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You are an expert at summarizing long articles. Proceed to summarize the following text:
multiple sclerosis ( ms ) is one of the most common chronic neurological diseases , affecting between 1 in 500 to 1 in 1500 people in australia , europe , and america [ 1 , 2 ] . the condition is characterised by neuronal demyelination and inflammation , leading to axonal injury as the disease progresses [ 2 , 3 ] . ms has several forms , the most common ( 7080% ) being relapsing - remitting . ms has a typical onset between 20 and 40 years of age , having a significant impact on quality of life ( qol ) over the duration of the disease . the disease manifests with a range of physical and neurological symptoms including sensory loss , optic neuritis , motor weakness , diplopia , and limb ataxia , as well as an increased risk of stress , anxiety , and depression . ms is considered incurable ; however , many strategies exist to help manage symptoms and alter the disease course including pharmacological agents , lifestyle risk factor modification , rehabilitation , and psychosocial support all of which have varying efficacy . although meditation in all its forms has been around for centuries , interest in the practice as a medical treatment in the western world has been more recent . the interest was particularly sparked by kabat - zinn 's research into mindfulness meditation suggesting that meditation could be a useful tool for treating chronic pain . meditation is a term that encompasses a wide range of techniques such as mindfulness - based meditation ( including mindfulness - based stress reduction ( mbsr ) and mindfulness - based cognitive therapy ( mbct ) ) , mantra meditation ( including transcendental and clinically standardised meditation ) , and many more [ 10 , 11 ] . in its various forms , meditation has been shown to be associated with symptom reduction in medical and psychiatric conditions , electroencephalography ( eeg ) changes , and beneficial structural brain changes on neuroimaging with long term use [ 1117 ] . while meditation has been shown to moderate stress , anxiety , and depression , studies have often been poorly designed , with biased patient selection , poor controls , and small sample sizes among other deficiencies detracting from the credibility of the literature . ms is associated with significant rates of depression [ 7 , 1921 ] , anxiety , stress , chronic pain [ 23 , 24 ] , and fatigue which are often important predictors of qol [ 7 , 26 ] . stress and other mental health - related comorbidities appear to play a role in increasing relapse rates and decreasing qol in ms [ 27 , 28 ] . yet , few studies have examined the effect of meditation on mental health outcomes or qol in ms , despite its great potential benefit . this integrative review aims to examine the existing peer - reviewed literature on the use of meditation in ms to reduce depression , anxiety , stress , chronic pain , and fatigue and whether meditation can lead to improved qol for those with ms . it also aims to gather evidence on the effectiveness of meditation as secondary prevention for ms morbidity . this review of literature published prior to march 2014 was undertaken using pubmed , psycinfo , the cochrane library , and google scholar . search terms used were multiple sclerosis , ms , meditation , and mindfulness ( all were searched as mesh terms ) . the boolean operator term or was used to search [ mindfulness or meditation ] ; these results were then combined with the boolean operator term and to search [ ( mindfulness or meditation ) and ( multiple sclerosis or ms ) ] . articles which cited a key randomised controlled trial by grossman et al . were searched on google scholar . only english language articles were included ; no other limits were set . primary literature was selected only if the patient sample had ms and the assessment of mindfulness or another form of meditation ( hypnoses and complementary and alternative therapies were excluded ) was examined in the study , including both observation and intervention studies . intervention studies were accepted regardless of whether or not there was a control group . in the context of an integrative review , all qualitative and quantitative research was selected ( assuming it met the criteria above ) regardless of whether validated tools were used to measure outcomes or not . in studies where ms and another medical condition were examined , the reference list of all studies that were accepted for the review was analysed to see if there were any additional relevant papers that had not been found using the search methods above . all search results were reviewed by a single reviewer ( al ) to determine relevance to the criteria ; included papers were then reviewed by a second reviewer ( eh ) and any discrepancies were resolved by a third reviewer ( tw ) . the assessment of intervention and observation studies was guided by the prisma guidelines and the strobe guidelines , respectively . selected papers were summarised in table 1 , highlighting study type , study focus , methodology , participant information , control details ( if applicable ) , outcomes , and limitations . using the search criteria 19 results were found on pubmed , psycinfo , and the cochrane library and 9030 results were found on google scholar . there were also 72 articles which cited grossman et al . on google scholar . using the selection criteria 12 pieces of primary literature were selected to be included in the literature review . of these , two were randomised controlled trials [ 29 , 32 ] , four were cohort or pre- and postinterventional studies [ 3336 ] , and six were surveys [ 3742 ] ( table 1 ) . grossman et al . conducted a randomised controlled trial of 150 patients residing in switzerland with relapsing - remitting or secondary progressive ms . cases were assigned : one 2.5 hour session of mindfulness meditation a week for eight weeks , a one - day seven - hour session , and 40 minutes of meditation homework daily . it was found that , using validated self - report measures , the meditation group had significant improvements in qol and significantly lower rates of fatigue ( mfis ) , depression ( ces - d ) , and anxiety ( stai ) when compared to the control group after intervention and at a six - month follow - up . this trial was soundly constructed and provides reliable evidence that meditation may have a beneficial effect on qol and mental health - related comorbidities . the major limitation of the study was the lack of a sham control group , which meant that the self - efficacy effect may have influenced the results . mills and allen conducted a randomised controlled trial on patients with secondary progressive ms , with 12 participants randomised to the meditation group and 12 to the control group . the meditation group received an hour of one - on - one meditation sessions for six weeks as well as handouts , tapes , and written material . the control group continued on normal therapy . at the completion of the mindfulness course and at three months after intervention , self - reported ms symptoms were found to have significantly improved in the intervention group . each participant also had a relative or a friend assess their symptoms before and after intervention and significant improvements were also noted with that measure . given the small size of the study and a relatively large dropout rate , the results must be treated with caution . additional limitations include not having any sham control and follow - up being limited to three months . in a longitudinal study , hadgkiss et al . and li et al . found that a five - day live - in course which focussed on lifestyle modifications including diet , sunlight exposure , exercise , and meditation improved physical and mental hrqol compared to baseline ; effects were followed and found to be significant at one , two and a half , and five years after the course was completed [ 33 , 34 ] . while it was conducted thoroughly , the effect of , and adherence to , meditation specifically was not differentiated from the other program recommendations . performed an intervention study of meditation in ms patients , with the intervention group undertaking weekly meditation classes for two months and control subjects receiving standard care . it was found that those who participated in meditation had significantly less pain , improvements in cognitive and psychosocial aspects of fatigue , and improved qol after intervention but no statistically significant change in mobility . the study was limited by a small sample size ( less than 20 patients with ms ) , lack of long term follow - up , and lack of a sham control group . furthermore , it was not randomised meaning that those who chose to be in the meditation group may have had an increased perception of the beneficial effects of meditation . pritchard et al . conducted a study which demonstrated decreased stress compared to baseline after a six - week meditation course . stress was assessed on a validated scale and shown to significantly decrease after the course , suggesting an association between meditation and decreased stress levels in ms . the study was limited by a small sample size ( only 12 ms patients ) , no control group , and lack of long term follow - up . in a survey of over 3000 ms patients conducted by nayak et al . , it was found that 12% of the survey responders had tried meditation , with modest efficacy ( determined by a self - reported five - point scale of reduction in perceived disease severity ) . participants reported pain , overall symptoms , and fatigue as the top three issues that meditation was helpful for . this is one of the largest studies of its type and probably represents the best estimate of meditation use in ms in the western world . berkman et al . found that 22.9% of survey responders had tried meditation , reporting benefits of stress relief , relaxation , lower fatigue , strengthened immune system , and , interestingly , slower