics including awareness of VCT services provided in the area; importance of VCT; reasons that influence people to seek VCT; VCT utilization by men; and socio-cultural beliefs that influence VCT utilization among men. The FGDs were chosen strategically to represent variations in the age and place of residence. The FGD participants were stratified by sex and included men, women, and VCT clients who were aged 18 years and above. Two FGDs for men, one FGD for women and one FGD for VCT clients at Bwera hospital were conducted. The women were included in this study in order to capture their opinions and views about the men since men have an influence on them in regard to decisions about VCT use. The FGDs were meant to provide a deeper understanding of the information on the perceptions and socio-cultural aspects of the study. The information shared was recorded on audio tapes and notes taken. The data collected was transcribed verbatim and translated from <italic>Lhukonzo </italic>into English and analyzed manually according to themes arising. The data was then triangulated with the quantitative findings from the individual household interviews.</p>", "<p>Ten key informant interviews were held with two Community Health Workers, one religious leader, two NGO managers, one Community Based Organization manager, two VCT counsellors, the District Director of Health Services and the in-charge of Bukonzo West HSD. The interviews were carried out using an open-ended questionnaire that was presented to the informant shortly before the interview. After transcription and translation, the data was coded manually. Data from the focus group discussions and key informant interviews was then triangulated with the quantitative findings.</p>", "<title>Ethics</title>", "<p>Makerere University Clinical Epidemiology Unit, the Faculty of Medicine Ethics and Research Committee and the Uganda National Council of Science and Technology approved the study. The research participants were enrolled in the study after written informed consent. The participants were informed that their participation in the study was voluntary and they could withdraw from the study at anytime. In addition, measures were taken to ensure the confidentiality of the data. In particular, the interviews were held in privacy, away from relatives and friends; information on HIV status was not collected; codes rather than names of participants were used; and the data was kept under lock and key by the principal investigator.</p>" ]
[ "<title>Results</title>", "<title>Characteristics of the respondents</title>", "<p>Between January and April 2005, a total of 780 men were enrolled into the study (Table ##TAB##0##1##). The men were aged between 18 and 90 years with a median age of 32 years. Of the 780 men, 45.3% were Roman Catholic, 43.7% were Anglican while 6.7% were Muslim. Most of the men (71.3%) were married. Of these, 76.3% were in monogamous unions. The majority of the men were peasant farmers (58.1%), and 32% were earning less than one dollar a day (1US$ ≈ 1700 Uganda Shillings).</p>", "<title>Prevalence of VCT use among men</title>", "<p>Of the 780 men interviewed, 46 % (95% CI 40.8–51.2) had pre-test counselling while 25.9 % (95% CI 19.9–32) had tested for HIV. Thus the VCT drop out was 43.7 %. Of the 201 men who tested for HIV, 96% received their HIV test results. The prevalence of complete VCT use (pre-test counselling, HIV testing, post test counselling and receiving HIV test results) was 23.3% (95% CI 17.2–29.4) (Table ##TAB##0##1##). The most common reason for taking an HIV test was to know the HIV sero-status (74.3%) followed by frequent illness (15.3%) and plans for marriage (15.3%). When asked if they were willing to seek VCT in the near future, the majority (79%) said they were willing to go for VCT. However, willingness to seek VCT in the future varied among the two groups being 74.9% among those who had never had VCT at all and 83.9% among those who had ever had VCT.</p>", "<title>Knowledge and perceptions about VCT</title>", "<p>The majority (80.1%) of the men said they were aware about the existence of the VCT program in their area. When asked what was involved in the VCT process, they mentioned pre-test counselling on HIV (78.6%), taking off blood for HIV testing (92.6%) and counselling on the prevention and control of HIV/AIDS (81.6%). Most men (98.7%) knew of a site offering VCT services in Bukonzo West. The majority of the men received information about VCT through the health workers (75.2%) but other sources of information included friends (12.8%), radio (13.9%), newspapers (6.9%), and community leaders (5.4%). Of the 780 participants interviewed, 94.9% said that VCT was important for the good of the individual and the family. However, most men were worried of taking an HIV test because to them having a positive result meant imminent death.</p>", "<p><italic>\"You can die very soon if you tested positive because of worries\" (Male, Nyakiyumbu FGD)</italic>.</p>", "<p>Only 9.1% of the men perceived themselves to be at a high risk of HIV and yet in the FGDs, the majority of the men, especially single men, considered themselves to be at high risk of HIV because of multiple sexual relationships.</p>", "<p><italic>\"I have tested several times and been found negative. But this time am worried of my status because of the fourth wife whom I married recently\" (Male 33 years, Karambi FGD)</italic>.</p>", "<p>Of the 780 respondents interviewed, 91.3% said that it was important to test together with their sexual partners. The findings from the FGDs showed that most men appreciated the importance of couple testing, with the majority saying that it would help them in family planning in the event of a positive HIV test result. On the contrary, men said that going to the health facility for HIV testing with a spouse was not common in their culture and would be considered as strange behaviour.</p>", "<p>On the issue of discordance, over half of the men (61.8%) did not believe that discordant HIV results could exist. This view was supported by the FGDs where most men said it was not possible for one of the cohabiting partners to test HIV positive and the other HIV negative. They believed that if one of the partners was found HIV positive, then it was automatic that the other also had HIV infection.</p>", "<p><italic>\"Some men have lost their wives due to HIV/AIDS but they lie to us that they were found HIV negative when they tested. This is not true\" (Male, 35 years old, Nyakiyumbu FGD)</italic>.</p>", "<title>Socio-cultural factors</title>", "<p>On disclosure of HIV test results, the majority of the men (81.8%) said they would disclose to their partners. There was, however, contradiction in the FGDs whereby the majority of the men felt that disclosure of HIV positive results would deny them their sexual rights and would imply that they brought the disease into the family.</p>", "<p>\"Some of our women are very emotional and if you disclosed to her, she might end up denying you your sexual rights\" (Male, 34 years old, Key informant)</p>", "<p>With regard to sexual partners, 35.1% of the men reported having two or more sexual partners. During the past one year, 24.9% of the men reported having had extramarital sex. Of those who had extra-marital sex, 76.8% used a condom on their last sexual encounter. Both men and women in the focus group discussions confirmed that multiple sexual relationships were common among the men and this could negatively affect VCT use. The participants felt that having VCT would not stop men from acquiring more sexual partners. Most participants said that the majority of the men live in doubt that they might be infected because of having multiple sexual partners.</p>", "<p><italic>\"Men have many sexual partners and fear that they might be infected with HIV already\" (Female, Kyasenda FGD)</italic>.</p>", "<p>Participants were asked if they had any fears about taking an HIV test and 41.3% said they had fear of taking the HIV test. When asked to mention the fears they had, they mentioned inaccuracy of the HIV testing (37.9%), stigma (57.1%), fear of divorce or separation from partner (38.8%) and lack of confidentiality (8.4%). The participants further mentioned that the majority of the men felt that if they tested positive, their families and the society would easily tell that they were infected. They expressed the feeling that the community might look at them differently and also deny them certain rights like holding political positions. Similar findings were obtained during the focus group discussions.</p>", "<p>The participants were asked if they would feel comfortable if they were seen at the VCT site and the majority (83.1%) said they would feel comfortable. The commonest reasons given for feeling uncomfortable were fear of being labelled an HIV victim (67.4%), stigma (36.4%), and meeting a relative or familiar people (11.4%). These findings were also supported by the focus group discussions.</p>", "<p>The participants were asked if they had any socio-cultural beliefs that might influence men to seek VCT services. Only 20% said that there were social beliefs that would influence men to seek VCT. Of those who said beliefs existed, the majority mentioned men's superiority over women (30.8%), if partner (wife) tested positive, then the man was also automatically positive (18.8%), widow inheritance/many sexual partners (17.3%) and witchcraft (14.7%). During the FGDs, the commonest belief mentioned was that of the superiority of men. Men superiority had a negative influence on the VCT seeking behaviour. Most decisions regarding health seeking behaviour among family members are dominated by the men and in most cases what the man decides is unchallenged by the entire family. Men also felt that once their sexual partners tested for HIV, they did not need to seek VCT because their results would be the same as those of their partners</p>", "<p>Participants were asked whom they would consult before deciding to go for VCT. Of the 780 respondents, 40.9% said they would consult their partners/spouses, followed by friends (15.8%), health workers (15.0%) and 22.7% said they would not consult any body. The participants were asked what action they would take if they tested HIV positive. The majority said they would abstain from sex (33.8%), live positively (29%), seek treatment/join The AIDS Support Organisation (TASO) (24.5%), or simply keep quiet (5.9%).</p>", "<title>Health service factors</title>", "<p>Most men expressed the feeling that they would go somewhere else other than in the HSD, the majority preferring VCT sites outside Bukonzo West where the VCT counsellors do not know them. They feared that they would easily be identified and labelled as HIV victims. Some men also said that the VCT staff were being bribed and hence giving negative results to HIV positive clients.</p>", "<p>\"I don't trust those health workers because they might not keep people's secrets\" (Male, Bwera FGD)</p>", "<p>\" I would prefer that the VCT Counsellors be people from other areas other than from Bukonzo\" (Male &amp; Single, FGD Karambi)</p>", "<p>A sizeable proportion of the respondents (44.7%) said they would be unwilling to pay for the VCT services.</p>", "<p>Of the 201 men who had ever undergone VCT, the main reasons given for choosing a particular VCT site were confidentiality (56.2%), proximity (37.8%), convenience (1.0%) and other (5.0%). The majority of the participants (91.6%) who had ever undergone VCT said they were satisfied with the VCT services. The majority of the participants (69%) lived within a radius of 5 kilometres from the nearest static VCT site. The commonest means of transport to the VCT sites was by walking (69.6%).</p>", "<p>During the FGDs, the majority of the participants said that the VCT sites were few (only two) and were located very far from the people, hence making the VCT services inaccessible. Most of the men wanted VCT services to be extended to the villages or to their homes in order to save them the costs of time and transport.</p>", "<title>Association between independent factors and VCT use among men</title>", "<p>On unadjusted analysis, age 35 years or less (OR = 2.97, 95% CI 2.00–4.43); secondary or tertiary education (OR = 2.10, 95% CI 1.50–3.00); non-subsistence occupation (OR = 2.54, 95% CI 1.68–3.85); willingness to test for HIV with partner (OR = 5.33, 95% CI 1.92–14.85); willingness to disclose HIV results to partner (OR = 2.19, 95% CI 1.43–3.38); feeling comfortable with VCT site (OR = 2.71, 95% CI 1.54–4.77); wanting to know HIV status (OR = 0.19, 95% CI 0.13–0.27); and a monthly income of more than 50,000 Uganda Shillings (OR = 1.84, 95% CI 1.23–2.76) were associated with VCT use among men (Table ##TAB##1##2##).</p>", "<p>On logistic regression with complete VCT as the outcome, age (OR = 2.89, 95% CI 1.77–4.07), occupation (OR = 2.37, 95% CI 1.52–3.71), fear of taking an HIV test (OR = 0.54, 95% CI 0.37–0.79), testing together with a spouse/partner (OR = 3.01, 95% CI 1.02–8.83), and willingness to disclose HIV test results to their sexual partners (OR = 1.64, 95% CI 1.04–2.60) were independently associated with VCT use among men (Table ##TAB##2##3##). Table ##TAB##3##4## shows results of logistic regression of factors associated with seeking pre-test counselling that included: to know the HIV sero-status (OR = 12.25, 95% CI 6.09–24.64); feeling comfortable with the VCT site (OR = 1.75, 95% CI 1.09–2.83); fear of being stigmatized (OR = 1.84, 95% CI 1.10–3.08); non-subsistence farmer (OR = 1.90, 95% CI 1.35–2.67); and confidentiality (OR = 15.91, 95% CI 5.54–45.74). Table ##TAB##4##5## shows results of logistic regression of factors associated with complete VCT among men who went for pre-test counselling that included: more than 7 years of education (OR = 2.9, 95% CI 1.1–7.8); to know the HIV sero-status (OR = 84.6, 95% CI 23.3 – 306.4); fear of inaccurate HIV test result (OR = 30.7, 95% CI 2.1 – 457.4); and confidentiality (OR = 53.1, 95% CI 10.8–261.8).</p>" ]
[ "<title>Discussion</title>", "<title>Prevalence of VCT use among men</title>", "<p>The prevalence of VCT use among men in Bukonzo West was low at only 23.3%. Our findings are similar to those of other population-based studies in sub-Saharan Africa. De Graft and colleagues examined use of VCT services in a rural district population of Malawi and found that 11% of men had ever been tested for HIV [##REF##16261266##18##]. Similarly, in an analysis of data from a population-based survey and a government clinic survey in the Eastern Cape Province of South Africa on VCT services, Hutchinson and Mahlalela reported that 17% of men had been tested for HIV [##REF##16777636##20##]. Our findings confirm that VCT uptake among men is low and reinforce the widely reported observation that men are not fully involved in HIV prevention programmes.</p>", "<title>Factors associated with VCT use among men</title>", "<title>Socio-demographic characteristics</title>", "<p>Consistent with the study by Hutchinson and colleagues in Eastern Cape, South Africa [##REF##16777636##20##], older men (35 years or more) were less likely to use VCT than younger men. In our study population, older men tended to be more conservative and may not have accepted VCT readily. They were more likely to be rural subsistence farmers, less educated, and less informed about HIV and VCT. Furthermore, they were more likely to be married, with less risky sexual behaviour, and thus with a lower perception of HIV risk. Because of their lower education and stronger roots to tradition, they were more likely to hold fatalistic attitudes about HIV and to prefer to remain in a state of denial regarding their HIV status. In Ghana, individuals with no formal education were more likely to stigmatize HIV/AIDS [##REF##17207292##13##].</p>", "<title>Knowledge and perceptions on VCT</title>", "<p>Although most men were familiar with the VCT programme and its procedures, this knowledge did not translate into high VCT use. Our findings contrast with those of Sherr and colleagues who reported that in a rural Zimbabwe cohort, motivation for VCT was driven by knowledge on VCT [##REF##17415040##25##]. The discrepancy between knowledge and practice in our study may be due to the frequently reported knowledge-practice gap. However, it could also be an indicator of critical knowledge gaps on HIV including VCT among the men. In support of this, most men in our study did not know about HIV discordance, although understanding of discordance even among discordant people is extremely low [##REF##16327323##3##,##REF##16176896##14##]. Men's gaps in HIV knowledge combined with superstition may explain why fatalism was a barrier against VCT use among men.</p>", "<p>AIDS related stigmas created barriers to seeking VCT among the men. More than half of the men feared to test for HIV because of stigma. Men were worried of being labelled HIV-infected because they would lose their social privileges. They expressed fear of meeting familiar people in HIV testing clinics, and preferred to test in distant clinics where they were not known by the people and staff. Our findings are consistent with research in sub-Saharan Africa which shows AIDS related stigmas are important barriers to VCT utilization [##REF##15746219##12##,##REF##16394855##15##,##REF##16777636##20##,##REF##14663117##21##,##REF##17415040##25##]. As demonstrated by Wolff and colleagues in Uganda, knowledge on HIV/AIDS is necessary but not sufficient to address AIDS related stigmas [##REF##15746219##12##]. Other interventions including public health laws; social marketing; anti stigma campaigns; community mobilization; social activism; and use of mass media are needed to change societal beliefs about people living with AIDS [##REF##14663117##21##]. Newer approaches including use of routine VCT integrated in health services, same-day mobile VCT services, and home-based VCT seem to offer promising results. In addition, availability of ART including use of home-based ART may also increase VCT use among men [##REF##17667332##10##, ####REF##16885775##11##, ##REF##15746219##12####15746219##12##,##REF##16394855##15##,##REF##17949401##24##].</p>", "<title>Risky sexual behaviour</title>", "<p>A significant proportion of the men engaged in risky sexual behaviour including having multiple sexual partners and engaging in unprotected sexual intercourse. Multiple sexual partners were a common practice and appeared to be a manifestation of male dominance in the society. Surprisingly, in spite of the risky sexual behaviour, perception for HIV risk, especially among older men, was very low. In Zambia, individuals willing to seek VCT were more likely to perceive themselves to be at high risk and were more likely to test HIV positive [##REF##15117300##30##]. However, in rural Zimbabwe, motivation for VCT was driven by knowledge and education rather than sexual risk [##REF##17415040##25##]. One possible explanation for the apparent discrepancy between multiple sexual partners and risk perception may be that multiple sexual partners are a societal norm and men do not perceive it as risky sexual behaviour. Secondly, and as discussed earlier, men may lack adequate knowledge on HIV to appreciate that multiple sexual partners increase the risk of HIV infection.</p>", "<title>Willingness for HIV testing and disclosure</title>", "<p>The strongest motivation for HIV testing was the desire to know the HIV status and most men were willing to test for HIV together with their wives. However, this willingness may not translate into action because, according to the men, society would consider a man going with his wife to a clinic for an HIV test as rather strange behaviour. The demand for couple testing is still low [##REF##12222368##31##,##UREF##7##32##]. In Zimbabwe, Machekano [##REF##9810402##33##] showed that only 7% of the men who tested for HIV brought in their wives for HIV testing. Similarly, although most men were willing to disclose their HIV results to their wives, the fear of negative consequences including sexual denial may ultimately pose a barrier [##UREF##8##34##, ####REF##7485664##35##, ##REF##7619122##36####7619122##36##]. Nevertheless, men's strong desire to know their HIV status and their positive attitude to PMTCT and disclosure of HIV results can be used as a springboard for strategies that target men so as to increase their utilization of VCT. Furthermore the VCT programme should make couple testing more attractive, for example, by improving confidentiality and using the home based VCT approach [##REF##16885775##11##].</p>", "<title>Health system factors</title>", "<p>The health system factors that influence VCT use among men were identified during the quantitative and qualitative studies. The most important concerns were access to VCT services, confidentiality and quality of HIV results [##REF##15746219##12##]. Access to VCT services and to health services in general is a nationwide problem [##UREF##9##37##]. The concerns of access overlap with those for confidentiality. Confidentiality was a major issue among the men, to the extent that many would prefer to test for HIV in a distant clinic where the staff did not know them, but which of course would raise issues of access. As has been reported elsewhere, confidentiality is an important factor that may reduce VCT utilization [##REF##15117300##30##,##REF##8970692##38##,##REF##9487418##39##]. Stigma and confidentiality are closely linked and the greater the stigma of a condition, the greater the need for confidentiality. Our findings suggest that there is strong stigma of HIV/AIDS in Bukonzo West prompting the men to seek VCT services that provide greater confidentiality, even if it means seeking services further away and at a higher cost.</p>", "<p>Men also have concerns about the quality of HIV results. This concern is not about the performance of the HIV test but rather about the integrity of the health workers in providing accurate HIV results. Specifically, men fear that health workers are bribed to falsify HIV results. To complicate the situation even more, men do not believe in discordant HIV results and view this as further evidence of corruption. Another angle to the issue of quality is the high drop out between pre-test counselling and HIV testing. This finding suggests that the group counselling that is routinely offered to the men in clinics may not be effective and there is need for one-to-one HIV counselling.</p>", "<title>Limitations of the study</title>", "<p>Household size could theoretically have biased the sampling strategy towards men in households with fewer eligible men. However, this was highly unlikely because of the social cultural practices of the Bakonzo. In most cases, a household contains one adult male because male children who are of age (usually 18 years or more) are not allowed to live under the same roof as their parents and have to build their own homes.</p>", "<p>Non-response was estimated to be 14% and may have introduced bias in the measurement of the prevalence of VCT and the associated behaviours. Over sampling in all the selected villages (clusters) by randomly selecting 25 respondents instead of the 21 (initially stated) was done in order to increase the sample size.</p>", "<p>The study was community-based and heavily relied on the self-report by the respondents. This may have led to reporting bias because of the reluctance to disclose sensitive behaviours such as sexual activity. The lack of an association between sexual behaviour and VCT use in our study may reflect the problem of measuring sexual behaviour. It is also possible that some respondents reported HIV testing behaviours, which they thought were socially desirable but that were not necessarily factual. In addition, recall bias may have occurred particularly with frequently occurring activities.</p>" ]