disease progression . in an online survey of 2529 patients with ms , which looked at a range of issues relating to the disease , simmons et al . found that 9% of respondents had ever practised meditation and reported that it improved their ms . interestingly , a small number of respondents ( 0.2% ) reported that meditation worsened their ms symptoms . in another large online survey skovgaard et al . also found that 5% of responders had used meditation in the previous 12 months to help treat their ms symptoms the survey found that respondents who had tried any form of complementary or alternative medicine were most likely to be females aged between 18 and 40 years old , which was not surprising given this is a significant proportion of the ms demographic . it was also found that meditators were more likely to have been university - educated and have a higher income . undertook a cross - sectional survey examining trait mindfulness and its effect on various outcomes , all assessed using validated tools . they found that having a mindful disposition ( as assessed by the five facet mindfulness questionnaire ( ffmq ) ) was significantly associated with decreased perceived stress ( model accounted for 25% variance ) , increased resilience ( 44% ) , increased adaptive coping ( 11% ) , decreased maladaptive coping ( 29% ) , and higher mental health qol ( 20% ) . it should be noted that participants ffmq scores were independent of whether they had participated in formal meditation practice or not , and as such the variation in the above mentioned scores can not be solely attributed to participation in meditation . in a mixed methodology study , incorporating a survey of attendees at an ms congress and focus group of attendees at a complementary therapies and ms workshop , conducted by esmonde and long , 25% were found to have participated in relaxation and meditation activities in the previous 12 months . reported benefits included improved sleep , reduced spasticity , easing of muscle tension , and increased sense of well - being . relaxation and meditation were reported to be at least somewhat helpful in relieving ms symptoms , with over a quarter rating it as extremely helpful . participants in the workshop may have been more likely to ascribe benefits to the practice of relaxation and meditation as they may have been more open to trying nonpharmacological therapies . the study was also limited by the use of a nonvalidated survey and having participants discuss their experiences in small groups rather than individually , which may have led to unease discussing personal experiences , especially if they were negative . nevertheless , the reports of benefits for some patients are an indication that meditation may be a useful augmentative treatment in ms . surveys were sent to large cohorts and were voluntarily - completed leading to a selection bias as patients who had tried meditation and found it effective may have been more likely to commence and complete the surveys . furthermore , results were limited by the self - reporting nature of surveys , which could have been biased by poor recall or under- or overestimation of responses . some useful analyses within these papers did not distinguish meditation from cam therapies , thus limiting the interpretation of findings . in some surveys it was difficult or even impossible ( in the case of online surveys ) to verify the ms diagnosis [ 37 , 39 , 40 ] . additionally , many of the survey populations were limited to one or two countries [ 37 , 39 , 41 , 42 ] , meaning that the findings may not be generalisable to the larger population of patients with ms . some of the studies did not employ validated instruments , so the reliability and validity of results are not clear [ 37 , 40 , 41 ] . additionally , some surveys had a small sample size [ 41 , 42 ] , which limited reliability and statistical precision of the findings . finally , all of the surveys were cross - sectional from which causation or change over time can not be deduced . past studies have found that 85% of ms exacerbations were associated with stressful life events , strongly suggesting a link between stress and disease course . further , multiple meta - analyses and systematic reviews have found that while there are other factors that contribute to morbidity , such as viral infections , stress plays an important role in the number of relapses patients with ms experience and is often underrecognised [ 27 , 47 , 48 ] . mohr et al . demonstrated that stressful life events were associated with new brain lesion formation ( assessed with mri ) four to eight weeks after a moderate stressor . cortisol , which is released by the hypothalamic pituitary axis in response to stress , is often chronically elevated in ms patients . as a result of chronically increased levels of cortisol , it has been proposed that ms patients form resistance to the hormone and as such do not benefit from the normal anti - inflammatory effects of the hormone , which would otherwise help to allay their symptoms . meditation has been shown to decrease cortisol levels and improve sleep for both novice and experienced meditators . given that cortisol plays a key role in stress modulation , the results suggest that the beneficial effects of meditation may operate partly through this mechanism . additionally , meditation has been shown to be associated with activation of the prefrontal cortex and the anterior cingulated cortex , which are areas of the brain associated with concentration as well as altering eeg tracings in many medical conditions . given that stress has been shown to be implicated in relapses in ms [ 46 , 49 ] and that meditation has been shown to relieve biological markers of stress [ 14 , 51 ] it is reasonable to hypothesise that meditation may have a direct impact on ms disease course through its effect of modulating the stress response , although this has not yet been studied in detail . because stress and depression affect qol in people with ms and may lead to disease exacerbation , mind - body interventions on disease course in ms as well as other neurological conditions [ 5355 ] , including a recent systematic review looking at the use of meditation in ms specifically . this latter systematic review however considered only randomised controlled trials ; given the paucity of these in the literature , the review may therefore be considered to have too narrow a focus . integrative reviews in contrast take a broader view including qualitative and quantitative research as well as discussion papers and grey literature . given the difficulties in conducting randomised controlled trials in this area , the broader approach we have adopted is necessary to better synthesise current knowledge and help develop a more realistic view of the effects of meditation on ms . integrative reviews on meditation as a therapeutic technique in ms have been lacking , thereby warranting the present review . our review shows that a substantial proportion of people with ms either have tried meditation or meditate regularly . the available body of research has consistently found meditation to be of benefit to those with ms , with very few or no harmful side effects . despite weaknesses in the evidence base , meditation appears to have important benefits in improving qol as well as potential benefits for the management of pain , stress , fatigue , and depression risk in people with ms . there is currently however limited evidence to suggest that meditation has an effect on relapse rates or other markers of disease course , although it is biologically plausible and this may be shown to be true in future better - designed studies . limitations of the review . given that meditation is an umbrella term for many different practices , there was a degree of heterogeneity between studies making it difficult to directly compare between different cohorts . many studies had limitations , most notably self - reported outcomes , failure to use well - validated tools , and , in the case of randomised controlled trials , the absence of sham intervention . the literature review was further limited by a small number of primary resources directly relating to meditation and multiple sclerosis . overall the evidence supports a beneficial effect of meditation on symptom management and qol among people with ms . it seems that meditation may have an important role to play in the integrated management of ms . further studies on meditation in ms are required , preferably randomised controlled trials utilising some sham form of therapy . ms is associated with a higher risk of depression , anxiety , stress , chronic pain , and fatigue , and stress has been shown to worsen ms course . meditation has been investigated as a possible beneficial intervention for stress and symptom relief for people with ms . the current literature suggests a beneficial effect of meditation in ms , particularly qol ; however , further research is needed to better understand the potential for meditation as an adjunct to the management of ms . | background . multiple sclerosis ( ms )
disease course is known to be adversely affected by several factors including stress . a proposed mechanism for decreasing stress and therefore decreasing ms morbidity and improving quality of life is meditation .
this review aims to critically analyse the current literature regarding meditation and ms .
methods .
four major databases were used to search for english language papers published before march 2014 with the terms ms , multiple sclerosis , meditation , and mindfulness .
results .
12 pieces of primary literature fitting the selection criteria were selected : two were randomised controlled studies , four were cohort studies , and six were surveys .
the current literature varies in quality ; however common positive effects of meditation include improved quality of life ( qol ) and improved coping skills .
conclusion .
all studies suggest possible benefit to the use of meditation as an adjunct to the management of multiple sclerosis .
additional rigorous clinical trials are required to validate the existing findings and determine if meditation has an impact on disease course over time . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
minimal change disease , formerly known as lipoid nephrosis , is responsible for approximately 7090% of childhood nephrotic syndrome and for 1015% of adult nephroses . generally speaking , it presents itself clinically as a primary renal disease , however eventually secondary to several other conditions such as : hodgkin 's disease , certain allergic states , as well as naiads ( a situation which is known as toxic interstitial nephritis . undoubtedly , fasciculation 's , even isolated ones , are still considered to be one of the most ominous clinical signs for the neurologist due to their relationship with amyotrophic lateral sclerosis and other motor neuron diseases . in view of this , clinical and neurophysiological reevaluation , as well as specific complementary laboratorial exams , unfortunately , attempts at precisely outlining the true meaning of benign fasciculation 's hit upon the absence of specific neurophysiological and clinical studies . happily , fasciculation 's are also present in completely normal individuals with no ther underlying pathologic processes involved . however , discontinuing the drug , even cautiously , is also potentially hazardous because of the possible occurrence of two events : tapering off syndrome ( abstinence ) and secondary adrenocortical failure . there is no research available in the current medical literature that associate use of corticosteroids with the ocurrence of bfs . thus , the objective of the study at hand is to present the case of a patient who began presenting with benign fasciculations at the moment of corticosteroid tapering , in this specific case , methypredsilone . a 28-year - old man , medical student , reports the onset in december of 2008 , after initiating tapering schedule for corticosteroids ( methyl - prednisolone,1 mg / kg / day ) , for treatment of mcgn started to present with fasciculations , diffuse in nature , migratory and intermittently . he says that the onset of symptoms coincided with the tapering of corticosteroids , which was initially at a dose of 60 mg / kg / day during 15 days , being gradually reduced to 20 mg / day on an alternate - day schedule , unitl its complete withdrawal in 8 weeks . he also says he had previously experienced the same clinical picture when his fasciculatory symptoms lasted for 3 months following abstinence . initially , the fasciculation 's were noticed in the proximal third of his arm and the distal third of his thigh . later on , intrinsic muscles of the hand and face as well as the shoulder girdle were also involved . they would last several days em specific regions , and , subsequently , would spread to other more distant parts of the body . physical exertional activity would worsen the fasciculations and would sometimes evolve into painful joint / arthritic - like symptoms . the patient was them submitted to a neurologic exam in december of 2008 , which revealed upon inspection , rapid , diffuse and visible contractions in face , trunk , upper and lower limbs . a cbc , electrolyte profile ( potassium= 4.2 meq / l ) , ( calcium= 5.1 meq / l , magnesium= 2.2 meq / l , phosphorus= 2.9 meq / l ) thyroid function , viral serology , and basal cortisol levels were ordered . serum creatinine ( 0.8 mg\dl ) and creatine kinase ( 102 u\l ) . the patient also denied neither preceding viral event , nor a bacterial one in the preceding days or weeks . an enmg at same month , revealed an interval between resting motor action potentials of 0.9 s. high amplitude and duration action potentials , positive sharp waves , and fibrillation potentials were also found upon testing . no medication was prescribed . in may of 2009 , after six months of its onset , the patient returned to the outpatient unit to inform the cessation and resolution of all symptoms . fasciculations are the result of spontaneous discharges of motor units , although the exact physiological mechanism involved is not yet known . fasciculation potentials may eventaully persist after distal nerve block , thus suggesting axonal or even anterior horn cell damage . experimental studies support the idea that the distal origino fasciculations may be reflected in their presence among distal axonal ramifications ( arborizations ) . this probably represents changes in conduction properties of ionic channels , such as fluctuations in ionic gradients along axonal membranes . the distinction between benign ( no association with serious neurologic conditions ) and malignant fasciculations ( commonly associated with degenerative neurologic conditions ) should be liberally used since the most important factor here is without a doubtless is the evaluation of the morphology , duration , and number of phases of the motor action potential as well as the presence or absence of other types of spontaneous activity ( acute positive waves , fibrillation potentials ) . it should also be noted that there are countless clinical situations in which fasciculations is a major chief complaint . among several causes of fasciculations , we may include , fasciculation 's in normal individuals , as a result of neurologic disorders , associated with metabolic diseases , medication - induced and fasciculation 's of systemic diseases ( table 1 ) . table 1fasciculations causes.1benign fasciculations ( healthy subjects)1.1 occasional ( mainly in forearms , calves and thumb).1.2 caused by coffee , stress , anxiety , cigarettes.1.3 strenuous and high intensity exercises .1.4 benign fasciculations ( isolated and persistent ) - duration for months and | or years.2neurogenic disorders resulting from fasciculations2.1 amyotrophic lateral sclerosis.2.2 spinal muscular atrophies.2.3 benign monomelic amyotrophy ( hirayama disease).2.4kennedy disease2.5 multifocal motor neuropathy2.6 acute anterior poliomyelitis2.7 radiculopathy2.8 peripheral neuropathies2.9 plexopathy2.10 syringomyelia2.11creutzfeldt - jakob disease2.12 spinal amyotrophy of upper limbs ( distal)3fasciculations for metabolic diseases3.1 thyrotoxicosis3.2 tetanus4fasciculations caused by drug use4.1 anticholinergic drugs4.2 * * use of steroids ( corticoids)4.3 organophosphates and other insecticides | pesticides ( commonly in the acute intoxication).4.4 lithium5fasciculations caused by systemic diseases5.1 infections , mainly viral . ( hiv-1 , 2 , htlv-1 , syphilis)5.2 neurosarcoidosis
the use of corticosteroides may lead to various manifestations such as tolerance for carbohydrates , capillary and vascular fragility , proximal myopathy , motor weakness , hypertension , osteoporosis , a greater susceptibility to infections and psychiatric symptoms among others6 . it should be noted that the side effects of the same dose of corticosteroides are variable and hence heterogeneous among individuals of the same population with pharmokinetics and different plasmatic protein concentrations being the principal reasons for this . nevertheless , it should also be mentioned that to the best of our knowledge now here in the current literature has it been registered that there actually exists a relationship between ( bfs ) and corticosteroid use , such as in our study . a hypothetical cause and effect relationship raised by our study group would be that effects of corticosteroids upon the hydro - eletrolitic balance , through changes in the calcium , sodium , magnesium and potassium channels however , our patient presented with normal values . attention should also be called to the fact that the earliest complaints of our patient began during the final phase of corticosteroid tapering , with a dose of 20 mg / day on an alternate - day schedule . total tapering of time was eight weeks lasted eight , with the first two weeks consisting of a 60 mg / day dose . in this case , these symptoms include : anorexia , arthralgias , general weakness , weight loss , and skin exfoliation coupled with fever . unfortunately , the exact mechanisms through which the withdrawal syndrome occurs are still unknown . however two main theories still are widely accepted : the changes in plasmatic concentration of corticosteroides brought on by going from a higher to a much lower dose of this drug , which will in turn induce a clinical syndrome very similar to adrenal failure ; and elative glucocorticoide resistance leading to a hypercortisolemic state . it should also be remembered that our patient , besides complaining of fasciculations , also complained of generalized arthralgias . another question raised was the presence of the syndromes not associated in any way with corticosteroid use , such as neurological diseases , medical drugs , intoxications / poisonings , metabolic and systemic diseases . in the case of als , a progressive and chronic degenerative neurologic disease which involves both upper and lower motor neuron neurons , seldom present fasciculation at onset . generally speaking , fasciculation 's in this case are associated clinically with other signs and symptoms such as paraparesis among other anterior horn cell lesion symptoms . such fasciculation 's in this case might be considered as vain , unsuccessful attempts at reinnervation on by surviving and still functioning motor neurons located in partially denervated muscle fibers . although the neurologic exam of our patient was entirely normal , enmg revealed signs of fasciculation potentials , positive waves as well as fibrillations in the various muscle groups tested . singh et al . , in a study of 4 patients who were initially diagnosed with benign cramping and fasciculation syndrome ( scfb ) , identified these same patients as progressing to amyotrophic lateral sclerosis . in view of this , the authors warn that a diagnosis of scfb should not be considered accurate without a 4 to 5 year follow up following onset of clinical symptoms . according to the above - mentioned considerations , a follow up of five years duration was thus requested for the proper characterization of bfs in our specific case . mills kr9 proposed distinctions between fasciculation 's and cramps presented in als and those presented in patients with bfs . this kind of distinction was sought through the firing of motor unit fibers as well as other neurophysiological parameters and standards . fasciculation potentials were recorded in 63 muscles of 28 patients with als and then compared to 21 muscles in patients with bfs . it should be noted that in each muscle , in a single site / motor unit / location up to 15 fasciculation potentials were identified . thus , the characteristics of 430 fasciculation potentials in patients diagnosed with als were compared to the 191 benign fasciculation 's and then analyzed . measured were the fasciculation potential itself , the area , duration and interval of firing , variation indexes of wave morphology , axonal conduction block testing , the variability of axonal conduction and the potential for producing double fasciculation potentials . the authors concluded that the potentials found in als were both shorter and of a much higher frequency than those with bfs . however , although firing patterns in both cases were irregular , the firing rate in the case of als was shown to be significantly higher . whenever muscular weakness and neurogenic alteration were to be found on enmg in the als group of patients , the firing rates in this case were much higher , however the variability indexes of wave form ( morphology ) , the potential itself as well as the amplitude of potential area were all significantly reduced in als . the author concluded that wave form and fasciculation potential morphology were not good markers for distinguishing between benign as opposed to malignant fasciculation potentials . the case was about a 43-year old male who developed progressive muscular weakness along with fasciculation in all his extremities of 4 months duration . the patient was treated with a combination of intravenous immuno - globulin and corticosteroids with almost complete resolution of symptoms following discharge . we have hypothesized that the granu - lomatous inflammatory process must have compressed the ventral ( motor ) root of the spinal cord , hence resulting in the motor symptoms . in our case , estimated the prevalence as well the distribution rate of fasciculation 's among healthy adults , evaluating the effects of age , caffeine and physical exercise . the fasciculation 's were studied