[ "<title>Conclusion</title>", "<p>Our study showed that VCT use among men in Bukonzo West, Kasese district was low. The major barriers to VCT use among men were poor utilization of VCT services due to poor access, stigma and confidentiality of services. In order to increase VCT use among men, the VCT programme needs to address HIV stigma and improve access and confidentiality of VCT services. Among the more promising interventions are the use of routine counselling and testing for HIV of patients seeking health care in health units, home based VCT programmes, and mainstreaming of HIV counselling and testing services in community development programmes.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Voluntary HIV counselling and testing (VCT) is one of the key strategies in the prevention and control of HIV/AIDS in Uganda. However, the utilization of VCT services particularly among men is low in Kasese district. We therefore conducted a study to determine the prevalence and factors associated with VCT use among men in Bukonzo West health sub-district, Kasese district.</p>", "<title>Methods</title>", "<p>A population-based cross-sectional study employing both quantitative and qualitative techniques of data collection was conducted between January and April 2005. Using cluster sampling, 780 men aged 18 years and above, residing in Bukonzo West health sub-district, were sampled from 38 randomly selected clusters. Data was collected on VCT use and independent variables. Focus group discussions (4) and key informant interviews (10) were also conducted. Binary logistic regression was performed to determine the predictors of VCT use among men.</p>", "<title>Results</title>", "<p>Overall VCT use among men was 23.3% (95% CI 17.2–29.4). Forty six percent (95% CI 40.8–51.2) had pre-test counselling and 25.9% (95%CI 19.9–31.9) had HIV testing. Of those who tested, 96% returned for post-test counselling and received HIV results. VCT use was higher among men aged 35 years and below (OR = 2.69, 95%CI 1.77–4.07), the non-subsistence farmers (OR = 2.37, 95%CI 2.37), the couple testing (OR = 2.37, 95%CI 1.02–8.83) and men with intention to disclose HIV test results to sexual partners (OR = 1.64, 95%CI 1.04–2.60). The major barriers to VCT use among men were poor utilization of VCT services due to poor access, stigma and confidentiality of services.</p>", "<title>Conclusion</title>", "<p>VCT use among men in Bukonzo West, Kasese district was low. In order to increase VCT use among men, the VCT programme needs to address HIV stigma and improve access and confidentiality of VCT services. Among the more promising interventions are the use of routine counselling and testing for HIV of patients seeking health care in health units, home based VCT programmes, and mainstreaming of HIV counselling and testing services in community development programmes.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>FMB participated in the conception, design, and implementation of the study, statistical analysis, interpretation and drafting of manuscript. SNS, SB, JNK and CASK participated in study conception, design, interpretation and drafting of the manuscript. All authors read, edited and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2458/8/263/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>We are indebted to the following people for their invaluable contribution and support that enabled us to bring this project to fruition. Our thanks go to all the staff and students of Makerere University CEU; to all the study participants, the team of interviewers and the translator; and to the family, relatives and friends of FMB for their love, care and perseverance that saw him through school.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Conceptual framework of VCT utilization by men and possible predictor variables among 780 men in Kasese district, Uganda, 2005.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Frequency of voluntary counselling and testing among 780 men in Kasese district, Uganda, 2005</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Variable</bold></td><td align=\"right\"><bold>Total</bold><break/><bold>n (%)</bold></td><td align=\"right\"><bold>Accepted </bold><break/><bold>VCT</bold></td><td align=\"right\"><bold>Percent</bold><break/><bold>(95% CI)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>Overall</bold></td><td align=\"right\"><bold>780</bold></td><td align=\"right\"><bold>182</bold></td><td align=\"right\"><bold>23 (17–29)</bold></td></tr><tr><td align=\"left\"><bold>Age</bold></td><td/><td/><td/></tr><tr><td align=\"left\">≤ 20</td><td align=\"right\">86(11)</td><td align=\"right\">13</td><td align=\"right\">15</td></tr><tr><td align=\"left\">21–29</td><td align=\"right\">237(30)</td><td align=\"right\">81</td><td align=\"right\">34</td></tr><tr><td align=\"left\">30–39</td><td align=\"right\">242(31)</td><td align=\"right\">62</td><td align=\"right\">26</td></tr><tr><td align=\"left\">40–49</td><td align=\"right\">121(16)</td><td align=\"right\">23</td><td align=\"right\">19</td></tr><tr><td align=\"left\">50+</td><td align=\"right\">94(12)</td><td align=\"right\">3</td><td align=\"right\">3</td></tr><tr><td align=\"left\"><bold>Education</bold></td><td/><td/><td/></tr><tr><td align=\"left\">None</td><td align=\"right\">178(23)</td><td align=\"right\">22</td><td align=\"right\">18</td></tr><tr><td align=\"left\">Primary</td><td align=\"right\">309(40)</td><td align=\"right\">56</td><td align=\"right\">18</td></tr><tr><td align=\"left\">Secondary</td><td align=\"right\">196(25)</td><td align=\"right\">53</td><td align=\"right\">27</td></tr><tr><td align=\"left\">Tertiary</td><td align=\"right\">97(12)</td><td align=\"right\">41</td><td align=\"right\">42</td></tr><tr><td align=\"left\"><bold>Religion</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Roman Catholic</td><td align=\"right\">353(45)</td><td align=\"right\">75</td><td align=\"right\">21</td></tr><tr><td align=\"left\">Anglican</td><td align=\"right\">341(44)</td><td align=\"right\">84</td><td align=\"right\">25</td></tr><tr><td align=\"left\">Muslim</td><td align=\"right\">52(07)</td><td align=\"right\">14</td><td align=\"right\">27</td></tr><tr><td align=\"left\">Other</td><td align=\"right\">97(12)</td><td align=\"right\">9</td><td align=\"right\">27</td></tr><tr><td align=\"left\"><bold>Occupation</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Subsistence farmer</td><td align=\"right\">453(58)</td><td align=\"right\">76</td><td align=\"right\">17</td></tr><tr><td align=\"left\">Salaried employee</td><td align=\"right\">119(15)</td><td align=\"right\">47</td><td align=\"right\">40</td></tr><tr><td align=\"left\">Businessman</td><td align=\"right\">105(14)</td><td align=\"right\">31</td><td align=\"right\">30</td></tr><tr><td align=\"left\">Student</td><td align=\"right\">94(12)</td><td align=\"right\">26</td><td align=\"right\">28</td></tr><tr><td align=\"left\">Other</td><td align=\"right\">9(01)</td><td align=\"right\">22</td><td align=\"right\">2</td></tr><tr><td align=\"left\"><bold>Marital status</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Single</td><td align=\"right\">209(27)</td><td align=\"right\">59</td><td align=\"right\">28</td></tr><tr><td align=\"left\">Married</td><td align=\"right\">556(71)</td><td align=\"right\">122</td><td align=\"right\">22</td></tr><tr><td align=\"left\">Separated/divorced</td><td align=\"right\">15(02)</td><td align=\"right\">1</td><td align=\"right\">7</td></tr><tr><td align=\"left\"><bold>Type of marriage</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Monogamy</td><td align=\"right\">432(55)</td><td align=\"right\">101</td><td align=\"right\">23</td></tr><tr><td align=\"left\">Polygamy</td><td align=\"right\">134(17)</td><td align=\"right\">21</td><td align=\"right\">16</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Unadjusted association between independent factors and voluntary counselling and testing among 780 men in Kasese district, Uganda, 2005</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Variable</bold></td><td align=\"right\"><bold>No VCT </bold><break/><bold>n (%)</bold></td><td align=\"right\"><bold>Accepted VCT </bold><break/><bold>n (%)</bold></td><td align=\"right\"><bold>OR (95% CI)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>Age in years</bold></td><td/><td/><td/></tr><tr><td align=\"left\">&gt;35</td><td align=\"right\">253(88)</td><td align=\"right\">36(13)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">≤ 35</td><td align=\"right\">345(70)</td><td align=\"right\">146(30)</td><td align=\"right\">2.97(2.00–4.43)</td></tr><tr><td align=\"left\"><bold>Education</bold></td><td/><td/><td/></tr><tr><td align=\"left\">None/Primary</td><td align=\"right\">399(82)</td><td align=\"right\">88(18)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Secondary/Tertiary</td><td align=\"right\">199(68)</td><td align=\"right\">94(32)</td><td align=\"right\">2.10(1.50–3.00)</td></tr><tr><td align=\"left\"><bold>Religion</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Muslim</td><td align=\"right\">38(73)</td><td align=\"right\">4(27)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Christian</td><td align=\"right\">560(77)</td><td align=\"right\">168(23)</td><td align=\"right\">0.81(0.43–1.54)</td></tr><tr><td align=\"left\"><bold>Occupation</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Subsistence farmer</td><td align=\"right\">526(80)</td><td align=\"right\">135(20)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Other</td><td align=\"right\">72(77)</td><td align=\"right\">47(23)</td><td align=\"right\">2.54(1.68–3.85)</td></tr><tr><td align=\"left\"><bold>Marital status</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Not married</td><td align=\"right\">164(73)</td><td align=\"right\">60(27)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Married</td><td align=\"right\">434(78)</td><td align=\"right\">122(22)</td><td align=\"right\">0.77(0.54–1.10)</td></tr><tr><td align=\"left\"><bold>Type of marriage</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Monogamy</td><td align=\"right\">331(77)</td><td align=\"right\">101(23)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Polygamy</td><td align=\"right\">133(84)</td><td align=\"right\">21(16)</td><td align=\"right\">0.61(0.36–1.02)</td></tr><tr><td align=\"left\"><bold>Fear of taking an HIV test</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">327(71)</td><td align=\"right\">131(29)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">271(84)</td><td align=\"right\">51(16)</td><td align=\"right\">0.47(0.33–0.67)</td></tr><tr><td align=\"left\"><bold>Fear of divorce or separation</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">144(82)</td><td align=\"right\">32(18)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">127(87)</td><td align=\"right\">19(13)</td><td align=\"right\">0.67(0.36–1.25)</td></tr><tr><td align=\"left\"><bold>Lack of confidentiality</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">245(83)</td><td align=\"right\">50(17)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">26(96)</td><td align=\"right\">1(04)</td><td align=\"right\">0.19(0.03–1.42)</td></tr><tr><td align=\"left\"><bold>Willingness to test for HIV with partner</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">64(94)</td><td align=\"right\">4(06)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">534(75)</td><td align=\"right\">178(25)</td><td align=\"right\">5.33(1.92–14.85)</td></tr><tr><td align=\"left\"><bold>Willingness to disclose HIV results to partner</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">181(86)</td><td align=\"right\">30(14)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">417(73)</td><td align=\"right\">152(27)</td><td align=\"right\">2.19(1.43–3.38)</td></tr><tr><td align=\"left\"><bold>Would feel comfortable at VCT site</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">117(88)</td><td align=\"right\">15(11)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">481(74)</td><td align=\"right\">167(26)</td><td align=\"right\">2.71(1.54–4.77)</td></tr><tr><td align=\"left\"><bold>Wanting to know HIV sero-status</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">115(54)</td><td align=\"right\">98(46)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">348(86)</td><td align=\"right\">55(14)</td><td align=\"right\">0.19(0.13–0.27)</td></tr><tr><td align=\"left\"><bold>Monthly income (Uganda Shillings)</bold></td><td/><td/><td/></tr><tr><td align=\"left\">≤ 50,000</td><td align=\"right\">482(79)</td><td align=\"right\">131(21)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">&gt;50,000</td><td align=\"right\">90(67)</td><td align=\"right\">45(33)</td><td align=\"right\">1.84(1.23–2.76)</td></tr><tr><td align=\"left\"><bold>Number of sexual partners</bold></td><td/><td/><td/></tr><tr><td align=\"left\">One</td><td align=\"right\">382(76)</td><td align=\"right\">141(24)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Two or more</td><td align=\"right\">216(79)</td><td align=\"right\">58(21)</td><td align=\"right\">0.83(0.58–1.18)</td></tr><tr><td align=\"left\"><bold>Had sex outside marriage in past 1 year</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">457(78)</td><td align=\"right\">129(22)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">141(73)</td><td align=\"right\">53(27)</td><td align=\"right\">1.33(0.92–1.93)</td></tr><tr><td align=\"left\"><bold>Used a condom on last sexual encounter</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">32(71)</td><td align=\"right\">13(29)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">109(73)</td><td align=\"right\">40(27)</td><td align=\"right\">0.90(0.43–1.20)</td></tr><tr><td align=\"left\"><bold>Social belief exists</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">481(77)</td><td align=\"right\">143(23)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">117(75)</td><td align=\"right\">39(25)</td><td align=\"right\">1.12(0.75–1.68)</td></tr><tr><td align=\"left\"><bold>Willingness to seek VCT</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">135(82)</td><td align=\"right\">29(18)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">463(76)</td><td align=\"right\">153(25)</td><td align=\"right\">1.54(0.99–2.39)</td></tr><tr><td align=\"left\"><bold>Distance to nearest VCT site 5 km or less</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">187(77)</td><td align=\"right\">55(23)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">411(76)</td><td align=\"right\">127(24)</td><td align=\"right\">1.05(0.73–1.50)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Logistic regression of independent factors associated with acceptance of VCT among 780 men in Kasese district, Uganda, 2005</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Variable</bold></td><td align=\"right\"><bold>No VCT </bold><break/><bold>n (%)</bold></td><td align=\"right\"><bold>Accepted VCT </bold><break/><bold>n (%)</bold></td><td align=\"right\"><bold>OR </bold><break/><bold>(95% CI)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>Age in years</bold></td><td/><td/><td/></tr><tr><td align=\"left\">&gt;35</td><td align=\"right\">253(88)</td><td align=\"right\">36(13)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">≤ 35</td><td align=\"right\">345(70)</td><td align=\"right\">146(30)</td><td align=\"right\">2.69(1.77–4.07)</td></tr><tr><td align=\"left\"><bold>Occupation</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Subsistence farmer</td><td align=\"right\">526(80)</td><td align=\"right\">135(20)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Other</td><td align=\"right\">72(77)</td><td align=\"right\">47(23)</td><td align=\"right\">2.37(1.52–3.71)</td></tr><tr><td align=\"left\"><bold>Fear of taking an HIV test</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">327(71)</td><td align=\"right\">131(29)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">271(84)</td><td align=\"right\">51(16)</td><td align=\"right\">0.54(0.37–0.79)</td></tr><tr><td align=\"left\"><bold>Willingness to test for HIV with partner</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">64(94)</td><td align=\"right\">4(06)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">534(75)</td><td align=\"right\">178(25)</td><td align=\"right\">3.01(1.02–8.83)</td></tr><tr><td align=\"left\"><bold>Willingness to disclose HIV results to partner</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">181(86)</td><td align=\"right\">30(14)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">417(73)</td><td align=\"right\">152(27)</td><td align=\"right\">1.64(1.04–2.6)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Logistic regression of independent factors associated with pre-test counselling among 780 men in Kasese district, Uganda, 2005</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Variable</bold></td><td align=\"right\"><bold>Pre-test counselled </bold><break/><bold>n (%)</bold></td><td align=\"right\"><bold>Not pre-test counselled </bold><break/><bold>n (%)</bold></td><td align=\"right\"><bold>OR (95% CI)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>To know HIV sero-status</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">411(65.2)</td><td align=\"right\">219(34.8)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">10(6.7)</td><td align=\"right\">140(93.3)</td><td align=\"right\">12.25 (6.09–24.64)</td></tr><tr><td align=\"left\"><bold>Feeling comfortable with VCT site</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">91(68.9)</td><td align=\"right\">41(31.1)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">330(50.9)</td><td align=\"right\">318(49.1)</td><td align=\"right\">1.75 (1.09–2.83)</td></tr><tr><td align=\"left\"><bold>Fear of being stigmatized</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">312(52.3)</td><td align=\"right\">284(47.7)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">109(59.2</td><td align=\"right\">75(40.8)</td><td align=\"right\">1.84 (1.10–3.08)</td></tr><tr><td align=\"left\"><bold>Occupation</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Subsistence farmer</td><td align=\"right\">279(61.6)</td><td align=\"right\">178(38.4)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Other</td><td align=\"right\">142 (43.4)</td><td align=\"right\">185(56.6)</td><td align=\"right\">1.90 (1.35–2.67)</td></tr><tr><td align=\"left\"><bold>Reason for choosing VCT site</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Other</td><td align=\"right\">417(62.5)</td><td align=\"right\">250(37.5)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Confidentiality</td><td align=\"right\">4(3.5)</td><td align=\"right\">109(96.5)</td><td align=\"right\">15.91 (5.54–45.74)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Logistic regression of independent factors associated with complete VCT among 369 men seeking VCT in Kasese district, Uganda, 2005</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Variable</bold></td><td align=\"right\"><bold>Pre-test counselling only </bold><break/><bold>n (%)</bold></td><td align=\"right\"><bold>Complete VCT </bold><break/><bold>n (%)</bold></td><td align=\"right\"><bold>OR (95% CI)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>Education in years</bold></td><td/><td/><td/></tr><tr><td align=\"left\">0 – 7</td><td align=\"right\">131(59.5)</td><td align=\"right\">89(40.5)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">More than 7</td><td align=\"right\">55(36.9)</td><td align=\"right\">94(63.1)</td><td align=\"right\">2.9 (1.1–7.8)</td></tr><tr><td align=\"left\"><bold>To know HIV sero-status</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">181(79)</td><td align=\"right\">48(21)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">5(3.6)</td><td align=\"right\">135(96.4)</td><td align=\"right\">84.6 (23.3 – 306.4)</td></tr><tr><td align=\"left\"><bold>Fear of inaccurate HIV test result</bold></td><td/><td/><td/></tr><tr><td align=\"left\">No</td><td align=\"right\">152(46.9)</td><td align=\"right\">172(53.1)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Yes</td><td align=\"right\">34(75.6)</td><td align=\"right\">11(24.4)</td><td align=\"right\">30.7 (2.1 – 457.4)</td></tr><tr><td align=\"left\"><bold>Reason for choosing VCT site</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Other</td><td align=\"right\">184(70.8)</td><td align=\"right\">76(29.2)</td><td align=\"right\">1.0</td></tr><tr><td align=\"left\">Confidentiality</td><td align=\"right\">2(1.8)</td><td align=\"right\">107(98.2)</td><td align=\"right\">53.1 (10.8–261.8)</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
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[ "<graphic xlink:href=\"1471-2458-8-263-1\"/>" ]
[]
[{"collab": ["MOH"], "source": ["Health Sector Strategic Plan: Mid Term Report"], "year": ["2003"], "publisher-name": ["Kampala: Ministry of Health Uganda"]}, {"collab": ["MOH"], "source": ["Uganda HIV/AIDS Sero-Behavioural Survey 2004\u20132005"], "year": ["2006"], "publisher-name": ["Kampala: Ministry of Health and ORC Macro, Calverton, Maryland, USA"]}, {"collab": ["World Health Organization"], "source": ["The Health Sector Response to HIV/AIDS: Coverage of Selected Services in 2001. Preliminary Assessment"], "year": ["2002"], "publisher-name": ["Geneva: World Health Organization"]}, {"collab": ["MOH"], "source": ["Uganda National Policy Guidelines for HIV Voluntary Counseling and Testing"], "year": ["2003"], "publisher-name": ["Kampala: Ministry of Health"]}, {"surname": ["Byomire"], "given-names": ["H"], "article-title": ["Prevalence and factors associated with male involvement in PMTCT, Mulago Hospital, Uganda"], "source": ["Dissertation"], "year": ["2003"], "publisher-name": ["Kampala: Makerere University"]}, {"collab": ["Uganda Bureau of Statistics"], "source": ["2002 Population and Housing Census Report for Uganda"], "year": ["2003"], "publisher-name": ["Entebbe: Ministry of Finance, Planning and Economic Development, Uganda"]}, {"collab": ["UBOS, ORC Macro"], "article-title": ["Uganda Demographic and Health Survey 2000\u20132001"], "source": ["Kampala"], "year": ["2001"]}, {"collab": ["UNAIDS"], "source": ["Behavioral and social factors driving sexual transmission of HIV/AIDS"], "year": ["2000"], "publisher-name": ["Geneva: UNAIDS"]}, {"surname": ["Baingana", "Choi"], "given-names": ["G", "K"], "article-title": ["Female partners of AIDS patients in Uganda: reported knowledge, perceptions and plans"], "source": ["AIDS"], "year": ["1995"], "volume": ["9"], "fpage": ["15"], "lpage": ["19"]}, {"collab": ["Uganda Bureau of Statistics"], "source": ["Uganda Demographic and Health Survey 2006"], "year": ["2006"], "publisher-name": ["Kampala, Uganda: Bureau of Statistics, Kampala, Uganda and ORC Macro International Inc., Calverton, Maryland, USA"]}]
{ "acronym": [], "definition": [] }
39
CC BY
no
2022-01-12 14:47:26
BMC Public Health. 2008 Jul 30; 8:263
oa_package/84/f4/PMC2529297.tar.gz
PMC2529298
18700043
[ "<title>Background</title>", "<p>Vast amounts of three-dimensional (3D) protein data from structural genomic studies and other individual efforts have been added to our knowledge, thereby enhancing our understanding of protein structures. To date, only two extremes of protein structural data have been studied. One extreme includes local features of proteins: those of short protein segments, typically of 10 residues long or less. The other extreme includes global features of proteins: protein folds or structural domains.</p>", "<p>Regarding the short protein segments, abundant research examples exist partly because of the existence of variations of methods to analyze the local features of proteins. Various measures, such as RMSDs after structural superposition [##REF##8481817##1##, ####REF##8229095##2##, ##REF##10899788##3####10899788##3##], <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>atomic distances coupled with the torsion angles [##REF##1438182##4##,##REF##2381907##5##], dihedral angles [##REF##11025540##6##], and so on have been used to define the conformational similarity of protein segments. Different clustering techniques, such as <italic>k</italic>-means clustering [##REF##9061789##7##,##REF##16405736##8##], hierarchical methods [##REF##2342110##9##], competitive learning [##REF##11025540##6##,##REF##8931122##10##], and other methods [##REF##12577264##11##], have been used to describe the organization of the segments' conformational space. The abundance of research results in this area is also partly attributable to various applications of the clustering results of the short segments. A set of representatives from the resulting clusters are often called structural building blocks (SBBs). Even when using different procedures, clustering resolutions of SBBs can be categorized into only a few levels depending mainly on their respective applications, such as structural modeling, verification, comparison, and prediction [##REF##11025540##6##,##UREF##0##12##]. The most dominant cluster of the short segments, which is common in all studies, corresponds to α-helices, whereas the variability of β-strands is observed at the high-resolution clustering. Regarding global features of proteins, understanding of their organization and analysis of the protein-fold (or structural domain) space studies are progressing well.</p>", "<p>As reviewed recently [##REF##16678402##13##], both hierarchical and continuous aspects of fold space have been realized. Regarding hierarchical classification, widely used databases such as CATH [##REF##9309224##14##] and SCOP [##REF##7723011##15##] have been constructed. Other databases such as FSSP [##REF##1304898##16##] and VAST [##REF##8804824##17##] have been developed. They are based on continuous measurements of protein structural similarity. Several studies have provided insights into the nature of fold space. Holm and Sander first described the conformational distribution of protein folds in a <italic>fold universe </italic>with multi-dimensional scaling methods based on an all-on-all comparison using the Dali program [##REF##8662544##18##]. Using the same measurement, Hou <italic>et al</italic>. [##REF##12606708##19##] showed visual representations of the protein fold universe and identified three major components which characterize the fold space: secondary structure compositions, chain topologies, and the protein domain size.</p>", "<p>Compared to these two extremes, limited surveys have been done on the conformational space of medium size segments between protein short segments and folds. Specifically, supersecondary structures such as α-hairpin, βαβ-unit, and β-hairpin are typical structural motifs of medium size; those motifs have been analyzed. For example, Salem <italic>et al</italic>. reported that most superfolds contain a higher proportion of their α-helical or β-strand residues in one such supersecondary structure [##REF##10222204##20##]. Szustakowski <italic>et al</italic>. built a dictionary of supersecondary structures [##REF##16204127##21##]. Kurgan and Kedarisetti studied regularity among twilight zone protein structures at the level of the sequence segments that correspond to the secondary structure fragments of varying length [##REF##17115254##22##]. However, the organization and statistical properties of the whole conformational space of medium-to-long segments remain unclear. Statistical and systematic analyses should be done on the 'segment universe' from short to long lengths to bridge this gap.</p>", "<p>Our previous study identified structural clusters and visualized the uneven distribution of short segments in the conformational spaces of 6–22 residues, where known and novel secondary-structure motifs are distributed as isolated clusters [##REF##15802651##23##]. The general features of the segment distribution were consistent for these lengths. However, the question we sought to answer is: Do spaces of long segments differ from those of short segments? In this study, we explore the relationships between the conformational distribution of segments and their length: 10–50 residues, thereby providing a global view of a 'segment universe' and showing critical dual changes (i.e. dual transitions) of the distribution shape in the conformational space of short to long segments. The critical changes might reflect changes of the protein structures' organization. Therefore, the present results suggest the adequacy and the possibility of further progress of the hierarchical treatment used in the recent <italic>de novo </italic>structure prediction methods. Furthermore, by comparing conformational components among structural classes (i.e., all-α, all-β, α/β, and α+β), we demonstrate the specificity and generality of protein fold classes.</p>" ]
[ "<title>Methods</title>", "<title>Preparing the segment libraries</title>", "<p>One representative from each fold group of the SCOP database (ver. 1.63) [##REF##7723011##15##] was chosen to obtain a segment library without a bias of usage of the folds. The representatives cover the four major structural classes (all-α, all-β, α+β, and α/β), because we are interested in and specifically examine characterization of the nature of segments embedded in usual size globular proteins. Small proteins of less than 50 residues and non-single chain proteins with less than 100 residues were excluded, as were membrane proteins. It is expected that those proteins possess different structural properties from those of usual size globular proteins and induce biased results. In all, 600 representatives were used for this study (all-α, 150; all-β, 116; α+β, 219; α/β, 115; see Additional File ##SUPPL##4##5##). Dividing the protein structures into segments with a sliding window by one residue along the sequence generated a segment library of arbitrary length. We prepared a segment library for each length of 10–50 residues to generate conformational spaces of short-to-long segments. In such cases, segments with incomplete coordinate data (e.g., having an unusual covalent-bond length or lacking main-chain atoms) were excluded. Furthermore, to elucidate differences among the conformational spaces derived from the four major structural classes, we generated a segment library for each class.</p>", "<title>Construction and visualization of conformational space</title>", "<p>We previously reported a method for constructing and visualizing the conformational space of protein segments using principal component analysis based on intra-segment <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>atomic distances [##REF##15802651##23##]. Briefly, atomic distances of all <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>pairs for each segment in a segment library of an arbitrary length were calculated first. A distance is designated as <italic>q</italic><sub><italic>i</italic></sub>, where <italic>i </italic>is the index for the <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>pair, <italic>i </italic>= 1, ..., <italic>n</italic>(<italic>n </italic>- 1)/2, and <italic>n </italic>is the segment length, as expressed by the number of residues in the segment. Subsequently, a set of eigenvectors and eigenvalues were obtained by diagonalizing a variance-covariance matrix, <bold>C</bold>, that was calculated as <bold>C</bold><sub><italic>ij </italic></sub>= (&lt;(<italic>q</italic><sub><italic>i </italic></sub>- &lt;<italic>q</italic><sub><italic>i</italic></sub>&gt;)(<italic>q</italic><sub><italic>j </italic></sub>- &lt;<italic>q</italic><sub><italic>j</italic></sub>&gt;)&gt; = &lt;(<italic>q</italic><sub><italic>i</italic></sub><italic>q</italic><sub><italic>j </italic></sub>- <italic>q</italic><sub><italic>i</italic></sub>&lt;<italic>q</italic><sub><italic>j</italic></sub>&gt;- &lt;<italic>q</italic><sub><italic>i</italic></sub>&gt;<italic>q</italic><sub><italic>j </italic></sub>+ &lt;<italic>q</italic><sub><italic>i</italic></sub>&gt;&lt;<italic>q</italic><sub><italic>j</italic></sub>&gt;)&gt; = &lt;<italic>q</italic><sub><italic>i</italic></sub><italic>q</italic><sub><italic>j</italic></sub>&gt;- &lt;<italic>q</italic><sub><italic>i</italic></sub>&gt;&lt;<italic>q</italic><sub><italic>j</italic></sub>&gt;- &lt;<italic>q</italic><sub><italic>i</italic></sub>&gt;&lt;<italic>q</italic><sub><italic>j</italic></sub>&gt; + &lt;<italic>q</italic><sub><italic>i</italic></sub>&gt;&lt;<italic>q</italic><sub><italic>j</italic></sub>&gt; =) &lt;<italic>q</italic><sub><italic>i</italic></sub><italic>q</italic><sub><italic>j</italic></sub>&gt;- &lt;<italic>q</italic><sub><italic>i</italic></sub>&gt;&lt;<italic>q</italic><sub><italic>j</italic></sub>&gt;, where the average &lt;...&gt; is taken over the segments. Two equations, <bold>C<italic>v</italic></bold><sub><italic>i </italic></sub>= <italic>λ</italic><sub><italic>i</italic></sub><bold><italic>v</italic></bold><sub><italic>i </italic></sub>and <bold><italic>v</italic></bold><sub><italic>i</italic></sub>·<bold><italic>v</italic></bold><sub><italic>j </italic></sub>= δ<sub><italic>ij</italic></sub>, are satisfied. Eigenvectors with larger eigenvalues are more important in the study of the conformational varieties of the segments. Eigenvalues are arranged in descending order: <italic>λ</italic><sub><italic>i </italic></sub>&gt; <italic>λ</italic><sub><italic>j </italic></sub>if <italic>i </italic>&lt;<italic>j</italic>. The contribution ratio of the <italic>i</italic>-th PCA element (i.e. the <italic>i</italic>-th eigenvector) to the whole conformational distribution is given as <italic>Q</italic><sub><italic>i </italic></sub>= <italic>λ</italic><sub><italic>i</italic></sub>/Σ<sub><italic>k</italic></sub><sup><italic>all </italic></sup><italic>λ</italic><sub><italic>k</italic></sub>. The eigenvectors, which are called <bold>PC<sup>x</sup>1</bold>, <bold>PC<sup>x</sup>2</bold>, <bold>PC<sup>x</sup>3</bold>, ...etc., were used as conformational axes to construct a segment conformational space, a PCA space, in which <italic>x </italic>indicates a segment dataset: <italic>x </italic>= α, β, α/β, α +β, or all). The indicator \"<italic>x </italic>= all\" is given when conformational axes are generated by the whole segment dataset. The origin of the PCA space is set on the average <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>atomic distances: &lt;<bold><italic>q</italic></bold>&gt; = [&lt;<italic>q</italic><sub>1</sub>&gt;, &lt;<italic>q</italic><sub>2</sub>&gt;, &lt;<italic>q</italic><sub>3</sub>&gt;, ..., &lt;<italic>q</italic><sub><italic>n</italic></sub>&gt;]. This enables ready comparison of conformational distributions between constructed universes. Any position (i.e. any segment structure) in the PCA space can be expressed using a linear combination of eigenvectors as <italic>c</italic><sub><italic>k </italic></sub>= Σ<sub><italic>n</italic></sub><sup><italic>all </italic></sup>(<bold><italic>q </italic></bold>- &lt;<bold><italic>q</italic></bold>&gt;)·<bold><italic>v</italic></bold><sub><italic>k </italic></sub><italic>λ</italic><sub><italic>k</italic></sub><sup>1/2</sup>, where <italic>c</italic><sub><italic>k </italic></sub>is a coordinate (i.e. projection of <bold><italic>q</italic></bold>) on the PC axis <italic>k</italic>. Using the first three eigenvectors (<bold>PC<sup>x</sup>1</bold>, <bold>PC<sup>x</sup>2</bold>, <bold>PC<sup>x</sup>3</bold>), a three-dimensional (3D) PCA space can be constructed.</p>", "<p>We defined a vector, <bold><italic>r</italic></bold>, to express the position of each segment in the 3D PCA space: <bold><italic>r </italic></bold>= [<italic>c</italic><sub>1</sub>, <italic>c</italic><sub>2</sub>, <italic>c</italic><sub>3</sub>]. After projection of the segments on the 3D PCA space, the distribution of segments in the 3D PCA space was visualized using the following procedure. The 3D space was divided into <italic>N </italic>bins (total <italic>N</italic><sup>3 </sup>cubes). The bin size was defined as (max [<italic>c</italic><sub>1</sub>] - min [<italic>c</italic><sub>1</sub>])/<italic>N</italic>, where <italic>N </italic>= 36, and max [<italic>c</italic><sub>1</sub>] and min [<italic>c</italic><sub>1</sub>] respectively signify the maximum and minimum of the coordinates of the segments along the first principal component axis. The number (i.e. frequency) of segments detected in a cube represents the density (i.e. probability) of segments to be found in the cube. The density of each cube, ρ was normalized by the maximum density, ρ<sub>max </sub>among the cubes so that the maximal value of normalized density (we call this <italic>density </italic>in the text) is set to 1 (refer to eq. (3) in [##REF##15802651##23##]). Four levels of contour surfaces (i.e. iso-density surfaces) were depicted to visualize the 3D PCA space. The density values for those surfaces were set respectively as 0.005, 0.01, 0.1, and 0.35.</p>", "<p>We also separately constructed the universe for four structural classes to assess differences among their conformational spaces. For this study, we specifically examined the first 10 PC axes of each structural class because the 10 PC axes are more important than the other axes with respect to capturing the differences in the conformational axes. Although the eigenvectors from the same structural class are mutually uncorrelated (i.e., <bold><italic>v</italic></bold><sup><italic>x</italic></sup><sub><italic>i</italic></sub>·<bold><italic>v</italic></bold><sup><italic>x</italic></sup><sub><italic>j </italic></sub>= 0, where <italic>i </italic>≠ <italic>j </italic>and <italic>x </italic>= α, β, α/β, or α+β), the eigenvectors from different structural classes might have some correlation (i.e., <bold><italic>v</italic></bold><sup><italic>x</italic></sup><sub><italic>i</italic></sub>·<bold><italic>v</italic></bold><sup><italic>y</italic></sup><sub><italic>j </italic></sub>≠ 0, where <italic>x </italic>≠ <italic>y</italic>). The PC axis is defined as the conformational component specific to the structural class when a PC axis from a structural class has no similarity to the first 20 PC axes from the other structural classes with a correlation coefficient &gt; 0.8 (i.e. <bold><italic>v</italic></bold><sup><italic>x</italic></sup><sub><italic>i</italic></sub>·<bold><italic>v</italic></bold><sup><italic>y</italic></sup><sub><italic>j </italic></sub>&gt; 0.8).</p>" ]
[ "<title>Results</title>", "<title>Transitions of segment distribution: short, medium, and long segments</title>", "<p>The <italic>coverage </italic>of segments in cluster(s) was calculated as described below. A densely populated region in the 3D principal component analysis (PCA) space was defined as a cluster [##REF##15802651##23##]. Given a density threshold, the segments are classifiable into two groups: those in regions of a density larger than the threshold and those outside the regions. The coverage of segments in clusters is defined as a ratio of the segments in the regions to all the segments.</p>", "<p>Figure ##FIG##0##1a## portrays the coverage of segments versus the density threshold for the conformational spaces of 10, 20, 30, 40, and 50 residue lengths. The coverage curves exhibited a transition from concave shapes for short lengths (10 and 20 residues long) to convex ones for long lengths (30, 40, and 50 residues long). Notably, the differences of coverage at a density of 0.2 or less show a transition between the short and long segments. For instance, at a density of 0.1, the coverage is only 16.3% for 10 residues, although the coverage is greater than 50% for 30 residues. In addition, at a density of 0.01, the coverage for 10 residues is 45.6%, although coverage for 30 residues is 91.9%. These quantitatively indicate that the density gradient of the conformational space changes markedly with segment elongation.</p>", "<p>Further analyses of the coverage graphs between the short and long segments were meaningful to discover the boundaries of distribution changes. Figure ##FIG##0##1b## shows coverage curves for lengths of 21–30 residues. The dual and critical transitions, with an intermediate phase for segment lengths of 23–26 residues, can be recognized clearly, as presented in Fig. ##FIG##0##1b##. The transitions at intermediate length are also characterized by the distributional alteration of the radius of gyration of segments in the populated region with density of 0.10–0.35 (Fig. ##FIG##1##2##). To adjust the effect of different segment lengths, we defined here the relative score (<italic>F_Rg</italic>) of the radius of gyration for a segment as (<italic>Rg</italic><sub>(<italic>i</italic>,<italic>j</italic>) </sub>- Min <italic>Rg</italic><sub>(<italic>j</italic>)</sub>)/(Max <italic>Rg</italic><sub>(<italic>j</italic>) </sub>- Min <italic>Rg</italic><sub>(<italic>j</italic>)</sub>), where <italic>Rg</italic><sub>(<italic>i</italic>,<italic>j</italic>) </sub>denotes the radius of gyration of a segment <italic>i </italic>with length <italic>j</italic>, and where Max <italic>Rg</italic><sub>(<italic>j</italic>) </sub>and Min <italic>Rg</italic><sub>(<italic>j</italic>) </sub>represent the maximal and minimal radius of gyration of the entire segment dataset with length <italic>j</italic>. Based on these observations, the segment length is categorized into the following three groups: short (10–22 residues), medium (23–26 residues), and long (27–50 residues). We were able to show that changes in the density gradient are associated with distributional alterations in the segment universe in subsequent analyses of visualizing the 3D PCA space. In fact, the difference in the coverage between lengths of 10 and 30 residues was attributable to the increase in the volume for the most populated region, as discussed below. The typical global images of segment universes from the three categories are depicted in Fig. ##FIG##2##3d##. The segment universes here were generated by the first three principal components derived from the entire segment dataset: <italic>PC</italic><sup><italic>all</italic></sup><italic>1</italic>, <italic>PC</italic><sup><italic>all</italic></sup><italic>2</italic>, and <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>(see <italic>Methods</italic>).</p>", "<title>Short length (10–22 residues long)</title>", "<p>The conformational space of short segments showed a distribution with an extreme density gradient that originated from secondary structure clusters: α-helix and β-strand clusters were discriminated using a density of 0.01 (shown in orange in Fig. ##FIG##2##3a##). Between the lengths of 10 and 20 residues, spatial arrangements of the segment distribution, especially for α-helical, β-strand, and β-hairpin clusters, were conserved in short conformational spaces. The highly populated core of the α-helix cluster exhibited a density of 0.1 (shown as magenta in Fig. ##FIG##2##3a##), consisting of completed α-helical segments. The surrounding area of the central region consisted of various types of helical conformations including helix-capping motifs [##UREF##0##12##]. The central region of the β-strand cluster consisted of fully extended segments that originated mainly from β-sheets and loop regions. The β-hairpin conformations were separated into several clusters at a density of 0.005. Then they were discriminated using the coordinate <italic>c</italic><sub>2 </sub>along <italic>PC</italic><sup><italic>all</italic></sup><italic>2 </italic>(see <italic>Methods </italic>for the definition of <italic>c</italic><sub>2</sub>). The β-hairpin clusters showed a symmetrical relationship related to the N-terminal and C-terminal halves. They were arranged symmetrically around an edge of segment universes of short length.</p>", "<title>Medium length (23–26 residues long)</title>", "<p>The segment distribution for medium lengths differed from that for short lengths. The distributional change from short to medium lengths is characterized using a diminishing β-strand cluster and a growing α-helix cluster. The overall distribution was shortened in the direction of <italic>PC</italic><sup><italic>all</italic></sup><italic>1</italic>, and enlarged in the direction of <italic>PC</italic><sup><italic>all</italic></sup><italic>2 </italic>and <italic>PC</italic><sup><italic>all</italic></sup><italic>3</italic>. In the segment universe of 26 residues, the α-helix cluster was discriminated using a density of 0.1 (magenta in Fig. ##FIG##2##3b##). Interestingly, the shape of the α-helix cluster was a ring (designated as a <italic>helix ring cluster</italic>). The helix ring cluster that is specific to the medium-length universe consisted not only of the extended α-helices but also of various α-helical conformations, as presented in the inset of Fig. ##FIG##2##3b##. This cluster included conformations that had originated mainly from all-α, α/β, and α +β proteins (Fig. ##FIG##3##4a##). The average content of the α-helical residues per segment in the helix ring cluster was about 50% (Fig. ##FIG##3##4b##); 24.9% of all segments were included within the helix ring cluster. The long-α-helical segments, whose conformation was not compact, were located near the origin of the conformational space (red in Fig. ##FIG##2##3b##). In contrast, the α-hairpin conformations with a small radius of gyration were located on the opposite side of the position on <italic>PC</italic><sup><italic>all</italic></sup><italic>1</italic>. The various α-hairpin conformations with the different turn positions were located symmetrically along <italic>PC</italic><sup><italic>all</italic></sup><italic>2</italic>. For medium lengths, the β-strand clusters were diminished because long extended β-strands are rarely found in proteins. The β-hairpin conformations were located symmetrically along <italic>PC</italic><sup><italic>all</italic></sup><italic>2</italic>, although the cluster separation of β-hairpins was not clear in medium lengths.