using ultrasound in 58 healthy adults and distributed among distinct categories according to age . questionnaires were used to determine the effects of caffeine as well as exercise on the genesis of fasciculation 's . finally , researchers tested the effects of intense of physical exercise on 10 healthy adults . of the total of individuals studied , 25 ( 43% ) presented with fasciculation 's on ultrasound , most of them located in the long abductor of the halux muscle . fasciculation 's were also identified to a lesser degree and intensity in the middle and proximal third of the lower limbs . the great majority of individuals who presented with fasciculation 's were older than those who had never complained of having fasciculation 's . it was also found that caffeine and physical activity did not play a role as far as the prevalence of fasciculation 's was concerned . however , excessive , extenuating physical exercise did , in fact , cause a temporary increase in fasciculation 's in the lower limbs . our patient admitted to the use of caffeine on a daily basis as well as to practicing regular aerobic exercise , three times a week for about 50 minutes . such data were considered for the present case at hand , since the majority of patients in this age range made use of caffeine regularly as well as practice physical exercise , although this pattern and distribution of fasciculation 's such as those found in our patient is not commonly seen under these conditions , which can provoke , in rare instances , fasciculation potentials , mainly in the eyelids , thumb and halux . we must keep in mind that daily exercise possibly causes bfs through the release of a higher than normal amount of free radicals during prolonged exercise . free radicals are a sub product of the cell 's energy production ; hence , the longer the activity and the more intense the exercise , the more free radicals are released . for this reason , athletes are subject to an overload of free radicals as well as oxidative stress . if prolonged exercise is actually related to benign fasciculation 's then certainly the mechanism must involve free radicals that accumulate within nerve endings and cause prolonged muscle excitability . singh g and khurana g17 pointed out the effects of acute organophosphate poisoning , one of the most common kinds of poisonings seen in clinical emergency medicine . among the principal effects are those found among the peripheral nervous system , which in turn results from the inactivation of the acetyl cholinesterase enzyme located among the nicotinic as well as muscarinic receptors , not only in the peripheral but also in the central nervous system as well . the main neurologic manifestations that usually occur late in the course of severe disease are fasciculation 's associated with neuromuscular paralysis . our patient did not present with an occupational history of such poisoning neither did he show any clinical manifestations of acute clinical poisoning . we also considered the possibility of our patient having made use of other medical drug that could induce bfs , such as lithium , for example . acute lithium neurotoxicity , according to azevedo et al.18 , manifests itselfs through dysarthria , ataxia and coarse tremors . associated with acute mental confusion , there may also be frequent muscular fasciculations ( myofasciculations ) as well as myoclonus , with blepharospasm and aparaxia of eyelid opening also having been described . our theory , therefore , is that he myofasciculations presented by our patient may have been related to corticosteroid use , not only through their systemic effects , but also through their varied effects upon polrization of ionic channels . thus , corticosteroids , so widely used by physicians in general as well as in diverse specialties , have the potential for substantial morbi - mortality within not adequately manipulated . in view of this , corticosteroids should always be used for the least period of time necessary as to avoid unwanted side effects6 . the treatment of benign myofasciculation is always symptomatic , based especially upon antiepileptic drugs , including carbamazepine , gabapentine , and phenytoine , mostly with only partial clinical control of symptoms . the interpretation of the effect of these drugs concerning their precise mechanism of action is still speculative , although believed to be through the decrease of distal peripheral nerve ending excitability . fasciculations require a meticulous clinical and neurologic evaluation in order to look for the cause behind them . the diagnosis frequently is difficult to reach due to the ample range and scope of potential clinical conditions likely to cause them . we have presented a case study of a patient whose chief complaint was fasciculations , both diffuse and migratory , with subsequent remission of symptoms within six months of tapering off of oral corticosteroids , which is our most likely hypothesis . | fasciculations are characterized by visible subtle and fast contractions of muscle , even wormlike in movement , by the contraction of a fascicle of muscle fibers .
the authors present the case study of a 28-year - old patient with the appearance of migratory and diffuse fasciculations with an onset after partial tapering off of oral corticosteroides ( 60 mg total dose ) indicated for treatment of minimal change glomerulopathy .
clinical neurological physical exam allied with an enmg , besides other complementary laboratory exams were used for screening the above - mentioned patient .
afterwards , current research relating to the topic at hand was made in order to update the data available in the bireme , scielo and pubmed data banks using the following key words : fasciculation 's , motor neuron disease , and benign fasciculations in the portuguese , english as well as spanish language .
although fasciculation 's are most commonly associated with motor neuron disease as well as with certain metabolic disorders , they may also be present in individuals with absolutely no underlying pathological disorders . in our case ,
fasciculation potentials that have been present for six months , with no other signs of a neurogenic disorder as well as absence of laboratory findings , the patient received a diagnosis of benign fasciculation syndrome ( bfs).we believe that the use of corticosteroides in high doses with subsequent tapering contributed to the fasciculation 's , especially due to the changes that this causes on the ionic channels .
fasciculation 's are symptoms seen in a large range of conditions , and also being the main symptom of the so - called benign fasciculation syndrome .
we have presented an example of this clinical syndrome in a patient whose complaint was fasciculation 's , with complete clinical remission of symptoms following complete tapering off of corticosteroid six months previously . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
this article is concerned with the possible rapid - equilibrium rate equations for enzyme - catalyzed reactions of the type a + b + c products . many reactions of this type are found among the oxidoreductases , and most ligase reactions are of this type.(1 ) four examples of these reactions are shown in table 1 . the fact that the change in binding of hydrogen ions rnh of the forward reaction is positive suggests that one or more hydrogen ions are bound in the rate - determining reaction.(2 ) the effects of ph in rapid - equilibrium enzyme kinetics have been discussed recently,(3 ) and the relation between rnh and the integer number n of hydrogen ions consumed in the rate - determining reaction has been derived for a + b products . there are five possible rapid - equilibrium rate equations for the enzyme - catalyzed reaction a + b + c products , with mechanisms from the completely ordered mechanism to the completely random mechanism . the completely ordered mechanism involves four kinetic parameters , and the completely random mechanism involves eight kinetic parameters . rapid - equilibrium rate equations are derived for each of these five mechanisms using a computer . these rate equations can be used to determine the kinetic parameters with the minimum number of measured velocities.(4 ) the concept that n kinetic parameters can be calculated from n velocity measurements was introduced by duggleby(5 ) in 1975 . before that , frieden,(6 ) in an article about malate dehydrogenase , showed that it was possible to determine the four kinetic parameters ( vab , ka , kb , and kab ) in the rate equation v = vab/(1 + ka/[a ] + kb/[b ] + kab/[a][b ] ) with four velocity measurements . this article is concerned with rapid - equilibrium rate equations because they are the simplest possible rate equations . steady - state rate equations have more terms , but the concept of determining the kinetic parameters with the minimum number of velocity measurements also applies . rapid - equilibrium rate equations can be derived with a computer because mathematica , maple , and matlab have the operation solve that solves sets of simultaneous polynomial equations . general recommendations are made here as to the choice of triplets of substrate concentrations { [ a],[b],[c ] } to be used to determine velocities . the objective is to use triplets of substrate concentrations that reduce the effects of experimental errors on the estimated kinetic parameters . the determination of kinetic parameters using the minimum number of velocity measurements makes it possible to study the kinetics of more complicated enzyme - catalyzed reactions . this method has already been illustrated with a + b products(4 ) when the experimental data follows rapid - equilibrium rate equations . another advantage of deriving rate equations with a computer is that complicated rate equations are obtained in computer - readable form so that they do not need to be typed into the computer . the effects of experimental errors in velocities on the kinetic parameters are smaller when the substrate concentrations are chosen according to the general recommendations given here . the objective in this article is not to find the best set of triplets of substrate concentrations but to apply some general principles that indicate what can be done . when the kinetic parameters for a + b + c products are to be determined for the first time , it is recommended that the program for the completely random mechanism be used because it can identify the mechanism and determine the kinetic parameters in one operation . when the enzyme catalysis follows the completely random mechanism , all of reactions 112 reactions are at equilibrium , not just those in the mechanism used to calculate the equilibrium concentration of eabc . when the values of kinetic parameters have been calculated with one minimal set of measured velocities , it is recommended that the calculation be repeated with a second set of substrate concentrations to be sure the same values ( within the expected experimental errors ) are obtained . the precision of kinetic parameters can be improved by making replicate measurements of the velocities . the steady - state rate equation for ordered a + b + c products has seven terms in the denominator,(9 ) but the rapid - equilibrium rate equation for the completely random mechanism has eight terms in the denominator . the number of terms in the denominator is useful in distinguishing between the five mechanisms discussed here . usually , rapid - equilibrium rate equations have fewer denominator terms than steady - state rate equations . cornish - bowden(10 ) gives a good discussion of reactions with three or more substrates and calls attention to the fact that frieden(11 ) was the first to call attention to the importance of the missing terms in the denominator of a rate equation . computer programs using the minimum number of velocities can be used to calculate kinetic parameters for five rapid - equilibrium mechanisms of the reaction a + b + c products . 2 . the effects on the kinetic parameters of experimental errors in the velocities and substrate concentrations can be calculated , one at a time . equilibrium constants can be calculated for reactions not included in the mechanism used to derive the rapid - equilibrium rate equation . more general computer programs can be used to identify a simpler mechanism and determine the kinetic parameters for the simpler mechanism . | rapid - equilibrium rate equations are derived for the five different mechanisms for the enzymatic catalysis of a + b + c products using a computer .