</p>", "<title>Long length (27–50 residues long)</title>", "<p>Conformational spaces for the long lengths were further shortened in the direction of <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>and enlarged in that of <italic>PC</italic><sup><italic>all</italic></sup><italic>3</italic>. The segment distribution converged on a large populated region that exhibited a density of 0.1 (magenta in Fig. ##FIG##2##3c##) in the conformational space. With a length of 30 residues, there were two clusters consisting of compact segments and long α-helical segments, respectively, with densities of 0.35 (red in Fig. ##FIG##2##3c##) in the populated region. The emergence of the compact-segment cluster was attributable to an increase in various types of segments with a small radius of gyration (see inset of Fig. ##FIG##2##3c##). Various types of conformations are mixed up in the compact-segment cluster. The α-hairpins are derived mainly from all-α proteins. The compact β-sheet structures are derived mainly from all-β proteins. Compact conformations of other types are derived from α/β and α +β proteins (Fig. ##FIG##3##4c##). About 2% of all segments were included in the compact-segment cluster for 27-residue length. In contrast, long α-helical segments with a large radius of gyration were located on the opposite side of the cluster of the compact segments along the <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>axis. For lengths greater than 30 residues, the proportion of the conformations with a small radius of gyration in the compact-segment cluster increased rapidly to around 14% for 50-residue lengths. Those conformations were derived from various folds (Fig. ##FIG##3##4c##). The supersecondary structures, such as βαβ units and β-sheets, were included in the compact-segment cluster (Fig. ##FIG##3##4d##).</p>", "<title>Contribution ratios of principal axes</title>", "<p>Distributional alterations were observed associated with the changes of segment length. For principal component analyses, the contribution ratios (see <italic>Methods </italic>for the contribution ratios) of the principal components (i.e. PC axes) to the entire distribution indicate how well the PC axes can cover the variation in the original data. Figure ##FIG##4##5## portrays contribution ratios of the first five PC axes (<italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>– <italic>PC</italic><sup><italic>all</italic></sup><italic>5</italic>) for segment lengths of 10–50 residues. Even with a length of 43 residues, the cumulative contribution ratio of the first three PC axes, <italic>Q</italic><sub>123 </sub>(= <italic>Q</italic><sub>1 </sub>+ <italic>Q</italic><sub>2 </sub>+ <italic>Q</italic><sub>3</sub>), was greater than 60%, although <italic>Q</italic><sub>123 </sub>decreased constantly with increased segment length. Each of <italic>Q</italic><sub>4 </sub>and <italic>Q</italic><sub>5 </sub>was always less than 8%. The contribution ratios for higher-order PC axes than <italic>PC</italic><sup><italic>all</italic></sup>5 did not exceed 5% for the examined segment lengths. Therefore, it is sufficient to use only the first three PC axes (or the first five PC axes occasionally) to explain the original structural variation.</p>", "<p>With respect to the individual contribution ratios (<italic>Q</italic><sub>1</sub>-<italic>Q</italic><sub>3</sub>) of the first three PC axes, <italic>Q</italic><sub>1 </sub>was overwhelmingly higher than those of the other PC axes up to 50-residue length (Fig. ##FIG##4##5##), which indicates that <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>is a meaningful and fundamental descriptor for segment conformation. Actually, <italic>Q</italic><sub>1 </sub>decreased rapidly, and <italic>Q</italic><sub>2 </sub>increased in the short segment lengths (i.e. 10–22 residues). Thereafter, both <italic>Q</italic><sub>1 </sub>and <italic>Q</italic><sub>2 </sub>decreased slowly. In addition, <italic>Q</italic><sub>3 </sub>increased gradually with lengths up to 33 residues, with a maximum value of 11.5%.</p>", "<title>Investigation of structural properties of conformational axes</title>", "<p>An eigenvector was analyzed for each PC axis with a triangle map to elucidate the physical and conformational meaning of the PC axes of the conformational space of the short to long segments. The eigenvector can be regarded as a collective variable to describe the segment conformation. Figure ##FIG##5##6## shows triangle maps of the first five PC axes (<italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>– <italic>PC</italic><sup><italic>all</italic></sup><italic>5</italic>) for short (10 residues), medium (26 residues), and long segments (30 residues). The triangle map clearly portrays residue pairs, with large or small deviations of <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>distances along each PC axis from the average distance &lt;<italic>q</italic><sub><italic>i</italic></sub>&gt;. In the triangle map, positive (red) and negative (blue) areas correspond to residue pairs with mutually inverse deviations. The patterns of red and blue areas are conserved in the universes of short to long segments, indicating that conformational deviations related to the PC axes are conserved among the universes. Figure ##FIG##6##7## depicts the conformational changes along the PC axis using colored arrows.</p>", "<p>Actually, <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>corresponds to the change of the radius of gyration (<italic>Rg</italic>). The triangle map for <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>has only one positive area, shown as red in Fig. ##FIG##5##6##, which is located near the residue pairs at the N-terminal and C-terminal sides. This single area indicates that the distant residue pairs in the sequence have a larger conformational deviation along <italic>PC</italic><sup><italic>all</italic></sup><italic>1</italic>. The correlation coefficient of the conformational deviation along <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>with <italic>Rg </italic>was greater than 0.9 in segment lengths of 10–50 residues (Fig. ##FIG##7##8##). The arrows in Fig. ##FIG##6##7## point to the center of the segment, which indicates clearly that the conformational changes along <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>are involved with expansions or compressions of the conformation. For short lengths, <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>also shows a strong correlation with the changes of the segment end-to-end distance (<italic>D</italic><sub><italic>end</italic></sub>), which is defined as the <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>distance between the first and last residues of segments. Correlation between <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>and <italic>D</italic><sub><italic>end </italic></sub>slowly weakened with increased segment length: 0.91 for 10 residues, 0.79 for 26 residues, and 0.77 for 30 residues.</p>", "<p>The <italic>PC</italic><sup><italic>all</italic></sup><italic>2 </italic>correlates to a degree of structural symmetry (<italic>D</italic><sub><italic>sym</italic></sub>) of a segment with respect to the N-terminal and C-terminal halves. The <italic>D</italic><sub><italic>sym </italic></sub>is defined as follows: Given a distance matrix for a segment, where element (<italic>i</italic>,<italic>j</italic>) is the distance (denoted as <italic>r</italic><sub><italic>ij</italic></sub>) between <bold>C</bold><sub>α </sub>atoms of residue <italic>i </italic>and <italic>j</italic>. Then, the degree of structural symmetry is defined as the sum of the squared differences of symmetric elements in a distance matrix for a segment: <italic>D</italic><sub><italic>sym </italic></sub>= Σ<sub>1 ≤ i &lt; j ≤ n </sub>(<italic>r</italic><sub><italic>ij </italic></sub>- <italic>r</italic><sub><italic>n</italic>-(<italic>j</italic>-1)<italic>n</italic>-(<italic>i</italic>-1)</sub>)<sup>2</sup>, where <italic>n </italic>is the segment length. The triangle map for <italic>PC</italic><sup><italic>all</italic></sup><italic>2 </italic>was separated into one positive area (red) and one negative area (blue). The correlation coefficient of the conformational deviation along <italic>PC</italic><sup><italic>all</italic></sup><italic>2 </italic>with structural symmetry, <italic>D</italic><sub><italic>sym </italic></sub>was greater than 0.90 in the segment lengths of 10–50 residues (Fig. ##FIG##7##8##). Both conformations displayed mirrored symmetry about a plane constructed by <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>and <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>when two conformations were picked from opposite positions along <italic>PC</italic><sup><italic>all</italic></sup><italic>2</italic>. The segment conformations picked up along <italic>PC</italic><sup><italic>all</italic></sup><italic>2 </italic>are shown in Figs. ##FIG##2##3a–3c##.</p>", "<p>The <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>correlated with a physical indicator that describes a conformational transition between structures with one turn and ones with two turns (<italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>in Fig. ##FIG##5##6##). The picked conformations along <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>indicate that segregation of a β-hairpin structure exists along with conformational changes by <italic>PC</italic><sup><italic>all</italic></sup><italic>3</italic>. We defined the physical indicator (<italic>D</italic><sub><italic>mn</italic>+<italic>mc</italic></sub>) of the β-hairpin formation: <italic>D</italic><sub><italic>mn</italic>+<italic>mc </italic></sub>is the sum of the norms of two vectors, which were generated by the middle point of the segment for both the N-terminal and C-terminal residues: <italic>D</italic><sub><italic>mn</italic>+<italic>mc </italic></sub>= , where and respectively denote the vectors from the midpoint to the N-terminal and C-terminal residues of the segment. Good correlation was found between <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>and <italic>D</italic><sub><italic>mn</italic>+<italic>mc </italic></sub>(Fig. ##FIG##7##8##). The correlation coefficient was greater than 0.7 for the 10–50 residues. The triangle map of <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>indicated a separation of one positive area (red) and two negative areas (blue). It is noteworthy that the triangle map of <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>for short segments differed slightly from those of medium and long segments. A positive area is visible near the residue pair of the N-terminal and C-terminal in the short map, suggesting that <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>has a (negative) correlation with <italic>D</italic><sub><italic>end</italic></sub>. For medium and long lengths, the positive area was close to the center of the triangle map. Therefore, the correlation between <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>and <italic>D</italic><sub><italic>mn</italic>+<italic>mc</italic></sub>/<italic>D</italic><sub><italic>end </italic></sub>was necessarily smaller in medium and long lengths.</p>", "<p>The triangle map of <italic>PC</italic><sup><italic>all</italic></sup><italic>4 </italic>had one negative area and one positive area. The positive area, located at the map center, suggests that <italic>PC</italic><sup><italic>all</italic></sup><italic>4 </italic>is correlated with the radius of gyration (<sub><italic>mid</italic></sub><italic>Rg</italic>) of the middle region of the segment – except for both the N-terminal and C-terminal quarter portions – in the medium and long segments. The respective correlation coefficients for the 26 and 30 residue lengths were 0.73 and 0.72. The <italic>PC</italic><sup><italic>all</italic></sup><italic>4 </italic>also has a weak (negative) correlation with <italic>D</italic><sub><italic>end</italic></sub>. The respective correlation coefficients between <italic>PC</italic><sup><italic>all</italic></sup><italic>4 </italic>and <italic>D</italic><sub><italic>end </italic></sub>for the 26 and 30 residue lengths were -0.45 and -0.42.</p>", "<p>We identified no simple physical indicator for conformational changes along <italic>PC</italic><sup><italic>all</italic></sup><italic>5</italic>. However, visual inspection from conformations picked along <italic>PC</italic><sup><italic>all</italic></sup><italic>5 </italic>suggests that <italic>PC</italic><sup><italic>all</italic></sup><italic>5 </italic>is a conformational axis that represents segregated β-sheet structures. Conformations picked up from both ends on <italic>PC</italic><sup><italic>all</italic></sup><italic>5 </italic>are depicted in Fig. ##FIG##5##6##. In the triangle map for <italic>PC</italic><sup><italic>all</italic></sup><italic>5</italic>, two positive and two negative areas exist along the diagonal line, which might indicate that <italic>PC</italic><sup><italic>all</italic></sup><italic>5 </italic>segregates segment conformations with double turns. The <italic>PC</italic><sup><italic>β</italic></sup><italic>5 </italic>contribution ratio, which was derived from all-β proteins, was higher than that derived from other structural classes, which suggests that <italic>PC5 </italic>is important for describing the structural variation of β-structures.</p>", "<title>Segment universes derived from different structural classes</title>", "<p>The segment universes described above are those derived from proteins of the four structural classes. Therefore, decomposition of the universe into four classes is helpful to evaluate the influence of each structural class on the segment universe. To this end, a segment universe was constructed for each structural class separately, and compared the PC axes derived from each universe with those of all segments (i.e., <italic>PC</italic><sup><italic>all</italic></sup><italic>1-PC</italic><sup><italic>all</italic></sup><italic>3</italic>). The first three largest eigenvectors of each structural class were also compared respectively with <italic>PC</italic><sup><italic>all</italic></sup><italic>1</italic>, <italic>PC</italic><sup><italic>all</italic></sup><italic>2</italic>, and <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>to elucidate the structural properties of PC axes derived from each universe.</p>", "<p>Figure ##FIG##8##9## depicts the contribution ratios of the first three PC axes, <italic>PC</italic><sup><italic>x</italic></sup><italic>1 </italic>-<italic>PC</italic><sup><italic>x</italic></sup><italic>3 </italic>(<italic>x </italic>= α, β, α +β, or α/β), in each structural class. The marks on the curves in Fig. ##FIG##8##9## indicate that the correlation coefficient (<bold><italic>v</italic></bold><sup><italic>x</italic></sup><sub><italic>i</italic></sub>·<bold><italic>v</italic></bold><sup><italic>all</italic></sup><sub><italic>i</italic></sub>) between <italic>PC</italic><sup><italic>x</italic></sup><italic>1 </italic>-<italic>PC</italic><sup><italic>x</italic></sup><italic>3 </italic>and <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>-<italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>(i.e., <italic>i </italic>= 1, 2, 3) is greater than 0.7, which was used here as a threshold of conservation of structural properties. The properties of the first two PC axes corresponding to the <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>and <italic>PC</italic><sup><italic>all</italic></sup><italic>2 </italic>were highly conserved in all four structural classes. The characteristics of <italic>PC</italic><sup><italic>all</italic></sup>3 were also conserved in all four structural classes, although exceptions were apparent for the 20-residue-long and 10–16-residue-long all-α and all-β classes. Therefore, it is confirmed that the first three PC axes (<italic>Rg</italic>, symmetry, and one/two turn(s)) are important in almost all cases to describe the conformation of segments embedded in globular proteins.</p>", "<p>However, the curves for the contribution ratios of both all-α and all-β classes (see two panels of Fig. ##FIG##8##9##) differ clearly from those of <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>– <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>(i.e. <italic>Q</italic><sub>1 </sub>– <italic>Q</italic><sub>3 </sub>in Fig. ##FIG##4##5##). The <italic>Q</italic><sup><italic>α</italic></sup><sub>1</sub>, contribution ratio was always higher than 40%, which indicates that the distribution of the all-α segments has a large deviation with respect to <italic>Rg</italic>. In contrast, the <italic>Q</italic><sup><italic>β</italic></sup><sub>1 </sub>contribution ratio decreased rapidly with increasing segment length. The value of <italic>Q</italic><sup><italic>α</italic></sup><sub>2 </sub>increased moderately with increasing segment length. In contrast, the <italic>Q</italic><sup><italic>β</italic></sup><sub>2 </sub>had a maximum value greater than 20% at a length of 22 residues. This rapid increase of <italic>Q</italic><sup><italic>β</italic></sup><sub>2 </sub>might reflect a typical feature for β-sheet conformations. For <italic>PC3</italic>, the curves for the contribution ratios of the all-α and all-β classes also mutually differed. Although <italic>Q</italic><sup><italic>β</italic></sup><sub>3 </sub>peaked at a length of 35 residues, <italic>Q</italic><sup><italic>α</italic></sup><sub>3 </sub>peaked with a short length, which indicates that the structural variable based on <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>is important for β-segments longer than 30 residues. In contrast, the behaviors of the contribution ratios for both α+β and α/β classes along with the segment length resembled each other. They were also similar to <italic>Q</italic><sub>1</sub>-<italic>Q</italic><sub>3 </sub>in Fig. ##FIG##4##5## because those structural classes are mixtures of α-helices and β-sheets.</p>", "<p>Subsequently, PC axes that were specific for each structural class were examined. For this analysis, the PC axis was defined as a \"class-specific\" one when a PC axis from a structural class showed no similarity with the first 20 PC axes from the other three structural classes (see <italic>Methods</italic>). The first 10 PC axes of each class were investigated for the short (10 residues), medium (26 residues), and long (30 residues) segments. Ten class-specific conformational axes were identified and consisted of one (<italic>PC</italic><sup><italic>β</italic></sup><italic>10</italic>) for the short length, eight for the medium, and one (<italic>PC</italic><sup><italic>α</italic></sup><italic>8</italic>) for the long. The eight class-specific axes for the medium-length segments are <italic>PC</italic><sup><italic>α</italic></sup><italic>5</italic>, <italic>PC</italic><sup><italic>α</italic></sup><italic>8</italic>, and <italic>PC</italic><sup><italic>α</italic></sup><italic>10 </italic>for all-α, <italic>PC</italic><sup><italic>β</italic></sup><italic>10 </italic>for all-β, <italic>PC</italic><sup><italic>α+β</italic></sup><italic>9 </italic>and <italic>PC</italic><sup><italic>α+β</italic></sup><italic>10 </italic>for α+β, and <italic>PC</italic><sup><italic>α/β</italic></sup><italic>8 </italic>and <italic>PC</italic><sup><italic>α/β</italic></sup><italic>10 </italic>for α/β. Four examples out of eight are depicted in Fig. ##FIG##9##10##. A clear correlation of these PC axes is difficult to discern according to simple physical or structural quantities. Figure ##FIG##9##10a## shows that the <italic>PC</italic><sup><italic>α</italic></sup><italic>8 </italic>describes a structural change of three (both ends and the middle portion) parts of α-segments. The <italic>PC</italic><sup><italic>α/β</italic></sup><italic>8 </italic>is related to βαβ motifs, which is the most fundamental structural unit for α/β proteins.</p>" ]
[ "<title>Discussion</title>", "<p>Investigation of the protein segment universe is an important subject for bioinformatics. Results of this study show that the segment universe can be categorized naturally into three regimes: short, medium, and long. A main finding of this study is that the three regimes are clearly demarcated by critical changes in the shape of the segment distribution in the conformational space. Preceding studies demonstrated that the average length of α-helix is 14 residues [##REF##8874031##24##] and that for β-strand is five residues [##REF##14983075##25##]. Results of the present study show that transitional segment lengths (22 and 26 residues long) do not coincide with these average lengths. Therefore, a single secondary structure element does not characterize the shape of the segment distribution. The appearance of the medium length regime segregates the segment fold universe into three. The combination of secondary-structure elements is important to characterize not only the medium-length segment universe but also the entire segment fold universe.</p>", "<p>Meanwhile, loops, which make up 30% of the protein structures [##REF##8976569##26##], are also expected to take a larger role to form some unique conformations by connecting secondary-structure elements in the medium to the long-length segment universe than short one. The segments in the cluster of the medium to long-length universe tend to contain more loop regions than those of the short segment universe, as shown in Figs. ##FIG##3##4b## and ##FIG##3##4d##, and have a wider variety of origins (Figs. ##FIG##3##4a## and ##FIG##3##4c##). For example, the segments in the cluster with density of 0.35–1.0 of the universe of 30 residues length are derived from 461 proteins out of all 600 representatives used for this study (see Additional File ##SUPPL##0##1##). Longer loops that possess extended conformations are located on the opposite side of the compact-segment cluster along <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>in the medium to long segment universe (Figs. ##FIG##2##3b## and ##FIG##2##3c##). Instead of discrete clusters, they appear to constitute a rather continuous distribution. Some analyses examine short loops with respect to their completeness [##REF##7809034##27##,##REF##9278278##28##] and elaborate classification [##REF##8976569##26##,##REF##10373380##29##]. In the analysis of short segments, our method also captured some loop conformation classes, such as joint loops connecting two helices, and exposed and extended loops participated in protein-protein interactions [##REF##15802651##23##].</p>", "<p>A natural boundary was identified, in this study, between the peptide-like and protein-like distributions between the lengths of 23 and 26 residues using actual conformations of protein segments. This observation with respect to the boundary is consistent with the results described by Shen et al. [##UREF##1##30##], even though they used a sphere-packing model to estimate a minimal domain size of about 20 residues. A recent study by Sawada and Honda [##REF##16731566##31##] also identified a boundary at 10–20 residue length by calculating the structural diversity of segments. They discretized the conformational space using a single-pass clustering method. In contrast, we observed the density distribution to uncover differences of conformational space between short and long segments. The segment conformational space for lengths of 10–22 residues provided a distribution with an extreme density gradient towards the secondary structure, such as the α-helix, β-strand, and β-hairpin clusters, which are expected to belong to the peptide-like conformational regime. This conformational variation reflects that short segments embedded in globular proteins are mainly stabilized by the physicochemical property of the peptide. On the other hand, the segment conformational spaces for lengths of 27 residues or more have a distribution that is dominated by compact segments, which suggests a protein-like distribution (protein-like conformational regime). This distribution arises from the hydrophobic effect imparted by the solvent molecules, which is of great importance for structural stability in long segments derived from globular proteins. If this is the case, our observations support the <italic>de novo </italic>structure prediction methods, so-called fragment assembling methods, that have been developed recently [##REF##12215415##32##, ####UREF##2##33##, ##REF##15752606##34##, ##REF##16317788##35####16317788##35##]. These approaches are usually based on the prediction of local segment conformations followed by assembly of segments, and are generally used to separate criteria at each step; sequence similarity or secondary structural propensity for the prediction of segment conformations, and non-local energy terms for the assembling step. These strategies used in the <italic>de novo </italic>prediction methods seems to be consistent with the results shown here. Results of our analyses clearly show such a hierarchical organization of protein structures, and indicate that preparing segment libraries up to around 20 residues long would be helpful for such methods.