these rate equations are used to determine the minimum number of velocities required to estimate the values of the kinetic parameters . the rate equation for
the completely ordered mechanism involves four kinetic parameters , and the rate equation for the completely random mechanism involves eight kinetic parameters .
therefore , the four to eight kinetic parameters can be estimated by determining four to eight velocities and solving four to eight simultaneous equations .
general recommendations are made as to the choices of triplets of substrate concentrations { [ a ] , [ b ] , [ c ] } to be used to determine the velocities .
the effects of 5% errors in the measured velocities , one at a time , are calculated and are summarized in tables .
calculations of effects of experimental errors are useful in choosing the triplets of substrate concentrations to be used to obtain the most accurate values of the kinetic parameters .
when the kinetic parameters for a + b + c products are to be determined for the first time , it is recommended that the program for the completely random mechanism be used because it can identify the mechanism and determine the kinetic parameters in one operation . |
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while brm scales with body mass ( bm ) , several other factors seem to influence brm , such as sociability , environmental and dietary specializations , as well as energetic costs of brain tissue ( mace et al . , 1981 ) . large brains contain more neurons and neural connections , and thus have greater potential for information processing . large brains also tend to be more modular , which allows a great amount of connections between neurons ( krubitzer and kaas , 2005 ) . hence , increased brain mass and potential for neural connections may have facilitated large brained mammals to colonize complex habitats , develop sensory systems and evolved complex societies ( e.g. , budeau and verts , 1986 ) . for example , the mass - specific metabolic rate of the human brain is nine times higher than that of the body as a whole ( martin , 1981 ) . the metabolic costs by having a large brain must be paid for by a direct mother metabolic constraint or by a trade - off mechanism between brain mass and energy consumption by other functions ( e.g. , gibbons , 1998 ; pitnick et al . , 2006 ) . other energy allocations such as relative costs of flight and reproductive strategy in birds may also be reduced to shunt energy to an enlarged brain ( isler and van schaik , 2006 ) . recent examples also show that brm may become decoupled from bm over short time spans . 2009 ) found that tanganyikan cichlid bm exhibited recent bursts of rapid evolution , a process that is consistent with divergence linked to ecological specialization , while brm showed no bursts of divergence but evolved in gradual manner , consistent with energetic constraints to rapid bm change . originally , aiello and wheeler ( 1995 ) proposed that a primate is able to meet the high metabolic cost of a large brain without incurring a compensatory increase in relative basal metabolic rate ( bmr ) by decreasing the amount of other metabolically expensive tissues ( i.e. , heart , lung , kidney , liver , and gastrointestinal tract ) . a similar hypothesis was recently proposed for fish ( kaufman et al . , 2003 ) . recently , isler and van schaik ( 2009 ) using a large compilation of brain size , bm , and life history data , found evidence that an energetically costly increase in brain size has to be met by either increasing the total energy budget of a species or by compensating changes of energy allocation to other maintenance functions , such as digestion or growth and offspring production , or a combination of these . martin , 1981 , 1996 ) , for example , found that the energetic investment of the mammalian mother during the development of the fetus and during the postnatal life up to the time of weaning resulted in a weak link between bmr and brain mass . similarly , jones and maclarnon ( 2004 ) showed that for certain clades of bats , maternal investment plays an important role in the adult brain mass . while a number of studies have thus focused on these mechanisms of development , little is known about the evolutionary relationship between bmr and brm . very few studies have tested the generality of the costly brain hypotheses across multiple species with different evolutionary histories , and using phylogenetic approaches . basal metabolic rate is a fundamental parameter in comparative studies and lineages - specific exponents characterize its allometric scaling ( white et al . , 2009 ) . recently , isler and van schaik ( 2006 ) controlling both for bm and phylogentic relationships found evidence that bmr correlated with brm in large groups of mammals . however , brm explained a small % of the variation in metabolic rate at the species and family level ( 2.6 and 10.4% , respectively ) . at higher taxonomic levels , independent contrast ( ic ) revealed a significant correlation only for primates . it is possible that the use of composite phylogenies lacking resolution and accurate estimates of branch lengths ( bl ) may have obscured the underlying patterns ( e.g. , malia et al . , 2003 ) . other potential confounding we generated a novel phylogeny of wild rodents using bayesian analysis of cytochrome b sequence data . we included species where high - quality bmr , brm , and bm data are available . we then used this analysis to test the hypothesis that bmr and brm are correlated within rodents , after taking into consideration both bm and phylogeny . cytochrome b for 132 rodent species and six rabbits as outgroups ( wilson and reeder , 2005 ) were downloaded from genbank , and one sequence donated ( table a1 in appendix ) . cytochrome b was chosen as that marker has proven to be of high utility for species level phylogenetics ( may - collado and agnarsson , 2006 ; agnarsson et al . , 2010 , 2011 ) sequences were aligned using clustalx 1.83 ( thompson et al . , the preferred model for the bayesian analyses was selected with modeltest ( posada and crandall , 2001 ) using the aic criterion ( posada and buckley , 2004 ) . the best - fitting model was gtr + + i ( yang , 1994 ) . bayesian analyses were carried out using mrbayes v3.12 ( huelsenbeck and ronchist , 2001 ) with the settings as specified in agnarsson and may - collado ( 2008 ) . the markov chain monte carlo search was ran with 10,000,000 generations sampling the markov chain every 1,000 generations , and the sample points of the first 7,000,000 generations were removed ( burnin ) , after which the chain had reached stationarity . data on the log of brm and bm ( g ) , and bmr ( cmo2/h ) were used in this study ( table a1 in appendix ) . for studies comparing traits among species , such as regression analyses , it is necessary to account for phylogenetic relationships among the compared species ( felsenstein , 1985 ) . ignoring phylogenetic relationships can lead to pseudoreplication as species are not independent data points , rather independent evolutionary changes in the traits being compared are the data points , or the ic ( see felsenstein , 1985 ; may - collado et al . , 2007 ) among species and lineages . to describe the evolutionary relationship between bmr and brm we performed various phylogenetic analyses . ( i ) the pdap module in mesquite ( midford et al . , 2008 ) was used to estimate ic ( felsenstein , 1985 ) . we used bl as estimated by mrbayes testing them for statistic appropriateness using pdap . to correct for bm we regressed bmr and brm against bm and subsequently regressed the residuals from these regressions ( garland et al . , 1993 ) . if the residuals are correlated then that is consistent with a relationship among these variables ( bmr and brm ) , that is independent of the bm of , and phylogenetic relationship among , species ( e.g. , may - collado et al . , 2007 ) . ( ii ) to evaluate the correlated evolution among bmr , brm , and bm , we assess the phylogenetic effect on the trends in character relationships between taxa ( i.e. , the observed pattern ) using the best model of evolution that was found for each character . to do this we evaluated the significance of the relationships between the pair of characters using a measure of correlated evolution ( corr ) in a bayesian framework implemented in bayestrait 1.0 ( pagel and meade , 2007 ) , assessing the probability of positively correlated ( corr > 0 ) and negatively correlated evolution ( corr < 0 ) . as the null hypothesis we used a model in which the covariance between characters was set to zero ( i.e. , complete character independence , corr = 0 ) , and the alternative hypothesis was , then , the observed covariance between characters ( pagel , 1999a , 1999b ) . if the null hypothesis was rejected ( i.e. , a significant historical relationship between characters exists ) , then we concluded that the phylogenetic relationship and the models of evolution of the characters influence the observed patterns , and we corroborate the hypothesis of correlated evolution between bmr , brm , and bm . we used a bayesian approach based on maximum likelihood with 10 test per tree and estimating pagel ( 1999a , b ) escalated phylogenetic parameters ( table a2 in appendix ) . the sign test was