</p>", "<p>These results indicate that the structural meanings for the conformational axes (i.e., the radius of gyration for <italic>PC</italic><sup><italic>all</italic></sup><italic>1</italic>, structural symmetry related to the N-terminal and C-terminal halves for <italic>PC</italic><sup><italic>all</italic></sup><italic>2</italic>, and a single-turn/two-turn structure for <italic>PC</italic><sup><italic>all</italic></sup><italic>3</italic>) are conserved in the different lengths and structural classes. This fact suggests that these conformational components are key structural variables for protein segments. On the other hand, when conformational axes among the four structural classes were compared, we were able to identify several conformational axes that were specific to each structural class, especially in the medium length range. In fact, a distribution change for medium lengths was observed, involving an increase in compact segments. Those segments included supersecondary structures such as α-hairpins, parts of the β-sheets, and βαβ units. These results might be related to the specificity of the structural class or fold of the contents of supersecondary structures [##REF##10222204##20##]. Typical supersecondary structural motifs, α-hairpin, β-hairpin, and βαβ are, respectively, the basic structural units for the all-α, all-β, and α/β proteins. These motifs are often shared within the structural classes. Therefore, the contribution ratios observed for the class-specific conformational axes were high. Class-specific conformational axes were rarely observed in short and long lengths, probably because short segments are too nonspecific and are often shared over different structural classes; long segments are too specific and have very low contribution ratios for conformational axes that are specific for each structural class.</p>", "<p>The currently found class-specific conformational axes provide a hint to solve a difficulty in classifying diverse sets of protein structures. Both α/β and α+β classes are known to show a substantial overlap. In the CATH classification, α/β and α+β classes are treated as one structural class as α-β class. Classifying α/β and α+β proteins is sometimes a difficult problem, although several classification [##REF##12606708##19##,##REF##8831787##36##,##REF##18427716##37##] and also prediction [##REF##16248794##38##,##REF##18452616##39##] schemes have been proposed. The present study showed that α/β and α+β classes have similar characteristics of universes, and also have unique ones at the same time. For example, our results show that <italic>PC</italic><sup><italic>α</italic>/<italic>β</italic></sup><italic>8</italic>, whose contribution ratio was 1.4%, was associated only with the βαβ motif. In the α+β class, no axis was strongly correlated with <italic>PC</italic><sup><italic>α</italic>/<italic>β</italic></sup><italic>8 </italic>(see Additional File ##SUPPL##1##2##), which is a clear example of the difference in structural variables between α+β and α/β classes originating from class-specific supersecondary structures. Consequently, projecting segments onto a conformational subspace using the axis <italic>PC</italic><sup><italic>α</italic>/<italic>β</italic></sup><italic>8 </italic>could be useful for objectively dividing protein domains of α-β class into α/β and α+β classes. A considerable localization of segments derived from α/β proteins in a PCA subspace is observed (see Additional Files ##SUPPL##2##3## and ##SUPPL##3##4##).</p>", "<p>An effective method must be developed for conformational sampling for <italic>de novo </italic>prediction methods. The resulting structural variables analyzed in this study would be helpful for additional progress in <italic>de novo </italic>structure prediction. For example, testing the distribution of segments or models in terms of the degree of symmetry using the descriptor (<italic>D</italic><sub><italic>sym</italic></sub>) might be useful to verify the completeness of sampling of the conformational space. Using a filtering threshold or function (generally used in fragment assembling methods for selecting proper models) that is tolerant of the radius of gyration might be useful for improving the prediction of all-α proteins because the contribution ratio, <italic>Q</italic><sup>α</sup><sub>1</sub>, of <italic>PC</italic><sup>α </sup><italic>1 </italic>corresponding to the radius of gyration (<italic>Rg</italic>) is larger than those of the other structural classes in the medium and long segments. Consequently, projecting segments of models onto a conformational subspace constructed by <bold>PC</bold><sup><italic>x </italic></sup>(where <italic>x </italic>= α, β, α/β, α+β, or all) axes might be helpful for filtering out models and assigning a protein to a structural class.</p>" ]
[ "<title>Conclusion</title>", "<p>In this study, the dual critical transitions in the protein segment universe from short to long length are shown. Our observations are related to the transitions proposed by the significance of two-stage treatment in <italic>de novo </italic>structure prediction. Considering the hierarchical organization of a protein segment universe that we have shown, we suggest the efficacy of using the evaluation functions that is secondary-structure-directed for sampling local structures less than 23 residues long. We also suggest the suitability of evaluating protein-like features of models using another function (e.g. <italic>Rg</italic>) for longer segments. Changing the criteria of filtering for each structural class will enhance the effectiveness of the conformation sampling process. Through these analyses, we have demonstrated that our clustering methodology is useful to identify a distinctive distribution shift of conformational space between short and long segments and that distribution changes depend on structural classes.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Many studies have examined rules governing two aspects of protein structures: short segments and proteins' structural domains. Nevertheless, the organization and nature of the conformational space of segments with intermediate length between short segments and domains remain unclear. Conformational spaces of intermediate length segments probably differ from those of short segments. We investigated the identification and characterization of the boundary(s) between peptide-like (short segment) and protein-like (long segment) distributions. We generated ensembles embedded in globular proteins comprising segments 10–50 residues long. We explored the relationships between the conformational distribution of segments and their lengths, and also protein structural classes using principal component analysis based on the intra-segment <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>atomic distances.</p>", "<title>Results</title>", "<p>Our statistical analyses of segment conformations and length revealed critical dual transitions in their conformational distribution with segments derived from all four structural classes. Dual transitions were identified with the intermediate phase between the short segments and domains. Consequently, protein segment universes were categorized. i) Short segments (10–22 residues) showed a distribution with a high frequency of secondary structure clusters. ii) Medium segments (23–26 residues) showed a distribution corresponding to an intermediate state of transitions. iii) Long segments (27–50 residues) showed a distribution converging on one huge cluster containing compact conformations with a smaller radius of gyration. This distribution reflects the protein structures' organization and protein domains' origin. Three major conformational components (radius of gyration, structural symmetry with respect to the N-terminal and C-terminal halves, and single-turn/two-turn structure) well define most of the segment universes. Furthermore, we identified several conformational components that were unique to each structural class. Those characteristics suggest that protein segment conformation is described by compositions of the three common structural variables with large contributions and specific structural variables with small contributions.</p>", "<title>Conclusion</title>", "<p>The present results of the analyses of four protein structural classes show the universal role of three major components as segment conformational descriptors. The obtained perspectives of distribution changes related to the segment lengths using the three key components suggest both the adequacy and the possibility of further progress on the prediction strategies used in the recent <italic>de novo </italic>structure-prediction methods.</p>" ]
[ "<title>Authors' contributions</title>", "<p>This study was conceived and carried out by KI, who also analyzed the results and drafted the manuscript. HT approved the study and participated in the discussion. JH participated in the design and coordination of the study. He also helped to write the manuscript. KT participated in the design and discussions of the study and wrote the manuscript. KI and JH developed the methodology. All authors read and approved the final manuscript.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>KI and JH were partly supported by <bold>BIRD </bold>of Japan Science and Technology Agency (<bold>JST</bold>).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Coverage versus the density threshold</bold>. Coverage of segments in clusters versus the density threshold for segment lengths of 10, 20, 30, 40, and 50 residues (A), and those of 21–30 residues (B). Density is presented on a logarithmic scale.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Probability density function of the <italic>F_Rg </italic>score</bold>. Distribution of <italic>F_Rg </italic>score of segments in the region with density from 0.10 (magenta in Fig. 3) to 0.35 (red). Distributions for segment lengths of 21, 23, 25, 27, and 29 residues are shown.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>3D representation of segment universes</bold>. A 3D representation of short (A, 10 residues), medium (B, 26 residues), and long (C, 30 residues) segment universes. The 3D representations were generated using the first three <italic>PC</italic><sup><italic>all </italic></sup>axes. They were expressed by four iso-density contours: 0.005 (blue), 0.01 (orange), 0.1 (magenta), and 0.35 (red). The PC axis numbers (1, 2, and 3) are given near the axes. Front and side views of images are shown, respectively, in the upper and lower areas of the figure. D: Schematic diagram of the relationship between properties of the cluster(s) in the segment universe and segment length. The properties of the cluster(s) in the segment conformational space changed according to increased segment length.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Relationships between segment properties and their length in the populated regions</bold>. Upper figures show that the percentages of structural classes from which segments in the clusters with density of 0.10–0.35 (A) and 0.35–1.0 (C) were derived. Lower figures show the percentages of structural properties of the segments in the clusters with density of 0.10–0.35 (B) and 0.35–1.0 (D). The magenta and green lines in lower figures represent the percentage of helical and strand segments in the cluster. A helical segment is defined as the segment with the rate of α-helical residues &gt;= 0.5. A strand segment is defined as the segment with the rate of β-strand residues &gt;= 0.5. The percentages of helix (H) and sheet (E), that are determined using the DSSP program [##REF##6667333##40##], are also represented by the red and blue lines, respectively.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Contribution ratios for the PC axes</bold>. Contribution ratios for the first five PC axes for segment lengths of 10–50 residues are shown here. The cumulative contribution ratio for the first three PC axes (<italic>Q</italic><sub>123</sub>, solid line with no symbol) and the individual contribution ratios of <italic>PC</italic><sup><italic>all</italic></sup><italic>1 </italic>(<italic>Q</italic><sub>1</sub>, crosses), <italic>PC</italic><sup><italic>all</italic></sup><italic>2 </italic>(<italic>Q</italic><sub>2</sub>, asterisks), <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>(<italic>Q</italic><sub>3</sub>, squares), <italic>PC</italic><sup><italic>all</italic></sup><italic>4 </italic>(<italic>Q</italic><sub>4</sub>, filled squares), and <italic>PC</italic><sup><italic>all</italic></sup><italic>5 </italic>(<italic>Q</italic><sub>5</sub>, circles) are shown.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Distance matrices for the PC axes</bold>. Distance maps express eigenvectors for the first five PC axes for 10, 26, and 30 residue lengths. Each map shows deviations of <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>distances along each eigenvector from the mean <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>distances. The scale bar indicates the relative deviation. The color indicates whether in this particular mode the distances are increasing (red) with respect to the mean value or decreasing (blue). The eigenvector values are scaled by the square root of eigenvalue of the PC axis <italic>k</italic>, <italic>λ</italic><sub><italic>k</italic></sub><sup>1/2</sup>. Residue numbers are displayed with horizontal and vertical sides of each triangle map. Two segment conformations were picked up from both ends on each PC axis; they are displayed under each triangle map.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p><bold>Visualization of collective variables for the first five PC axes of 26 residue lengths</bold>. Collective variables (eigenvectors) for the first five PC axes of 26 residue lengths are visualized by the vectors onto <bold>C</bold><sub>α </sub>atoms of a segment. The vector for the <bold>C</bold><sub>α </sub>atom of residue <italic>i </italic>is calculated as <italic>r</italic><sub><italic>i </italic></sub>= Σ<sub><italic>i </italic>≠ <italic>j</italic></sub><bold><italic>q</italic></bold><sub><italic>ij</italic></sub><italic>e</italic><sup><italic>vk</italic></sup><sub><italic>ij</italic></sub><italic>λ</italic><sub><italic>k</italic></sub><sup>1/2</sup>, where <bold><italic>q</italic></bold><sub><italic>ij </italic></sub>is the vector from residue <italic>i </italic>to <italic>j</italic>, <italic>e</italic><sup><italic>vk</italic></sup><sub><italic>ij </italic></sub>is the element of eigenvector of the PC axis <italic>k </italic>(<bold><italic>v</italic></bold><sub><italic>k</italic></sub>) corresponding to the <bold>C</bold><sub>α</sub>-<bold>C</bold><sub>α </sub>pair between residue <italic>i </italic>and <italic>j</italic>, and <italic>λ</italic><sub><italic>k </italic></sub>is the eigenvalue of the PC axis <italic>k</italic>. Positive (blue) and negative values (red) are shown for elements of eigenvectors. The reference segment used in this figure is designed to clarify the difference between structural variables.</p></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Figure 8</label><caption><p><bold>Correlation coefficients between conformational deviation along each PC axis and physical indicator</bold>. The radius of gyration (<italic>Rg</italic>) for <italic>PC</italic><sup><italic>all</italic></sup><italic>1</italic>, structural symmetry related to the N-terminal and C-terminal halves (<italic>D</italic><sub><italic>sim</italic></sub>) for <italic>PC</italic><sup><italic>all</italic></sup><italic>2</italic>, a simple indicator of β-hairpin (<italic>D</italic><sub><italic>mn</italic>+<italic>mc</italic></sub>) and <italic>D</italic><sub><italic>mn</italic>+<italic>mc</italic></sub>/end-to-end distance (<italic>D</italic><sub><italic>end</italic></sub>) for <italic>PC</italic><sup><italic>all</italic></sup><italic>3</italic>, and the radius of gyration (<sub><italic>mid</italic></sub><italic>Rg</italic>) around the midpoint of the segment for <italic>PC</italic><sup><italic>all</italic></sup><italic>4 </italic>were used in these analyses. Correlation coefficients were calculated at every 10 residues of 10–50 residues.</p></caption></fig>", "<fig position=\"float\" id=\"F9\"><label>Figure 9</label><caption><p><bold>Contribution ratios for the PC axes for each structural class</bold>. Contribution ratios (<italic>Q</italic><sub>1</sub>, crosses; <italic>Q</italic><sub>2</sub>, asterisks; and <italic>Q</italic><sub>3</sub>, squares) of <italic>PC</italic><sup><italic>x</italic></sup><italic>1</italic>-<italic>PC</italic><sup><italic>x</italic></sup><italic>3 </italic>vs. segment lengths of 10–50 residues for each structural class, where <italic>x </italic>= α, β, α+β, or α/β. The correlation coefficient between <italic>PC</italic><sup><italic>x</italic></sup><sub><italic>i </italic></sub>and <italic>PC</italic><sup><italic>all</italic></sup><sub><italic>i </italic></sub>(<italic>i </italic>= 1, 2, 3) is 0.7 or less if no mark is present at a segment length. For the all-β class, no axis exhibited a correlation coefficient greater than 0.7 up to <italic>PC</italic><sup><italic>all</italic></sup><italic>3 </italic>for segment lengths of 10–16 residues.</p></caption></fig>", "<fig position=\"float\" id=\"F10\"><label>Figure 10</label><caption><p><bold>Examples of class-specific conformational axes</bold>. Conformational axes specific to structural class for 26-residue segments are shown. Eigenvector maps and conformations picked up from both ends on each PC axis are shown: A for <italic>PC</italic><sup><italic>α</italic></sup><italic>8</italic>, B for <italic>PC</italic><sup><italic>α/β</italic></sup><italic>8</italic>, C for <italic>PC</italic><sup><italic>α</italic></sup><italic>10</italic>, and D for <italic>PC</italic><sup><italic>β</italic></sup><italic>10</italic>. The color contrast of the maps is enhanced to aid viewing. The anti-phase of the blue color is shown in red. Residue numbers are displayed with horizontal and vertical sides of each triangle map. The respective contribution ratios of <italic>PC</italic><sup><italic>α </italic></sup><italic>8</italic>, <italic>PC</italic><sup><italic>α/β</italic></sup><italic>8</italic>, <italic>PC</italic><sup><italic>α</italic></sup><italic>10</italic>, and <italic>PC</italic><sup><italic>β</italic></sup><italic>10 </italic>were 1.3%, 1.4%, 0.9%, and 0.7%.</p></caption></fig>" ]
[]
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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Origins of segments in the cluster of 30 residue length. Distributions of the origins of segments in the cluster of the universe of 30 residues length are shown.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional file 2</title><p>Correlation with the first 10 PC axes of α/β class of the medium (26 residue) segments. Maximal correlation coefficients between the first 10 PC axes of α/β class and PC axes of the other three structural classes are shown.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional file 3</title><p>Class-specific region for α/β segments on the <italic>PC</italic><sup><italic>α</italic>/<italic>β</italic></sup><italic>8</italic>-<italic>PC</italic><sup><italic>α</italic>/<italic>β</italic></sup><italic>3 </italic>plane. Distributions of segments of α/β structural class proteins for the medium length are shown.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S4\"><caption><title>Additional file 4</title><p>Discrimination of segments from the α/β structural class in the <italic>PC</italic><sup><italic>α</italic>/<italic>β</italic></sup><italic>8</italic>-<italic>PC</italic><sup><italic>α</italic>/<italic>β</italic></sup><italic>3 </italic>plane. Specificity and coverage rates of segments of α/β structural class proteins in the <italic>PC</italic><sup><italic>α</italic>/<italic>β</italic></sup><italic>8</italic>-<italic>PC</italic><sup><italic>α</italic>/<italic>β</italic></sup><italic>3 </italic>plane are presented.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S5\"><caption><title>Additional file 5</title><p>List of PDB ids used in this study. The PDB and SCOP IDs of proteins used in this study are listed.</p></caption></supplementary-material>" ]
[]
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[{"surname": ["Tomii", "Kanehisa", "Wang JTL, Shapiro BA, Shasha D"], "given-names": ["K", "M"], "article-title": ["Systematic detection of protein structural motifs"], "source": ["Pattern discovery in biomolecular data"], "year": ["1999"], "publisher-name": ["New York: Oxford University Press"], "fpage": ["97"], "lpage": ["110"]}, {"surname": ["Shen", "Davis", "Sali"], "given-names": ["M-y", "FP", "A"], "article-title": ["The optimal size of a globular protein domain: A simple sphere-packing model"], "source": ["Chemical Physics Letters"], "year": ["2005"], "volume": ["405"], "fpage": ["224"], "lpage": ["228"], "pub-id": ["10.1016/j.cplett.2005.02.029"]}, {"surname": ["Chikenji", "Fujitsuka", "Takada"], "given-names": ["G", "Y", "S"], "article-title": ["A reversible fragment assembly method for de novo protein structure prediction"], "source": ["The Journal of Chemical Physics"], "year": ["2003"], "volume": ["119"], "fpage": ["6895"], "lpage": ["6903"], "pub-id": ["10.1063/1.1597474"]}]
{ "acronym": [], "definition": [] }
40
CC BY
no
2022-01-12 14:47:26
BMC Struct Biol. 2008 Aug 13; 8:37
oa_package/fe/8f/PMC2529298.tar.gz
PMC2529299
18673567
[ "<title>Background</title>", "<p>Studies completed in the 1980s and early 1990s all found that in the United States African-American adults have twice the prevalence of CMV infections than have age-matched Caucasians [##REF##2552316##1##, ####REF##2542418##2##, ##REF##2826605##3##, ##UREF##0##4##, ##REF##9195071##5##, ##REF##2162370##6##, ##REF##1847399##7####1847399##7##]. In these studies African-Americans had seropositivity rate of between 75% and 100% [##REF##1847399##7##]. In Richmond VA in the 1980s we also observed a higher rate of CMV infections among the African-American population as compared to Caucasians [##REF##2552316##1##]. Two studies performed at different times reported the prevalence of CMV infections between 1986 and 1994 among African-Americans between ages 6 and 22 years [##REF##2542418##2##,##UREF##0##4##]. The rate of CMV seropositivity increased with age and ranged from 30 to 60%. These rates were also nearly twice the rate for age-matched Caucasians. The reasons for the increased rate of CMV infections among African-Americans as compared to Caucasians are unknown. Therefore we performed a study among African-American-American children and adolescents to determine the possible sources of infection.</p>" ]
[ "<title>Methods</title>", "<title>Study population</title>", "<p>Subjects were African-American 157 children and adolescents (between 5 months and 20 years of age) and their 113 family members who were recruited from 121 families at one private pediatric practice (106 families) and one public pediatric practice (15 families), each of which served inner city families in Richmond VA. The average family size was 4.28 members (range 1 to 6). Baseline demographics of subjects recruited from each practice type were similar. Recruitment occurred from November, 2005 until July, 2006. Only 4 subject families declined to participate. The index child was the one seeking medical consultation. When two or more children were present, the oldest child was the index child. The parent or guardian of each index child provided written informed consent for CMV testing and the completion of a demographic questionnaire. All of the household members and siblings present were tested for seropositivity. The questionnaire included: household size, income level, whether the family and/or child had health insurance, breastfeeding for the children, and the highest level of education within the immediate household. Index subjects greater than 11 years of age were interviewed privately by the attending physician and a second time by one of the investigators (I.W.) about sexual activity, birth control, and condom use. US 2000 census data were obtained from the US Census Bureau, Department of Commerce, Washington, DC.</p>", "<title>Laboratory assays</title>", "<p>Serologic status to CMV was determined using saliva. At least 100 uL of saliva was collected in 15 mL conical tubes by either expectoration, or for those unable to expectorate, via bulb suction syringe. Saliva was stored at -20°C. The saliva samples were assayed by enzyme immunoassay for IgG to CMV gB as previously described [##REF##8699071##8##,##UREF##1##9##]. IgG antibodies to gB move passively from serum to saliva and are present in direct proportion to their concentration in serum [##REF##8699071##8##]. The salivary assay detects IgG to gB when serum neutralizing titers to CMV are ≥ 1:64 in the serum. Serum neutralizing titers in seropositive sera are ≥ 1:256. To avoid measuring maternal antibodies enrolled children were greater than 5 months of age. Passively acquired maternal antibody has a half life of approximately 28 days thus by 5 months of age only 3% of maternal antibody levels are present in an infant serum. This level would be too low to detect in saliva [##REF##8699071##8##].</p>", "<title>Statistical analysis</title>", "<p>Subjects are referred to as adults if they were either one of the caregivers (four caregivers were between 19 and 21 years of age) in the family or were 21 years of age or older. Subjects were referred to as children if they were less than 21 years of age (two children were 19 and 20 years of age). The presence of antibodies to CMV (seropositivity) was modeled using repeated-measures logistic regression (GENMOD procedure in SAS version 9.1.3 SP3) with an exchangeable correlation structure to account for within-family correlation. The final model included the following terms: a linear and quadratic trend for the age of the children, a linear trend for the age of the adults, with a different intercept for children and adults. Likelihood-ratio chi-square tests were used to test for significance.</p>", "<p>This study was approved by the Virginia Commonwealth University Institutional Review Board. Informed written consent or assent was obtained from all subjects and studies were conducted in accordance with human experimentation guidelines of the US Department of Health and Human Services.</p>" ]
[ "<title>Results</title>", "<p>The characteristics of the study population are listed in Table ##TAB##0##1##. CMV seropositivity and age were determined for 157 children in 121 families. The average age of the children was 8.37 years (standard deviation = ± 5.33 years). For all subjects a multiple logistic regression analysis was used to determine the p-value for the association between each subject characteristic listed in table ##TAB##0##1## and age and CMV seropositivity. For children, the only characteristic associated with CMV seropositivity was age (chi-square = 5.76, df = 2, p = 0.056), with both the linear (p = 0.023) and quadratic trends (p = 0.042) both significant (Figure ##FIG##0##1##).</p>", "<p>For adults age was also associated with the CMV seropositivity rate (Figure ##FIG##0##1##). The average age of the 103 adults who reported their age was 33 years (range 19 to 53 years, SD = 8.75). Age was linearly related to CMV seropositivity (p = 0.027) but no quadratic trend was identified (p = 0.30). After age was taken into account no other characteristics were related to CMV seropositivity.</p>", "<p>Of 62 adolescents, only 6 reported being sexually active. Sexual activity was unrelated to CMV seropositivity (chi-square = 0.05, p = 0.82). Of the six sexually active adolescents each had only a single partner and condoms were used.</p>", "<p>For the index subjects there were 105 female (104 mothers and 1 aunt) and 8 male caregivers. There was no evidence for a relationship between the CMV seropositivity rate of the index subject and the mother or the female caregiver (data not shown) or between the seropositivity rate of all the children in each household and the mother or caregiver (Table ##TAB##1##2##).</p>", "<p>For families with 2 or more children living at home, CMV seroprevalence among siblings was associated (Table ##TAB##2##3##). This was independent of the number or ages of the children in the household. This within-family dependence was taken into account in all of the primary analyses.</p>", "<p>In the final regression model, CMV seroprevalence of adults was higher than children (chi-square = 18.8, p &lt; 0.0001) and for children CMV seropositivity increased with age (Figure ##FIG##0##1##). At age one year the CMV seropositivity rate was 11% (95%CI = 4% – 24%) and it increased approximately 1.8% each year until approximately age 13 years. Between ages 13 years and 20 years the CMV seropositivity rate remained between 22% and 33% (Table ##TAB##1##2## and Figure ##FIG##0##1##). For younger adults, the CMV seropositivity rate was approximately 84% (95%CI = 69%–92%) in 21 year olds (Figure ##FIG##0##1##).</p>", "<p>Finally, we compared the data from the 2000 US census for all African-Americans residing in Richmond, VA, the state of Virginia and the US to our subject group. The groups were similar for gender distribution, average household size, education levels, and income (Table ##TAB##3##4##).</p>" ]
[ "<title>Discussion</title>", "<p>Two novel observations emerge from this study. The first is that the cohort of the current generation of African-American children and adolescents in Richmond VA has lower rates of CMV seroprevalence than the older generation comprising their parents and caretakers. We observed a rate of CMV infections for female adults and caregivers that averaged 70%. This rate of seropositivity is similar to those previously reported by us for Richmond and is similar to those reported for adults in other US cities [##REF##2552316##1##, ####REF##2542418##2##, ##REF##2826605##3##, ##UREF##0##4##, ##REF##9195071##5##, ##REF##2162370##6####2162370##6##]. Further, in a population-based survey of African-Americans for the entire US, the average rate of seropositivity for African-American adults was 75.8% [##UREF##0##4##]. All of the published adult data were obtained in the late 1980s and early 1990s. A study conducted in the late 1980s in Houston reported infection rates for African-Americans between 6 and 22 years of age which increased with age from 30% to 75% [##REF##2542418##2##]. Similarly, in the US population-based study, infection rates for African-Americans tested between 1988 and 1994 from 6 to 20 years of age ranged from 40% to 65% [##UREF##0##4##]. These rates are similar to those we observed for the current generation of adult females.</p>", "<p>Ours is the first recent study to determine CMV seroprevalence rates for the current generation of lower socioeconomic African American children and adolescents. We did not measure the seroprevalence rates in age-matched Caucasians. However, the low rates we observed for lower socioeconomic African American children and adolescents were nearly identical to the low seroprevalence rates reported for Caucasians children and adolescents measured for the entire US population between 1988 and 1994 and for Caucasian children measured in Houston Texas in the late 1980s [##REF##2542418##2##,##UREF##0##4##]. In contrast, in these studies the seroprevalence rates for African-American children were significantly higher than age-matched Caucasians. Thus our data suggest the current generation of African-American and Caucasian children will have similar CMV seroprevalence rates.</p>", "<p>Our observations may be explained by two factors. First, our subject sample may not be representative of the population of African-Americans residing in Richmond, VA or elsewhere especially since we selected subjects seeking medical consultation. This seems unlikely for two reasons. As discussed above, we observed seroprevalence rates for the adults who were predominantly female which were similar to those previously reported for Richmond and other US cities [##REF##2552316##1##, ####REF##2542418##2##, ##REF##2826605##3##, ##UREF##0##4##, ##REF##9195071##5##, ##REF##2162370##6####2162370##6##]. Second, our population was similar in demographic characteristics to that described by the US census for African-Americans in residing the City of Richmond, the state of Virginia, and the nation.</p>", "<p>A second more likely reason we observed a lower rate of seroprevalence among African-American children may relate to the infrequent sexual activity reported by our adolescents. The preadolescent seroprevalence rates we observed were similar to those reported in the Houston study (30%–40%) and in the population-based US study (40%) [##REF##2542418##2##, ####REF##2826605##3##, ##UREF##0##4####0##4##]. However, in both these studies there was a marked increase in infections rates among adolescents. Sexual activity during adolescence has been frequently associated with increased rates of CMV infection [##REF##2162370##6##,##REF##1847399##7##,##UREF##2##10##]. Hence our results suggest a decrease in CMV infection in the current generation of African-Americans may have occurred due to decreased sexual activity, increased condom use, or a reduced number of sexual partners during adolescence. AIDS awareness may in part be responsible for this and it will be important to determine if similar changes in sexual behavior and CMV infection rates is also occurring among lower socioeconomic African-American children and adolescents in other cities.</p>", "<p>Other factors such the number of children per household and/or changes in hygienic practices may also account for the reduced rate of CMV seroprevalence infections we observed in the current generation of African-American adolescents and children as compared to the higher rates observed in their parents and in older studies.</p>", "<p>We observed a decline in the seropositivity rate with age for African-American adults between 40 and 45 years of age. The significance, if any, of this observation is uncertain This decline was not due to reduced antibody levels in saliva associated with aging since antibody titers to CMV persist for life and actually increased throughout adulthood with highest levels in the elderly [##REF##2543705##11##]. Although this decline may represent another cohort of older African-Americans with lower infection rates, the number of subjects in this age range was low and thus the 95% confidence intervals were wide and as shown in figure ##FIG##0##1##, the 95% confidence intervals overlapped.</p>", "<p>The second novel observation from our study was that sibling-to-sibling transmission may have been the primary mode of CMV acquisition among African-American children and adolescents. This was very different than we previously observed among Caucasian children in day care [##REF##1653938##12##]. For those Caucasian children, child-to-child transmission of CMV acquired in day care was very common but sibling-to-sibling transmission at home seldom occurred. In the current study only 19 children reported day care attendance. Children not in day care probably spend more time at home with their siblings than occurs for children attending day care. Since child-to child transmission of CMV requires prolonged and frequent contact; the association of seroprevalence rates among African-American siblings, most of whom received home care, may represent sibling-to sibling transmission.</p>", "<p>In our study 91% per cent of the caregivers were the biological mothers and over 70% were seropositive. Thus our data further indicate CMV acquisition by children from maternal sources such cervical-vaginal secretions, breast milk, or saliva is unlikely to account for the high rate of CMV infections among African- American adults. If infection from maternal sources occurred frequently, the children we observed should have had high CMV seroprevalence rates.</p>" ]
[ "<title>Conclusion</title>", "<p>We observed that the current generation of African-American children had CMV seroprevalence rates by age 20 years of less than one-half of that of their mothers. Our data suggest that sibling-to-sibling transmission was a possible source of CMV infections for the children. If our observations for Richmond VA represent what has occurred nationwide, a large proportion of the next generation of African-American women will be susceptible to a primary CMV infection during pregnancy and will benefit from a CMV vaccine. Exposure to household children is a major risk factor for congenital CMV infection [##UREF##2##10##]. Since we observed that 92% of infants are seronegative, infant immunization may be optimal.</p>" ]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Since African-Americans have twice the prevalence of cytomegalovirus (CMV) infections as age-matched Caucasians we sought to determine the ages and possible sources of infection of African-American children.</p>", "<title>Methods</title>", "<p>Subjects were 157 African-American healthy children and adolescents and their 113 household adults in Richmond VA. Families completed a questionnaire, provided saliva for antibody testing, and adolescents were interviewed regarding sexual activity.</p>", "<title>Results</title>", "<p>Regardless of age CMV seropositivity was not associated with gender, breast feeding, health insurance, sexual activity, or household income, education, or size. In the final regression model, prior CMV infection in adults was over two-fold higher than in children (chi-square = 18.8, p &lt; 0.0001). At one year of age the CMV seropositivity rate was 11% (95%CI = 4% – 24%) and increased 1.8% each year until age 13 years. Between ages 13 and 20 years the CMV seropositivity rate remained between 22% and 33%. For adults, the CMV seropositivity rate was 84% in 21 year olds (95%CI = 69%–.92%). There was no association between CMV infections of the children and their mothers but CMV infections among siblings were associated.</p>", "<title>Conclusion</title>", "<p>We observed that African-American children had CMV seroprevalence rates by age 20 years at less than one-half of that of their adult mothers and caregivers. Sibling-to-sibling transmission was a likely source of CMV infections for the children. The next generation of African-American women may be highly susceptible to a primary CMV infection during pregnancy and may benefit from a CMV vaccine.</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>IRW, AMB and SPA all contributed to the design of the study and the data analysis. IRW performed subject enrollment, sample collection, and laboratory assays. All authors read and approved final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2334/8/107/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>We thank Sanofi Pasteur for the gift of gB antigen and Drs. Sandra Bell, Ghouse Hyder, Allan D. Friedman, and Richard Brookman for their help. No extramural funding source was used for this study.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Relationship between age and the rate of CMV seropositivity for 103 adults and 157 children. 95% confidence intervals.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Characteristics of the Study Subjects by CMV seropositivity.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\" colspan=\"5\">No. of Children (N = 157 CMV status<sup>+</sup></td><td align=\"left\" colspan=\"5\">No. of Adults (N = 113) CMV status<sup>+</sup></td></tr></thead><tbody><tr><td align=\"left\">Characteristic</td><td align=\"left\">Pos.</td><td align=\"left\">Neg.</td><td align=\"left\">Total</td><td align=\"left\">% Pos.</td><td align=\"left\">p-value</td><td align=\"left\">Pos.</td><td align=\"left\">Neg.</td><td align=\"left\">Total</td><td align=\"left\">% Pos.</td><td align=\"left\">p-value</td></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"left\">Gender</td><td/><td/><td/><td/><td align=\"left\">0.54</td><td/><td/><td/><td/><td align=\"left\">098</td></tr><tr><td align=\"left\"> Male</td><td align=\"left\">23</td><td align=\"left\">58</td><td align=\"left\">81</td><td align=\"left\">28</td><td/><td align=\"left\">73</td><td align=\"left\">32</td><td align=\"left\">105</td><td align=\"left\">70</td><td/></tr><tr><td align=\"left\"> Female</td><td align=\"left\">16</td><td align=\"left\">60</td><td align=\"left\">76</td><td align=\"left\">21</td><td/><td align=\"left\">5</td><td align=\"left\">3</td><td align=\"left\">8</td><td align=\"left\">63</td><td/></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"left\">Breast fed</td><td/><td/><td/><td/><td align=\"left\">0.54</td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> No</td><td align=\"left\">16</td><td align=\"left\">68</td><td align=\"left\">84</td><td align=\"left\">19</td><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">16</td><td align=\"left\">32</td><td align=\"left\">48</td><td align=\"left\">33</td><td/><td/><td/><td/><td/><td/></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"left\">Yearly family income</td><td/><td/><td/><td/><td align=\"left\">0.66</td><td/><td/><td/><td/><td align=\"left\">0.59</td></tr><tr><td align=\"left\"> Under $20,000</td><td align=\"left\">14</td><td align=\"left\">40</td><td align=\"left\">54</td><td align=\"left\">26</td><td/><td align=\"left\">24</td><td align=\"left\">14</td><td align=\"left\">38</td><td align=\"left\">63</td><td/></tr><tr><td align=\"left\"> $20,000 to &lt; $40,000</td><td align=\"left\">9</td><td align=\"left\">21</td><td align=\"left\">30</td><td align=\"left\">30</td><td/><td align=\"left\">16</td><td align=\"left\">4</td><td align=\"left\">20</td><td align=\"left\">80</td><td/></tr><tr><td align=\"left\"> $40,000 to &lt; $60,000</td><td align=\"left\">6</td><td align=\"left\">14</td><td align=\"left\">20</td><td align=\"left\">30</td><td/><td align=\"left\">10</td><td align=\"left\">7</td><td align=\"left\">17</td><td align=\"left\">59</td><td/></tr><tr><td align=\"left\"> $60,000 to &lt; $80,000</td><td align=\"left\">1</td><td align=\"left\">5</td><td align=\"left\">6</td><td align=\"left\">17</td><td/><td align=\"left\">3</td><td align=\"left\">2</td><td align=\"left\">5</td><td align=\"left\">60</td><td/></tr><tr><td align=\"left\"> $80,000 or more</td><td align=\"left\">2</td><td align=\"left\">2</td><td align=\"left\">4</td><td align=\"left\">50</td><td/><td align=\"left\">2</td><td align=\"left\">1</td><td align=\"left\">3</td><td align=\"left\">67</td><td/></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"left\">Health insurance</td><td/><td/><td/><td/><td align=\"left\">0.89</td><td/><td/><td/><td/><td align=\"left\">0.41</td></tr><tr><td align=\"left\"> No</td><td align=\"left\">2</td><td align=\"left\">6</td><td align=\"left\">8</td><td align=\"left\">25</td><td/><td align=\"left\">5</td><td align=\"left\">3</td><td align=\"left\">8</td><td align=\"left\">63</td><td/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">31</td><td align=\"left\">80</td><td align=\"left\">111</td><td align=\"left\">28</td><td/><td align=\"left\">54</td><td align=\"left\">26</td><td align=\"left\">80</td><td align=\"left\">68</td><td/></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"left\">Family education</td><td/><td/><td/><td/><td align=\"left\">0.96</td><td/><td/><td/><td/><td align=\"left\">0.41</td></tr><tr><td align=\"left\"> Completed 9<sup>th </sup>or less</td><td align=\"left\">1</td><td align=\"left\">3</td><td align=\"left\">4</td><td align=\"left\">25</td><td/><td align=\"left\">2</td><td align=\"left\">2</td><td align=\"left\">4</td><td align=\"left\">33</td><td/></tr><tr><td align=\"left\"> Completed 10<sup>th </sup>or 11<sup>th</sup></td><td align=\"left\">4</td><td align=\"left\">7</td><td align=\"left\">11</td><td align=\"left\">36</td><td/><td align=\"left\">4</td><td align=\"left\">4</td><td align=\"left\">8</td><td align=\"left\">50</td><td/></tr><tr><td align=\"left\"> Graduated High School</td><td align=\"left\">14</td><td align=\"left\">30</td><td align=\"left\">44</td><td align=\"left\">32</td><td/><td align=\"left\">23</td><td align=\"left\">8</td><td align=\"left\">31</td><td align=\"left\">74</td><td/></tr><tr><td align=\"left\"> Post high school work</td><td align=\"left\">10</td><td align=\"left\">36</td><td align=\"left\">46</td><td align=\"left\">22</td><td/><td align=\"left\">23</td><td align=\"left\">11</td><td align=\"left\">34</td><td align=\"left\">68</td><td/></tr><tr><td align=\"left\"> Graduated college</td><td align=\"left\">4</td><td align=\"left\">12</td><td align=\"left\">16</td><td align=\"left\">25</td><td/><td align=\"left\">9</td><td align=\"left\">4</td><td align=\"left\">13</td><td align=\"left\">69</td><td/></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"left\">Household size</td><td/><td/><td/><td/><td align=\"left\">0.20</td><td/><td/><td/><td/><td align=\"left\">0.89</td></tr><tr><td align=\"left\"> 2</td><td align=\"left\">13</td><td align=\"left\">44</td><td align=\"left\">57</td><td align=\"left\">23</td><td/><td align=\"left\">28</td><td align=\"left\">17</td><td align=\"left\">45</td><td align=\"left\">62</td><td/></tr><tr><td align=\"left\"> 3</td><td align=\"left\">12</td><td align=\"left\">47</td><td align=\"left\">59</td><td align=\"left\">20</td><td/><td align=\"left\">27</td><td align=\"left\">11</td><td align=\"left\">38</td><td align=\"left\">71</td><td/></tr><tr><td align=\"left\"> 4</td><td align=\"left\">6</td><td align=\"left\">16</td><td align=\"left\">22</td><td align=\"left\">27</td><td/><td align=\"left\">16</td><td align=\"left\">1</td><td align=\"left\">17</td><td align=\"left\">94</td><td/></tr><tr><td align=\"left\"> 5+</td><td align=\"left\">8</td><td align=\"left\">11</td><td align=\"left\">19</td><td align=\"left\">42</td><td/><td align=\"left\">7</td><td align=\"left\">6</td><td align=\"left\">13</td><td align=\"left\">54</td><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>The Association of CMV Status between 104 Mothers and their Children.