used for statistical comparisons ( zar , 1996 ) with statistica 6.0 ( statsoft , 2001 ) . the novel phylogeny finds support for the monophyly of each currently recognized taxonomical bat family with the exception that heteromyidae contains geomyidae , and one species of muridae , sigmodon hispidus , groups with cricetidae ( figure a1 in appendix ) . the phylogeny overall agrees well with recent rodent phylogenies at higher levels ( e.g. , jansa and weksler , 2004 ; montgelard et al . , 2008 ) and thus represents an reasonable hypothesis for to study the evolution of characters ( e.g. , pagel and harvey , 1988 ) independent contrast revealed associations between bm and bmr ( p < 0.0001 ; r = 0.77 ) , and with brm ( p < 0.0001 ; r = 0.85 ) , and between bmr and brm ( p < 0.0001 ; r = 0.71 ) . when using a single value to represent species mass we also found significant correlation between bmr and brm , after accounting for bm . when using a single individual weight to represent the species , bmr explained 9.7% of the variation in brm ( p = 0.0003 ; r = 0.097 ; figure 1a ) . two extreme outliers affected the regression and removing these outliers resulted in much stronger regression ( p < 0.0001 ; r = 0.20 ; figure 1b ) . the outliers represent 3 species of small rodents ( cricetidae , arvicolinae ) , that inhabit circumpolar northern hemisphere biome ( nowak , 1999 ) . these lemmings seem to have higher metabolic rate than typical rodents with similar brm , which may be related to living in extreme climates requiring higher metabolism . this demonstrates that the climate and habitat as well as other potentially confounding factors ( bm , food habits , substrate , a restriction to islands or highlands , use of torpor , and type of reproduction ) make it difficult to demonstrate a significant correlation between bmr and brm , even when it exists ( e.g. , mcnab , 2008 ) . ( a ) independent contrast regression analysis between bmr and brm residuals , corrected by bm . ( b ) ic regression analysis between bmr and brm residuals , corrected by bm and removing outliners ( myopus schisticolor , lemmus sibiricus , and l. lemmus ) . where bm = body mass , bmr = basal metabolic rate , and brm = rodent brain mass . when using species mean bm , bmr explained approximately 3% of variation in brm ( p < 0.05 , r = 0.029 ) . this correlation , however , disappears when using two bm values for each species , one , the estimated species mean , to calculate residuals of brm , and the second , of the individuals used for the bmr experiments , to calculate residuals of bmr ( p > 0.05 ) . corr indicated significant correlations between all variables ( p < 0.0001 ; corr 1 ) , with the highest correlation recorded between bm and brm ( p < 0.0001 ; corr = 2.86 ; r = 0.95 ; figure 2 ) , followed by bm and bmr ( p < 0.0001 ; corr = 2.24 ; r = 0.92 ; figure 2 ) , and finally bmr and brm ( p < 0.0001 ; corr = 1.86 ; r = 0.91 ; figure 2 ) . the series of arrows in the middle of the figure indicates the model of correlated evolution between the studied characters in a bayesian framework ( for more details see materials and methods ) , where bm = body mass , bmr = basal metabolic rate , and brm = rodent brain mass . each inset labeled with a lower - case letter contains : on the left - hand side a line graph of a sample of 1,000 markov chain estimations ( x - axis ) of corr ( y - axis ) between pairs of characters , where the continuous black line represents corr = 0 , and the continuous gray line indicates corr = 1 ; and on the right - hand side a histogram of the probability distribution of covariance between pairs of characters , where the black bar indicates the average value and the gray bars indicate the lower 5% percentile ( lp ) and upper 95% percentile ( up ) . in particular , the analyzed characters were ( a ) bmr and brm ( lp = 1.33 , up = 2.46 ) ; ( b ) bm and bmr ( lp = 1.50 , up = 3.12 ) ; and ( c ) bm and brm ( lp = 2.08 , up = 3.77 ) . brains are the centers of the nervous system of vertebrates , controlling the organ systems of the body and coordinating responses to changes in the ecological and social environment ( shultz , 2010 ) . although brain mass per se does not capture the complexity of brain function , there is general evidence that relative brain size roughly correlates with cognitive ability ( e.g. , barton and harvey , 2000 ) . hence the evolution of brain size is of broad interest , including what factors may favor and constrain the evolution of relatively large , modular and complex brains ( sol , 2009 ) . basal metabolic rate is influenced by a variety of factors ( bm white and seymour , 2003 ; climate lovegrove , 2000 ; demography kurta and ferkin , 1991 ) . furthermore , an increase in brm results in increased costs for maintenance and information processing ( niven and laughlin , 2008 ) . for example , karbowski ( 2007 ) found that in volume - specific cerebral glucose metabolic rate of different brain structures closely scales with brain volume . these results confirm that information processing in the brain requires large amounts of metabolic energy . here , isler and van schaik ( 2006 ) found support for this relationship across all mammals combined , and within primates ( ic p = 0.025 , r = 0.20 ) , but not within other orders , such as rodents . adjustments appear to be clade - specific , the slopes of best - fit lines for brm against bm tend to be higher in analyses of more inclusive taxa ( e.g. , orders and suborders ) and lower in analyses of less inclusive taxa ( families , subfamilies , and genera ; e.g. , finarelli and flynn , 2009 ) . we hypothesize that the discrepancy between our findings and previous studies is potentially caused by two factors . first , the use of a composite versus primary - data based phylogenies , and second , differences in accounting for bm . composite phylogenies often reflect taxonomy , not necessarily phylogeny , and typically lack accurate bl estimates , two aspects that reduce the efficiency of comparative tests . accounting for bm is a complicated problem , but we argue that using more than a single value for a species , as have prior studies , may introduce confounding variables . thus using an estimate of species bm , such as average species bm to generate brm residuals , and the bm of the individual that happened to be used to evaluate bmr to generate bmr residuals , can likely adds noise that may obscure real patterns . if , for example , the evaluation of bmr happened to have been done on an atypically small or large animal , this would strongly affect the bm - bmr residual , and could readily obscure subtle patterns across two or more variables that are both highly correlated with bm . instead using a single value , whether mean bm of a species , or the weight of a single individual seems at least to be a reasonable alternative . furthermore , species mean bm is a measure independent of the available measures for bmr and brm ( typically single individuals ) , and as such provides a relatively neutral control unlikely to result in systematic error . here , we used a novel primary - data - based phylogeny with bl estimates and single estimates of species bm ( species average or single individuals ) . considering the compendium of factors that may contribute to bmr , the up to 20% of variation explained by brm in rodents is high . clearly , though , further research is necessary to understand the interplay between these variables , and ideally accurate estimates of species means based on multiple individuals would be available for each of these variables ( e.g. , smith and jungers , 1997 ) . our study is also consistent with two evolutionary paths favoring an increase in brm across rodent species . path in which an increase in bmr correlates with an increase in brm ( figure 2a ) . alternatively , indirect and additive path in which the effect of bm on bmr allows brm to increase ( figures 2a , b ) . comparatively , the direct scaling effect of bmr on brm is the least important relationship ( figure 2a ) , but the importance of direct scaling increases when considering the indirect path . dunbar and shultz ( 2007 ) supported the indirect path scenario in primates , where bmr had a limiting effect on brm , while bm had an effect on brm through bmr . here , we corroborate the hypothesis of isler and van schaik ( 2006 ) that an increase in brain mass is accompanied by an increase in basal metabolic rate , and suggests that this pattern may be general across mammals . our findings corroborate the hypothesis that large brains evolve when the payoff for increased brain mass is greater than the energetic cost they incur ( niven and laughlin , 2008 ) . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . aic , akaike information criterion ; bl , branch lengths in phylogenies ; bm , body mass ; bmr , basal metabolic rate ; brm , brain mass ; corr , measure of correlated evolution between characters in a bayesian framework ; gtr , general time reversible models of nucleotide substitution ; ic , phylogenetic independent contrasts ; pdap , phenotypic diversity analysis programs . | brains are the centers of the nervous system of animals , controlling the organ systems of the body and coordinating responses to changes in the ecological and social environment .