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"4\">Mothers</td></tr></thead><tbody><tr><td align=\"left\">Household children</td><td align=\"left\">CMV positive</td><td align=\"left\">CMV negative</td><td align=\"left\">Total tested</td><td align=\"left\">Per cent positive</td></tr><tr><td align=\"left\">Number CMV positive</td><td align=\"left\">30</td><td align=\"left\">24</td><td align=\"left\">54</td><td align=\"left\">55*</td></tr><tr><td align=\"left\">Number CMV negative</td><td align=\"left\">50</td><td align=\"left\">36</td><td align=\"left\">86</td><td align=\"left\">58</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>The Association of CMV Status between Index Children and their Siblings<sup>+</sup>.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"4\">Index children</td></tr></thead><tbody><tr><td align=\"left\">Household siblings</td><td align=\"left\">Number CMV positive</td><td align=\"left\">Number CMV negative</td><td align=\"left\">Total tested</td><td align=\"left\">Per cent positive</td></tr><tr><td align=\"left\">Number CMV positive</td><td align=\"left\">8</td><td align=\"left\">2</td><td align=\"left\">10</td><td align=\"left\">80*</td></tr><tr><td align=\"left\">Number CMV negative</td><td align=\"left\">1</td><td align=\"left\">26</td><td align=\"left\">27</td><td align=\"left\">3</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Comparison of Study Subjects to African-Americans Locally, State-wide, and Nationally.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"4\">Group</td></tr></thead><tbody><tr><td align=\"left\">Characteristic</td><td align=\"left\">Study subjects</td><td align=\"left\">City of Richmond *</td><td align=\"left\">Virginia (entire state)*</td><td align=\"left\">US population*</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\">Average number of persons/household</td><td align=\"left\">2.9</td><td align=\"left\">2.5</td><td align=\"left\">2.7</td><td align=\"left\">3.0</td></tr><tr><td align=\"left\">Median household income</td><td align=\"left\">$29,000</td><td align=\"left\">$25,292</td><td align=\"left\">$32,080</td><td align=\"left\">$29,423</td></tr><tr><td align=\"left\">Per cent of female children</td><td align=\"left\">51.6</td><td align=\"left\">51.5</td><td align=\"left\">49.8</td><td align=\"left\">49.3</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\">Percent of Household adults by education level</td><td/><td/><td/><td/></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\">&lt; 9<sup>th </sup>grade</td><td align=\"left\">1</td><td align=\"left\">5</td><td align=\"left\">5</td><td align=\"left\">8</td></tr><tr><td align=\"left\">9<sup>th </sup>to 12<sup>th </sup>grade</td><td align=\"left\">13</td><td align=\"left\">24</td><td align=\"left\">19</td><td align=\"left\">12</td></tr><tr><td align=\"left\">High school or equivalency</td><td align=\"left\">39</td><td align=\"left\">34</td><td align=\"left\">32</td><td align=\"left\">29</td></tr><tr><td align=\"left\">Some college or associate degree</td><td align=\"left\">34</td><td align=\"left\">27</td><td align=\"left\">30</td><td align=\"left\">27</td></tr><tr><td align=\"left\">Bachelor's or postgraduate degree</td><td align=\"left\">13</td><td align=\"left\">10</td><td align=\"left\">14</td><td align=\"left\">24</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><p>* p-values are reported from repeated-measures logistic regression to account for within family correlation. All p-values test for the significance of the characteristic after adjusting for age, in both children and adults, and the quadratic trend in age for children.</p><p><sup>+ </sup>Pos = CMV seropositive. Neg. = CMV seronegative. %Pos = the percentage of all test results that were positive.</p></table-wrap-foot>", "<table-wrap-foot><p>*Not significantly different from the rate of seronegative children, Fisher's exact p-value = 0.86</p></table-wrap-foot>", "<table-wrap-foot><p><sup>+</sup>From 30 families.</p><p>*Significantly different from the rate of seronegative siblings, Fisher's exact p-value &lt; 0.0001.</p></table-wrap-foot>", "<table-wrap-foot><p>*From the 2000 US Census.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1471-2334-8-107-1\"/>" ]
[]
[{"surname": ["Staras", "Dollard", "Radford", "Flanders", "Pass", "Cannon"], "given-names": ["SAS", "SC", "KW", "WD", "RF", "MJ"], "article-title": ["Seroprevalence of cytomegalovirus infection in the United States, 1988\u20131994"], "source": ["CID"], "year": ["2006"], "volume": ["43"], "fpage": ["1143"], "lpage": ["51"], "pub-id": ["10.1086/508173"]}, {"surname": ["Wang", "Adler"], "given-names": ["JB", "SP"], "article-title": ["Salivary antibodies to cytomegalovirus glycoprotein B accurately predict CMV infection among preschool children"], "source": ["J Clin Micro"], "year": ["1996"], "volume": ["34"], "fpage": ["2632"], "lpage": ["34"]}, {"surname": ["Fowler", "Pass"], "given-names": ["KB", "RF"], "article-title": ["Risk factors for congenital cytomegalovirus infection in the offspring of young women: Exposure to young children and recent onset of sexual activity"], "source": ["Pediatric"], "year": ["2006"], "volume": ["118"], "fpage": ["286"], "lpage": ["92"], "pub-id": ["10.1542/peds.2005-1142"]}]
{ "acronym": [], "definition": [] }
12
CC BY
no
2022-01-12 14:47:26
BMC Infect Dis. 2008 Aug 1; 8:107
oa_package/e6/69/PMC2529299.tar.gz
PMC2529300
18664276
[ "<title>Background</title>", "<p>Knee osteoarthritis (KOA) is a growing problem in the elderly, resulting in pain, functional limitations, disability and decreased quality of life leading to lost productivity and increased health care costs [##UREF##0##1##,##REF##9144852##2##]. The pathophysiological basis of KOA is multifaceted and includes intra-articular inflammation and collagen degradation, impaired muscle function, reduced proprioceptive acuity and the psychological traits of chronic pain [##REF##9458215##3##, ####REF##9230035##4##, ##REF##11522807##5##, ##REF##3348824##6####3348824##6##]. Currently, there are neither feasible preventive intervention strategies nor effective medical remedies for the management of KOA.</p>", "<p>Over the past 2 decades, Tai Chi, a form of mind-body therapy, has spread worldwide for health and fitness [##REF##15188733##7##]. Tai Chi combines deep diaphragmatic breathing and relaxation with many fundamental postures that flow imperceptibly and smoothly from one to the other through slow, gentle, graceful movements. Significant improvements have been reported in balance, strength, flexibility, cardiovascular and respiratory function, and reduction of pain, depression, anxiety and arthritic symptoms in a variety of patient populations including KOA [##UREF##1##8##].</p>", "<p>Thus, Tai Chi has the potential to become a novel, logistically feasible way of providing standardized exercises with a complementary mind-body approach to the management of KOA. The physical component provides exercise that is consistent with recommendations for OA (range of motion, flexibility, muscle conditioning, and aerobic cardiovascular exercise) [##REF##14644851##9##], while the mind component has the potential to increase psychological well-being, life satisfaction, and perceptions of health [##REF##18440398##10##]. These effects are especially pertinent for the treatment of older adults who have OA with knee pain and poor physical function.</p>", "<p>To date, only five randomized controlled trials (RCTs) conducted between 2000 and 2007 have compared the effect of Tai Chi with various controls in patients with OA [##REF##18323713##11##, ####REF##17264104##12##, ##REF##12966613##13##, ##REF##17443749##14##, ##REF##11129742##15####11129742##15##]. The results of three RCTs suggested significant pain reduction compared to controls [##REF##18323713##11##, ####REF##17264104##12##, ##REF##12966613##13####12966613##13##], but the other two found no significant changes [##REF##17443749##14##,##REF##11129742##15##]. Significant improvements in physical function were also reported in three RCTs compared with controls [##REF##18323713##11##,##REF##12966613##13##,##REF##17443749##14##], but no effects were seen in the other two [##REF##17264104##12##,##REF##11129742##15##]. Of the two RCTs [##REF##17443749##14##,##REF##11129742##15##] that evaluated the effects of Tai Chi on quality of life, only one reported positive results for Tai Chi compared with controls [##REF##11129742##15##]. In addition, only two RCTs reported significant differences between Tai Chi and control in improvements in flexibility or balance [##REF##18323713##11##,##REF##12966613##13##]. Heterogeneity of controls, different Tai Chi styles, doses and duration in addition to multiple OA sites prohibit a meaningful comparison across these trials. Furthermore, the absence of radiographic evidence of KOA as specified by the ACR criteria for OA [##REF##17443749##14##], high dropout rates [##REF##12966613##13##,##REF##17443749##14##], small sample size [##REF##18323713##11##, ####REF##17264104##12##, ##REF##12966613##13####12966613##13##,##REF##11129742##15##], the lack of standardized outcome measures and short follow up [##REF##18323713##11##, ####REF##17264104##12##, ##REF##12966613##13##, ##REF##17443749##14##, ##REF##11129742##15####11129742##15##] limit widespread applicability of the results from these studies.</p>", "<p>Because the overall findings from these RCTs suggest some favorable effects of Tai Chi on pain, physical function, quality of life, balance and flexibility in patients with KOA, a well designed study may be able to overcome the limitations of the previous studies and provide a more useful treatment. We hypothesize that Tai Chi may be beneficial to patients with KOA as a result of an effect on muscle strength, flexibility, pain, stress and anxiety as well as \"mind-body\" interactions. We therefore designed a 12 week trial with long term 1 year follow up to obtain data on the effects of Tai Chi on pain (as a marker of disease activity), functional independence (a marker of impairment), disability, joint proprioception and health status in elderly people with KOA.</p>", "<p>In this paper, we present the design and detailed protocol of a single-blinded, randomized controlled trial as well as a discussion of the overall challenges of conducting this trial with respect to strategies for recruitment, avoidance of selection bias, the actual practice of Tai Chi, and the maximization of adherence/follow-up. We report ways to overcome the theoretical and logistic limitations and problems of conducting such a clinical trial. The results from this trial will be reported at the completion of the study in accordance with the Consolidation of Standards for Reporting Trials guidelines [##REF##18215107##16##].</p>" ]
[ "<title>Methods/Design</title>", "<title>Study design</title>", "<p>This study is a single-blinded, randomized, attention-controlled, clinical trial to evaluate the physical and psychological effects of Tai Chi for patients suffering from tibiofemoral KOA. Our goal is to compare the safety and effectiveness of Tai Chi with an attention control (wellness education and stretching program) in 40 patients with KOA. (The \"single-blind\" study design has been decided upon because of the inability to conceal Tai Chi assignment allocation from participants and the instructors in Tai Chi clinical trials. However, all study evaluators will be masked to treatment assignment throughout the duration of the study.)</p>", "<p>Outcome parameters compare changes in knee pain, stiffness, and physical function using the well-validated Western Ontario and McMaster University Index (WOMAC), patient and physician global assessments, lower-extremity function, knee proprioception, depression, self-efficacy, social support, health-related quality of life, outcome expectation, adherence and occurrence of adverse events. Outcome measurements are performed at baseline, every week during the intervention period, and on completion of the 12-week program, in addition to the 24 week and 48 week follow-up. Study evaluators (study rheumatologist, study coordinator, exercise physiologist and statistician) are masked to treatment assignment.</p>", "<p>The study setting is located in an urban tertiary care academic hospital (Tufts Medical Center) in Boston Massachusetts. The study has received Ethics approval from the Tufts Medical Center/Tufts University Human Investigation Review committee and will be conducted in the Clinical Research Center and Division of Rheumatology at Tufts Medical Center.</p>", "<title>Study population</title>", "<p>This study comprises individuals with age &gt; 55 years, Body Mass Index (BMI) ≤ 40 kg/m<sup>2</sup>, and with knee pain on most days of the previous month during at least one of the following activities: walking, going up or down stairs, standing upright, or in bed at night [##REF##3741515##17##]. Furthermore, patients must have joint crepitus, morning stiffness lasting over 30 min. Participants are also required to have positive KOA radiological signs. Radiographic entry criteria are Kellgren and Lawrence (K/L) grade ≥ 2, defined as the presence of osteophytes in the tibiofemoral compartment and/or the patellofemoral compartment, assessed on standing anterior/posterior and lateral views (American College of Rheumatology criteria) [##REF##3741515##17##]. In addition, eligible participants must have a WOMAC pain subscale score (visual analog version) of &gt; 40 (range 0 to 500). Subjects are excluded if they have: 1. prior experience with Tai Chi or other similar types of complementary and alternative medicine such as Qi Gong, yoga or acupuncture; 2. cardiovascular or other severe disease limitations precluding full participation, as determined by the patient's primary care provider; 3. any intra-articular steroid injections in the previous 3 months or reconstructive surgery on the affected knee; 4. any intra-articular Synvisc or Hyalgan injections in the previous 6 months.</p>", "<p>Patients can continue routine medications such as non-steroidal anti-inflammatory drugs (NSAIDS) and acetaminophen, and maintain their usual treatment visits with their primary care physician or rheumatologist throughout the study. The investigators record any changes made to treatment but do not change or recommend change in medical therapy.</p>", "<title>Recruitment strategies</title>", "<p>Significant gaps in research participation do exist among ethnic minorities and thus limit the generalizability of findings. To ensure adequate enrollment of underrepresented groups, we place advertisements in the media (radio, local television, Internet, SAMPAN – a Chinese newspaper, the Boston Metro and Boston Globe newspapers). We also use the rheumatology clinic patient database at Tufts Medical Center for identifying patients with KOA. For interested respondents, the investigators provide information about the study and administer a brief, scripted interview to determine the caller's eligibility for the trial. This screening poses questions whose predictive values for KOA are known from population-based data. The lists of applicants who screen positive on the telephone interview are provided daily to the trial staff to schedule eligibility visits.</p>", "<p>During the 5-month recruitment period, we screened 366 patients from the greater Boston area using telephone interviews from which 62 potential patients were brought into the Clinical Research Center at the Tufts Medical Center for further eligibility screening. Of these, 40 (65%) were eligible after baseline evaluation and randomized to the Tai Chi or attention control group (wellness education and stretching) (See Figure ##FIG##0##1##). The major reason for ineligibility was the absence of radiographic evidence for KOA.</p>", "<title>Strategies to maximize adherence</title>", "<p>Adherence in clinical research is vital and can determine study quality and the validity of results. In order to maximize adherence, several procedures are performed: 1. Select a population of individuals who are both interested and reliable; 2. Screen patients using a well-designed questionnaire which in our previous studies consistently identified reliable individuals; 3. Discuss the proposed project in detail with each patient, especially the time commitment. A verbal and written commitment will be obtained from all participants, in which they state they will adhere to the program; 4. Schedule the visits at a time that is convenient for both patients and staff; 5. Recruit and assemble sufficient patients for baseline evaluation so that we have a large enough pool of patients to provide replacements if needed; 6. Perform the randomization after the baseline evaluation; 7. Provide friendly personal contact with participants under Institutional Review Board approval; 8. Organize entertaining Tai Chi and education intervention classes. Patients receive useful information that they could not reasonably expect to obtain through regular clinical care.</p>", "<title>Sample size</title>", "<p>Our empiric sample size is guided by numbers and outcomes of an RCT conducted at Tufts University that tested an exercise intervention among older adults with KOA [##REF##11469475##18##]. That study enrolled 46 patients and randomized them to either 4-month home-based progressive strength training or an attention control group of similar duration. The results were that the strength training group experienced a 36% decrease in the WOMAC index pain subscale (the primary outcome) compared to an 11% decrease in the attention control group. Thus, the exercise group had a mean change in the WOMAC pain score of -79 (SD = 91), while the control group had a mean change of -20 (SD = 77). Based on those numbers, a sample size of 20 per group and alpha = 0.05, would have power of 60% to detect a between-group difference of -59.0. While we recognize that the study is underpowered for a definitive comparison, we are primarily concerned with gathering preliminary data in order to plan and evaluate this innovative research direction.</p>", "<title>Randomization</title>", "<p>Randomization assignments are made using computer-generated random numbers to randomize permuted blocks of size 2 and 4 so that each block is complete. They are provided in a sealed, opaque envelope in two groups of 10 and opened upon the patient's agreement to participate. The block size is randomly assigned to minimize correct prediction of assignments while preserving approximate balance between groups. Specially designed software developed by Tufts Medical Center is used to generate the list of random numbers and treatment assignment. Randomization envelopes are not opened unless a patient meets eligibility criteria and completes the informed consent and baseline assessment. All study envelopes are saved in the individual patient's study notebook. The statistician (CS) closely supervises the preparation process with treatment assignment packets being prepared and checked on specific days. The maximum period between screening and randomization is 3 months.</p>", "<title>Intervention</title>", "<p>Forty ambulatory patients with KOA are randomly assigned to receive Tai Chi (n = 20) or attention control (wellness education and stretching) (n = 20) in twice-weekly one-hour group sessions for 12 weeks (Figure ##FIG##0##1##).</p>", "<title>Tai Chi</title>", "<p>The Tai Chi program is based on the classical Yang Style [##UREF##2##19##] with some modifications as described below. Patients participate in two 60-minute Tai Chi sessions conducted weekly for 12 weeks. Each session includes: 1. warm up and review of Tai Chi principles and techniques; 2. Tai Chi exercises; 3. breathing techniques; and 4. various relaxation methods. The teachings are carried out by a Tai Chi master (RR) who has over 20 years experience conducting Tai Chi Mind-Body exercise programs. Several modifications are developed by the Tai Chi master to achieve the physical (body) and mental (mind) goals of the study for KOA, accommodate KOA symptoms and limit dropouts. For example, we eliminate the 90 degree knee-flexor joint stance used in the traditional Tai Chi exercise that would place stress on the knees and replace it by having patients rest their backs on the wall to strengthen the quadriceps muscles and also by performing sitting and standing exercises from a chair. Patients perform 2 sets: one with the legs together and one with legs apart to further strengthen various muscles around the knee. In addition, the ability to tap into the power of the mind is developed further by having patients perform visualizations while sitting on the edge of a chair. Subjects are instructed to practice Tai Chi at least 20 minutes a day at home and encouraged to maintain their usual physical activities, but not to participate in additional new strength training other than their Tai Chi exercises.</p>", "<title>Attention control (wellness education and stretching)</title>", "<p>We use a wellness education and stretching program for the control group because this approach has been successfully used in other studies [##REF##11469475##18##,##UREF##3##20##, ####UREF##4##21##, ##UREF##5##22####5##22##]. The program provides an active control for the attention being paid to the Tai Chi group. There is adequate personal contact with the subjects (for attention) with little anticipated effect on the main outcomes. The control groups also attend two 60-minute sessions per week for 12 weeks. Each session starts with 40 minutes of didactic sessions on 1) OA knowledge; 2) diet and nutrition; 3) therapies to treat OA; and 4) physical and mental health education (recognizing and dealing with stress and depression, etc). The final 20 minutes of the hourly session consists of stretching exercises involving the upper body, trunk and lower body, each stretch being held for 10 to 15 seconds. Subjects are also instructed to practice at least 20 minutes of stretching exercises per day at home. All subjects are encouraged to maintain their usual physical activities, but not to participate in additional strength training other than their class stretching exercises. Throughout the 12-week period, we track the number of and reasons for any missed sessions in both groups.</p>", "<title>Measurements</title>", "<p>The Osteoarthritis Research Society currently recommends a core set of 4 domains (pain, physical function, patient's global assessment, and, for studies of at least 1 year, joint imaging) for outcome measurement for assessing KOA in clinical trials [##REF##3741515##17##,##REF##9101522##23##]. This core set is used in this study (except for joint imaging). Every participant is evaluated at baseline (prior to starting either intervention), after completing the intervention (12 weeks later), and at a 24 week and 48 week follow-up (see Table ##TAB##0##1##).</p>", "<title>Global knee pain</title>", "<p>The degree of self-reported global knee pain and function is recorded using the WOMAC index [##REF##3068365##24##]. The WOMAC is a self-reported instrument used to assess lower extremity pain, stiffness and physical function. The primary endpoint of the study is the change in the pain subscale of the WOMAC between baseline and the 12-week assessment.