the evolution of traits that correlate with cognitive ability , such as relative brain size is thus of broad interest .
brain mass relative to body mass ( bm ) varies among mammals , and diverse factors have been proposed to explain this variation .
a recent study provided evidence that energetics play an important role in brain evolution ( isler and van schaik , 2006 ) . using composite phylogenies and data drawn from multiple sources
, these authors showed that basal metabolic rate ( bmr ) correlates with brain mass across mammals .
however , no such relationship was found within rodents .
here we re - examined the relationship between bmr and brain mass within rodentia using a novel species - level phylogeny .
our results are sensitive to parameter evaluation ; in particular how species mass is estimated .
we detect no pattern when applying an approach used by previous studies , where each species bm is represented by two different numbers , one being the individual that happened to be used for bmr estimates of that species .
however , this approach may compromise the analysis . when using a single value of bm for each species ,
whether representing a single individual , or available species mean , our findings provide evidence that brain mass ( independent of bm ) and bmr are correlated .
these findings are thus consistent with the hypothesis that large brains evolve when the payoff for increased brain mass is greater than the energetic cost they incur . |
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t - tube choledochotomy for common bile duct ( cbd ) exploration was first described more than 100 years ago and has since been used by surgeons around the world for the management of biliary lithiasis . when laparoscopic cholecystectomy became the gold standard of treatment for calculous cholecystitis , laparoscopic techniques for cbd exploration became necessary and t - tube choledochotomy continued to be a useful approach . presently , however , many other options exist for the management of cbd stones , which do not require t - tube placement ( transcystic exploration , intraoperative endoscopic retrograde cholangiopancreatography [ ercp ] , postoperative ercp , and primary closure of the cbd ) . because the laparoscopic placement of a t - tube may be technically demanding and because both placement and removal of t - tubes may lead to severe complications , surgeons must question whether this should continue to be a routine approach or whether other , less morbid , techniques may be used . we present a case of severe biliary peritonitis after t - tube removal , which illustrates the grave consequences that may occur from this procedure and discuss other reported complications as well as therapeutic alternatives . four weeks after an uneventful laparoscopic cholecystectomy with cbd exploration and t - tube placement , a 31-year - old patient attended his follow - up visit where a cholangiogram showed no contraindications for removal of the t - tube . immediately upon removal , the patient experienced severe abdominal pain and tachycardia that did not respond to analgesics and he required admission to the hospital . an abdominal ultrasound showed a small fluid collection that was not considered to merit percutaneous drainage . after 24 hours of conservative management , the patient still complained of abdominal pain , and therefore an ercp was indicated to identify and treat the site of a possible bile leak . ercp showed a small rupture at the point where the fistulous tract joined the abdominal wall and contrast material leaking into the abdominal cavity ( figure 1 ) . endoscopic sphincterotomy was performed , and a biliary stent was placed in an attempt to reduce leakage through the ruptured fistulous tract . fortyeight hours after the ercp , the patient 's condition worsened , with fever , diaphoresis , tachycardia , leucocytosis , and an acute abdomen occurring . an emergency laparoscopy showed a severe generalized biliary - purulent peritonitis that required extensive lavage and placement of abdominal drains . the rupture of the fistulous tract was sutured to avoid further bile leakage . during the procedure , the patient developed hemodynamic instability and bacteremia and had to be transferred to the intensive care unit ( icu ) . the patient 's condition progressed to severe respiratory distress and systemic inflammatory response syndrome due to septic shock and required 4 weeks of aggressive treatment in the icu with ventilatory support , multiple antibiotics , vasoactive agents , and activated c protein . the patient was eventually discharged 6 weeks after the removal of the t - tube . this case is a clear example of the catastrophic events that may occur after t - tube removal in a patient who had previously undergone an uneventful laparoscopic cholecystectomy and t - tube choledochotomy . the ultrasound findings of a small fluid collection mislead clinical judgment and delayed a more aggressive approach such as percutaneous drainage . endoscopic treatment with sphincterotomy and stent placement , which is an accepted method for the resolution of such cases , also failed to control the problem . fortunately , the abdominal lavage procedure and the management in the icu eventually led to the patient 's recovery , but only after high health and financial costs resulted from the removal of the t - tube . cholecystectomy due to gallstone disease remains one of the most common surgical procedures performed , and cbd exploration is frequently required for concomitant choledocholithiasis . the incidence of choledocholithiasis in patients who undergo cholecystectomy increases with age , and in those with acute cholecystitis and jaundice it may reach up to 70% . unsuspected cbd stones ( found in routine intraoperative cholangiography ) are present in approximately 5% of cases . in 1889 , thornton described the first successful surgical cbd exploration , and since then , treatment for choloedocholithiasis has consisted mainly of stone extraction through a choledochotomy with placement of a t - tube for postoperative drainage and radiologic control . in the 1970s , the endoscopic approach to biliary stones became popular due to the good extraction results and low incidence of complications . in the 1990s , when laparoscopic cholecystectomy became the gold standard of treatment for calculous cholecystitis , laparoscopic cbd exploration became necessary , and t - tube choledochotomy continued to be used as a standard procedure . the placement of a t - tube during open cholecystectomy is usually a simple procedure that is used to control biliary drainage and can be helpful for radiologists , once the fistulous tract is formed , to remove stones that may remain in the biliary ducts . laparoscopic choledochotomy , although technically more demanding , is also commonly performed by surgeons who are familiar with advanced laparoscopic procedures . latex rubber tubes are usually preferred to other materials , and construction of small and flexible t ends is thought to facilitate placement and removal of the tube . in our review of the literature regarding t - tube complications , we found that most of the reports are isolated case presentations , and very few are larger case series that focus on this problem . this leads us to believe that , although t - tubes continue to be placed frequently in many countries , the complications associated with their placement or removal may be underestimated . according to some authors , the difference in complication rates does not seem to be related to whether the approach is open or laparoscopic ( 15.5% vs. 13.8% , respectively ) , but rather to the presence and removal of the tube . these include fluid and electrolyte imbalance , early dislodgment , tube dislocation , tube retention , and bacteremia . those associated with removal of the t - tube include bile leaks , biliary peritonitis , sepsis , and death . most authors conclude that these complications result in longer hospital stay , increased rate of reoperations , and higher financial costs . when reviewing larger series of cbd exploration , it has been found that complications are often related directly to the t - tube choledochotomy with reports showing approximately 2% to 15% morbidity and 1% to 6% mortality in these cases ( table 1 ) . most severe complications are due to rupture of the cbd or fistulous tract . although the cause of this is not well understood , it could be related to inadequate placement , incomplete fistulous tract formation , or forced removal . the incidence of bile leakage caused by removal of the t - tube has been reported from 0.45% to 10% , and that of biliary peritonitis may be higher than expected . to prevent bile leakage due to t - tube removal , the surgeon should pay particular attention to the technical aspects of placement , such as assuring small and flexible t ends , care not to suture the tube to the bile duct , and adequate percutaneous exteriorization of the tube . removal of the tube is usually an outpatient procedure that is recommended at least 15 days after its placement , once a proper fistulous tract has been formed and the risk of bile leaks and choledochal rupture is reduced . t - tube related complications morbidity and mortality of bile peritonitis due to biliary surgery have been reported to be as high as 57% and 14% , respectively . treatment of biliary peritonitis varies according to the severity of the case and may include from simple procedures , such as percutaneous drainage and endoscopic nasobiliary drainage , to more complex ones , such as endoscopic sphincterotomy with