</p>", "<p>In addition, the physician provides a global score summarizing both knees using a visual analog scale version at baseline and follow up. In the baseline assessment, we use a self-reported knee-specific global pain visual analog scale (VAS) with range 0–100 mm to compare pain severity in each knee. In the event that an enrollee has identical pain scores in both knees, our statistician randomly assigns one of these to be the 'study knee' (see statistical analysis). The 'study knee' will be the knee on which primary outcome assessments are determined and used for analysis of strength outcomes.</p>", "<title>Radiographs</title>", "<p>Anterior-posterior (AP) and lateral standing knee radiographs are obtained at the initial screening examination (the baseline assessment) using the Framingham study protocol [##REF##3632732##25##] and are screened by the study rheumatologist (RK) for acceptance criteria. AP knee radiographs are also obtained with the patient in the fully extended standing position and lateral images with the patients in the supine position with the knee in 30 degrees of flexion, as previously suggested [##REF##11085800##26##,##REF##8581753##27##]. The study rheumatologist (RK) and a musculoskeletal radiologist independently screen the radiographs using the Kellgren and Lawrence (K/L) grading system for global tibiofemoral radiographic severity, and discrepancies are resolved with a third reviewer (TM). The K/L score is determined for each knee compartment based on osteophyte formation, joint space width, and subchondral bone scleroses [##REF##13498604##28##]. All scores reported are for the most severely affected knee (study knee).</p>", "<title>Knee examination</title>", "<p>Knee examination is performed at baseline and during each follow up visit. The study rheumatologist (RK), who is blinded with respect to the patient's treatment assignments, assesses the presence and severity of the knee joint abnormalities relevant to KOA. The knee examination components were selected based on recent data demonstrating the potential for reproducibility across examiners [##REF##8581753##27##]. The study rheumatologist examines for swelling and tenderness of the knee joint and asks each subject to rate pain on a scale of none, mild, moderate or severe. The examination sequence is as follows: 1. Palpate study knee joint swelling (bulge sign, balloon test and patellar tap); 2. Palpate for patella tendon tenderness; 3. Palpate for patella tenderness; 4. Palpate for anserine bursa tenderness; 5. Palpate for popliteal space tenderness; 6. Rate pain in the knee/knee joint when limb is in motion; 7. Scale the general crepitus sign as none, fine or coarse; 8. Palpate for tibiofemoral tenderness laterally; 9. Palpate for tibiofemoral tenderness medially. Evidence of inflammation (e.g. joint effusion), joint deformity, and joint contractures will also be noted. Assessment of the patient's global status is also performed.</p>", "<title>Range of motion (flexibility)</title>", "<p>The passive range of motion for both knees (at full extension and flexion) is measured with a plastic goniometer (Whitehall Manufacturing, model G300,) marked in 1 degree increments. Patients are positioned on their sides with the lower extremity to be examined resting on a stabilizing board. The fixed arm of the goniometer is placed to align with the femur, pointing towards hip joint and the other arm in alignment with the fibula. With one hand holding the goniometer and supporting the leg, taking care to maintain axis of goniometer with knee axis, patients are asked to flex and extend their leg as far as they can. The maximal degrees of full flexion and extension are then read and recorded.</p>", "<title>Knee joint proprioception</title>", "<p>Knee proprioception, which is reduced in KOA [##REF##11097448##29##], is measured using a Biometrics™ electrogoniometer with an ADU301 angle display unit during each assessment visit. Three test angles (30, 45 and 60 degrees) are evaluated with each subject in a sitting position taken as neutral (0 degree). The electrogoniometer is placed longitudinally in alignment with the femur and tibia with a double-sided medical tape and used to determine each of the three test angles. Patients are first shown one of the angles, which is held for a few seconds, then they are asked to close their eyes and attempt to reproduce the angle; this is repeated for all three test angles.</p>", "<title>Physical performance</title>", "<p>Physical performance assessments include the timed stand, standing balance and 6 minute walk tests. Timed stand tests measure time taken to complete ten full stands from a sitting position [##REF##3966492##30##]. This is a measure of lower extremity muscle power. Patients are instructed to complete chair stand time as quickly as possible and are timed to the nearest 0.001 seconds. The same chair is utilized for testing before and after the interventions. Patients begin the chair stand seated with their arms folded across their chests, then rise to a standing position and sit back down with their back against the back rest of the chair. The test is completed when the patient stands for the 10<sup>th </sup>repetition. Chair stand time is recorded using the best (lowest) score of 2 trials.</p>", "<p>The standing balance tests include tandem, semi-tandem, side-by side, and one-legged stands. Patients are asked to maintain each position for 30 seconds. For each task, the research staff first demonstrates the task, asks the patient if they feel comfortable and ready and then supports the patient while positioning themselves [##REF##10682941##31##, ####REF##8126356##32##, ##REF##7838189##33####7838189##33##]. One point is given if they exceed 30 seconds and none if they can not do or do not attempt the test.</p>", "<p>The six minute walking test is a reliable measure of functional exercise capacity [##REF##3978515##34##]. Patients are asked to walk as fast and as far as possible within the 6-minute period and are accompanied by the research staff using a wheel measure (Redi measure, Redington, Windsor, CT) that measures distance covered in inches. Patients are given verbal encouragement throughout the 6 minutes and are informed of the remaining time throughout the 6 minutes. The distance covered at the end is noted and recorded.</p>", "<title>Health Related Quality of Life (HRQL)</title>", "<p>HRQL assessments are made using the Medical Outcome Study Short Form 36 (SF-36) [##REF##1593914##35##], and the EuroQol (EQ-5D) [##REF##10109801##36##] instruments. The SF-36 measures 8 domains: physical functioning, role-physical, bodily pain, general health, vitality, social function, emotional health and mental health. The EQ-5D measures 5 dimensions: mobility (disability), self-care (disability), usual activities (handicap), pain/discomfort (impairment), and anxiety/depression (impairment). There is also a visual analogue thermometer rating scale to evaluate the overall patient perception of health on a 0 to 100 scale. Higher scores indicate a better health state.</p>", "<title>Depression</title>", "<p>The Center for Epidemiology Studies Depression (CES-D) index is used to assess depressive symptoms [##UREF##6##37##]. It includes a 20-item Likert-type scale with scores ranging from 0 to 60. Higher scores indicate greater dysphoria.</p>", "<title>Outcome expectation</title>", "<p>Outcome expectations are beliefs that carrying out a specific behavior such as physical activity will lead to a desired outcome. The brief outcome expectations scale (OES) [##REF##11078112##38##] contains questions that ask about physical and mental benefits and will be used to assess outcome expectations. The measure is scored by summing the ratings for all the items and dividing by 9 to get the average of all 9 items. Scores can range from 1 to 5, with 1 indicating low outcome expectations for the exercise and 5 suggesting high outcome expectations.</p>", "<title>Self-efficacy</title>", "<p>Self-efficacy is important for individuals to adopt and maintain a program of regular physical activity. It is assessed using the self-efficacy instrument developed by Marcus et al [##REF##1574662##39##]. The patient rates his/her confidence of being physically active in different types of situations on a 5-item scale with responses ranging from \"not at all confident\" to \"extremely confident\". The total score is computed by calculating the average of all 5 questions. A higher score indicates greater self-efficacy.</p>", "<title>Social support</title>", "<p>This is assessed using the social support for physical activity scale adapted from Cohen and colleagues [##UREF##7##40##]. Thirteen questions rated from 0 to 5 were used to assess the influences of family and friends on patients in the last 3 months as they performed regular physical activity. Higher scores reflect more perceived social support from these individuals.</p>", "<title>Physical Activity Enjoyment</title>", "<p>The Physical Activity Enjoyment Scale adapted from Kendzierski and DeCarlo [##UREF##8##41##] is used to evaluate enjoyment of physical activity. It is a self-administered 18-item, Likert scale used by patients to rate their current feelings about physical activity. The total scale score, computed after recoding, is obtained by summing the scores of all the items. High scores correspond to increased enjoyment, while low scores correspond to little enjoyment.</p>", "<title>Adherence</title>", "<p>Participants' attendance is monitored during each session for 12 weeks for both interventions via signing of attendance sheets. Patients are also asked to maintain daily Tai Chi or stretching exercise activity logs during the 12 week intervention and are encouraged with phone calls from the research staff to continue throughout the follow up period. At the 24 and 48 week visit, the research staff will ask about the average number of times per week and the number of minutes per session the subject practices at home.</p>", "<title>Safety</title>", "<p>Study patients are monitored weekly for the occurrence of adverse events defined as any undesirable experience during the duration of the study. Lack of effect of Tai Chi or stretching/education is not considered an adverse event. Patients are monitored weekly to determine whether an adverse event has occurred. All adverse events are recorded on an adverse event case report form.</p>", "<title>Statistical Analysis</title>", "<p>The primary outcome is the measurement of change in knee pain between baseline and 12 weeks. Secondary outcomes are measurements of change at 24 weeks and 48 weeks. These will be analyzed both as individual time points and in longitudinal analyses. Analyses will be intention-to-treat with secondary analyses based on completers.</p>", "<p>We will also explore a knee-based approach to the study that will use linear mixed models to adjust for correlations in outcome between knees. The most disabled knee will be used as the unit of analysis. One knee will be chosen at random if two knees are equally affected. Considering both knees would require accounting for the correlation between knees and for the presence of disability in only one knee for many participants. However, we will characterize individuals on the basis of unilateral vs. bilateral KOA and explore any possible influences of this characterization in secondary analyses.</p>", "<p>For the patients who withdraw during the study, every effort will be made to complete a follow-up WOMAC and clinical knee examination at the time of withdrawal. If a withdrawal assessment cannot be made, we will carry forward the last assessment of knee pain, physical function or as a conservative assessment to bias towards the null hypothesis.</p>", "<p>We will also perform a cost-effectiveness analysis to evaluate whether Tai Chi is a cost-effective therapy for patients with KOA. The costs will be estimated from health care utilization reported by subjects at the end of the trial using the Health Assessment Questionnaire that includes medication usage, hospitalization and health care professionals' visits. The differences in direct and indirect health care cost between the Tai Chi and attention control (wellness education and stretching) groups will also be compared.</p>" ]
[]
[ "<title>Discussion</title>", "<p>In this article, we present the challenges of designing a randomized controlled trial with long-term follow up. The challenges encountered in this design are: strategies for recruitment, avoidance of selection bias, the actual practice of Tai Chi, and the maximization of adherence/follow-up while conducting the clinical trial for the evaluation of the effectiveness of Tai Chi on KOA.</p>", "<p>A total of 40 eligible patients, 20 with Tai Chi and 20 with an attention control (wellness education and stretching) have already completed the study including 3 month recruitment, 12 week intervention, as well as 24 and 48 week follow-up at the Tufts Medical Center. We will perform analysis of the data and report the findings. Therefore, this project will provide important preliminary data on physical and psychological effects of Tai Chi for KOA. It will establish rigorous methods for future research for testing the mechanisms by which Tai Chi may influence pain, disability, and health related quality of life in people with KOA.</p>" ]
[]
[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Knee Osteoarthritis (KOA) is a major cause of pain and functional impairment among elders. Currently, there are neither feasible preventive intervention strategies nor effective medical remedies for the management of KOA. Tai Chi, an ancient Chinese mind-body exercise that is reported to enhance muscle function, balance and flexibility, and to reduce pain, depression and anxiety, may safely and effectively be used to treat KOA. However, current evidence is inconclusive. Our study examines the effects of a 12-week Tai Chi program compared with an attention control (wellness education and stretching) on pain, functional capacity, psychosocial variables, joint proprioception and health status in elderly people with KOA. The study will be completed by July 2009.</p>", "<title>Methods/Design</title>", "<p>Forty eligible patients, age &gt; 55 yr, BMI ≤ 40 kg/m<sup>2 </sup>with tibiofemoral osteoarthritis (American College of Rheumatology criteria) are identified and randomly allocated to either Tai Chi (10 modified forms from classical Yang style Tai Chi) or attention control (wellness education and stretching). The 60-minute intervention sessions take place twice weekly for 12 weeks. The study is conducted at an urban tertiary medical center in Boston, Massachusetts. The primary outcome measure is the Western Ontario and McMaster Universities (WOMAC) pain subscale at 12 weeks. Secondary outcomes include weekly WOMAC pain, function and stiffness scores, patient and physician global assessments, lower-extremity function, knee proprioception, depression, self-efficacy, social support, health-related quality of life, adherence and occurrence of adverse events after 12, 24 and 48 weeks.</p>", "<title>Discussion</title>", "<p>In this article, we present the challenges of designing a randomized controlled trial with long-term follow up. The challenges encountered in this design are: strategies for recruitment, avoidance of selection bias, the actual practice of Tai Chi, and the maximization of adherence/follow-up while conducting the clinical trial for the evaluation of the effectiveness of Tai Chi on KOA.</p>", "<title>Trial registration</title>", "<p>ClinicalTrials.gov identifier: NCT00362453</p>" ]
[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>CW obtained funding for the study. CW, TM, PH, RoR, CS, RK, and RaR designed the randomized controlled trial. CW, TM, PH, CS, RK and RaR conducted the research. CW and AO wrote the first draft of the manuscript. CW, TM, CS, PH, RoR, AO, RK and RaR participated in the revision of subsequent draft. All authors approved the final version of the manuscript. None of the authors declared any conflicts of financial interest.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2474/9/108/prepub\"/></p>" ]
[ "<title>Acknowledgements</title>", "<p>Dr. Wang is supported by R21AT002161 from the National Center for Complementary and Alternative Medicine (NCCAM) of the National Institutes of Health. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NCCAM or the National Institutes of Health. The authors gratefully acknowledge the Data Safety Monitoring Board members: Drs. Karen H. Costenbader, Kristin Baker, Wenjun Li and Ms. Resa Arovas for their insightful suggestions and comments for the study protocol.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Study flow chart.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Sequence of trial measurements for primary and secondary outcomes*</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>VISIT</bold></td><td align=\"center\"><bold>Baseline</bold></td><td align=\"center\"><bold>Intervention** (for 12 weeks)</bold></td><td align=\"center\"><bold>Week 12</bold></td><td align=\"center\"><bold>Week 24 Follow up</bold></td><td align=\"center\"><bold>Week 48 Follow up</bold></td></tr><tr><td align=\"left\"><bold>Time (months)</bold></td><td align=\"center\"><bold>-1</bold></td><td align=\"center\"><bold>0</bold></td><td align=\"center\"><bold>1 – 3</bold></td><td align=\"center\"><bold>6</bold></td><td align=\"center\"><bold>12</bold></td></tr></thead><tbody><tr><td align=\"left\" colspan=\"6\"><bold>Primary outcome measure</bold></td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\">WOMAC-Pain</td><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\" colspan=\"6\"><bold>Secondary outcome measures</bold></td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>WOMAC-Physical function</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>WOMAC-Stiffness</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Physicians' Global KOA Severity</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Patients' Global KOA Severity</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>SF-36 &amp; EQ-5D &amp; CES-D</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Self-efficacy</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Outcome Expectation</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Social Support</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Enjoyment Questionnaire</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Physical Functional Tests***</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Physical Activity Questionnaire</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Joint Proprioception</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"><bold>Medications</bold></td><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Adverse Events</bold></td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Body Mass Index</bold></td><td align=\"center\">X</td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Weekly Update</bold></td><td/><td align=\"center\">X</td><td/><td/><td/></tr><tr><td align=\"left\"><bold>Knee X-Ray</bold></td><td align=\"center\">X</td><td/><td/><td/><td/></tr><tr><td align=\"left\"><bold>Adherence</bold></td><td/><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td><td align=\"center\">X</td></tr><tr><td align=\"left\"><bold>Follow-up Questionnaire</bold></td><td/><td/><td/><td align=\"center\">X</td><td align=\"center\">X</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>* KOA = Knee Osteoarthritis, WOMAC = Western Ontario and McMaster Universities Osteoarthritis Index, SF-36 = Medical Outcome Survey Short-Form 36, EQ-5D = EuroQol self-repot questionnaires, CES-D = Center for Epidemiology Studies Depression index.</p><p>** Randomization to Tai Chi or Attention Control intervention.</p><p>*** Physical Function Tests include range of motion, standing balance, 6-minute walk, and chair standing.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"1471-2474-9-108-1\"/>" ]
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[{"surname": ["Cooper", "Klippel JH DPA"], "given-names": ["C"], "article-title": ["Epidemiology"], "source": ["Rheumatology"], "year": ["1994"], "volume": ["1"], "publisher-name": ["St. Louis (MO), Mosby"], "fpage": ["3.1"], "lpage": ["3.4"]}, {"surname": ["Wang", "Collet", "Lau"], "given-names": ["C", "JP", "J"], "article-title": ["The effect of Tai Chi on health outcomes in patients with chronic conditions: a systematic review"], "source": ["Archive Internal Medicine"], "year": ["2004"], "volume": ["164"], "fpage": ["493"], "lpage": ["501"], "pub-id": ["10.1001/archinte.164.5.493"]}, {"source": ["China Sports. Simplified \"Taijiquan\""], "year": ["1983"], "edition": ["2"], "publisher-name": ["Beijing, China, China Publications Center"]}, {"surname": ["Buckelew", "Conway", "Parker", "Deuser", "Read", "Witty", "Hewett", "Minor", "Johnson", "Van Male", "McIntosh", "Nigh", "Kay"], "given-names": ["SP", "R", "J", "WE", "J", "TE", "JE", "M", "JC", "L", "MJ", "M", "DR"], "article-title": ["Biofeedback/relaxation training and exercise interventions for fibromyalgia: a prospective trial"], "source": ["Arthritis Care Research"], "year": ["1998"], "volume": ["11"], "fpage": ["196"], "lpage": ["209"], "pub-id": ["10.1002/art.1790110307"]}, {"surname": ["Buckelew", "Huyser", "Hewett", "Parker", "Johnson", "Conway", "Kay"], "given-names": ["SP", "B", "JE", "J", "JC", "R", "DR"], "article-title": ["Self-efficacy predicting outcome among fibromyalgia subjects"], "source": ["Arthritis Care Research"], "year": ["1996"], "volume": ["9"], "fpage": ["97"], "lpage": ["104"], "pub-id": ["10.1002/1529-0131(199604)9:2<97::AID-ANR1790090205>3.0.CO;2-F"]}, {"surname": ["Wang"], "given-names": ["C"], "suffix": ["Lau, J., Roubenoff, R., Kalish, R., Christopher, S., Hibberd, P"], "article-title": ["Tai Chi improves pain and functional status in adult with rheumatoid arthritis"], "source": ["Arthritis & Rheumatism"], "year": ["2003"], "volume": ["48"], "fpage": ["3656"], "lpage": ["3658"]}, {"surname": ["Kohout", "Berkman", "Evans", "Cornoni-Huntley"], "given-names": ["FJ", "LF", "DA", "J"], "article-title": ["Two shorter forms of the CES-D (Center for Epidemiological Studies Depression) depression symptoms index"], "source": ["Journal of Aging Health"], "year": ["1993"], "volume": ["5"], "fpage": ["179"], "lpage": ["193"], "pub-id": ["10.1177/089826439300500202"]}, {"surname": ["Cohen", "Mermelstein", "Kamarck", "Hoberman", "Sarason IIGSBR"], "given-names": ["S", "R", "T", "HM"], "article-title": ["Measuring the functional components of social support."], "source": ["Social support: Theory, research and applications"], "year": ["1985"], "publisher-name": [", The Hague: Martinus Nijhoff."], "fpage": ["73"], "lpage": ["94"]}, {"surname": ["Kendzierski", "DeCarlo"], "given-names": ["D", "KJ"], "article-title": ["Physical activity enjoyment scale: Two validation studies"], "source": ["Journal of Sport & Exercise Psychology"], "year": ["1991"], "volume": ["13"], "fpage": ["50"], "lpage": ["64"]}]
{ "acronym": [], "definition": [] }
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BMC Musculoskelet Disord. 2008 Jul 29; 9:108
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