endobiliary stent placement , re - operation with t - tube reinsertion , or reoperation with bilioenteric reconstruction . early recognition is essential for adequate treatment , and some authors have suggested performing a fistulogram immediately after removal of the t - tube to confirm the presence or absence of a leak . with the advent of other , less - invasive techniques , the risks and benefits of t - tube choledochotomy must now be compared with the risks and benefits of other therapeutic measures . the management of cbd stones can usually be determined by whether the diagnosis is made preoperatively , transoperatively , or postoperatively ( table 2 ) . in all cases , options that avoid choledochotomy and t - tube placement note also , that with the widespread performance of these therapeutic measures , the need for postoperative radiologic stone extraction is becoming less frequent , and therefore , this indication for t - tube placement may not be necessary in the future . treatment options for choledocholithiasis cbd = common bile duct ; ercp = endoscopic retrograde cholangiopancreatography . ercp and sphincterotomy , although shown in some studies to be associated with complications and death , have proven to be effective and safe methods for cbd stone extraction . in the hands of experienced endoscopists , however , ercp may have certain limitations , such as the presence of multiple intrahepatic stones , stones > 1.5 cm , the presence of a nondilated cbd , or a concomitant duct stenosis . in these circumstances minimally invasive surgery provides options to explore the cbd without having to place a t - tube . laparoscopic transcystic exploration with biliary balloon catheters or dormia baskets should always be attempted first and when not feasible , due to the presence of large or impacted stones , pneumatic dilatation of the cystic duct and use of a choledochoscope may be required . success of laparoscopic cbd exploration and stone clearance has been reported at rates of 85% to 95% with impacted stones being the most likely cause of failure . in these difficult cases , placement of a transcystic stent or guidewire followed by postoperative ercp may be a good option to avoid choledochotomy and t - tube placement . since laparoscopic cbd exploration requires less manipulation and devascularization of the bile duct , smaller and more precise choledocotomies can be performed . this has led some authors to advocate the use of primary closure of the cbd after choledochotomy . primary closure can be complimented with transcystic biliary decompression or endoscopic sphincterotomy and endobiliary stenting to reduce the risk of bile leakage . although this method has not yet been widely accepted due to fear of complications , recent cochrane reviews were unable to find sufficient evidence to recommend t - tube placement over primary closure of the cdb in either open or laparoscopic procedures . although not statistically significant , this interesting review does show that the deaths of the patients in the t - tube group were directly related to surgery and sepsis and that bile peritonites was higher ( 2.9% ) in the t - tube group than in the primary closure group ( 1% ) finally , although most long - term biliary strictures are due to iatrogenic events during cholecystectomy or other hepatobiliary procedures , some have been associated with severe scarring at the site of a previous t - tube choledochotomy . it has already been mentioned that the laparoscopic approach , in experienced hands , may cause less devascularization , and it allows for smaller choledochotomies , which will probably reduce these scarring effects and the number of postoperative strictures . needless to say , when analyzing the differences between surgical and endoscopic treatment of biliary lithiasis , a cochrane review found that laparoscopic bile duct clearance is providing a safe and efficient method with no statistical difference in morbidity or mortality compared with ercp in specialized centers ( table 3 ) . however , it is difficult to establish a comparison between the morbidity and mortality rates of laparoscopic t - tube choledochotomy and ercp for the treatment of bile duct stones because most studies on laparoscopic cbd exploration report on the transcystic approach and not on the more demanding choledochotomy procedure . comparison between ercp and surgical treatment of bile duct stones ns = no statistical significance ; ercp = endoscopic retrograde cholangiopancreatography . many reports found in the literature show that both placement and removal of the t - tube may have complications that result in longer hospital stay , increased rate of reoperations , higher financial costs , and some degree of deaths . because most of these reports are isolated case descriptions and only a few of them present large series of patients , the morbidity and mortality associated with the use of t - tube choledochotomy in the treatment of bile duct lithiasis may be underestimated . this procedure has been performed for many years and continues to be a useful resource in other areas of hepatobiliary surgery , but the tendency towards less invasiveness , in all areas of general surgery , should prompt surgeons to consider other possibilities with equal or better results for the resolution of choledocholithiasis . although the authors of this article are experienced in laparoscopic cbd exploration and had not previously had complications associated with t - tube choledochotomy , this case served to encourage us to reconsider the therapeutic options in future cases . we now believe that a precise case selection is necessary before determining which procedure will be better for each patient . clinical findings and pre - existing conditions , intraoperative findings and anatomical or inflammatory variations , surgical and endoscopist experience and success rate or even technological availability are all important factors that should be taken into consideration to provide the best possible treatment . | t - tube choledochotomy has been an established practice in common bile duct exploration for many years .
although bile leaks , biliary peritonitis , and long - term postoperative strictures have been reported and are directly associated with the placement or removal of the t - tube , the severity of these complications may often be underestimated by surgeons .
we present the case of a 31-year - old male patient who developed biliary peritonitis and septic shock after removal of a t - tube and illustrate one of the catastrophic events that may follow such procedures .
literature shows that these complications may occur more frequently and have higher morbidity and mortality than other less invasive procedures .
this article reviews the advances in laparoscopic and endoscopic techniques , which provide alternative therapeutic approaches to choledocholithiasis and allow the surgeon to avoid having to perform a choledochotomy with t - tube drainage . |
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there are about 350 million people worldwide suffering from chronic hepatitis b virus ( hbv ) infection . china is one of the countries with a high prevalence of hbv infection . the hbv vaccination program for all newborns was implemented by the chinese government in 1992 , which increased the yearly national hbv vaccine inoculation rate . based on this achievement , the prevalence of hbv surface antigen ( hbsag ) carrier in the general population ( 159 years old ) of china was decreased from 9.75 % in 1992 to 7.18 % in 2006 . despite the reduction in the incidence of hbv infection , there are still about 93 million people chronically infected with hbv and an estimated 20 million cases of symptomatic chronic hepatitis b in china now . therefore , the healthcare burden from chronic hbv infection is still a big challenge for china . patients with chronic hbv infection may develop liver fibrosis , cirrhosis , hepatic decompensation , and hepatocellular carcinoma ( hcc ) and eventually die from liver failure and other complications . numerous studies have shown that active hbv replication leads to liver injury and disease progression . as pointed out by the guidelines for prevention and treatment of chronic hepatitis b from the american association for the study of liver disease ( aasld ) , european association for the study of the liver ( easl ) , asia pacific association for the study of the liver ( apasl ) , and chinese association for the study of liver disease ( casld ) , the ultimate long - term goal of treatment is to prevent hepatic decompensation , reduce progression to cirrhosis and/or hcc , and prolong survival . therefore , antiviral therapy plays a central role in controlling chronic hbv infection and preventing the disease progression . due to economic and medical system issues , , we will discuss some key issues in the area of antiviral treatment for chronic hepatitis b in china . patients with positive serum hbsag for more than 6 months or patients with positive hbsag and chronic liver disease proven by biopsy can be diagnosed with chronic hbv infection . based on the information from the guidelines for management of chronic hepatitis b virus infection published by casld in 2010 , the indications for antiviral therapy are as follows : general indications : ( 1 ) for hbeag positive patients with serum hbv dna 10 copies / ml ( equivalent to 20,000 iu / ml ) and for hbeag negative patients with hbv dna 10 copies / ml ( equivalent to 2,000 iu / ml ) ; ( 2 ) alanine transaminase ( alt ) 2 upper limit of normal ( uln ) , but for patients treated with interferon- ( ifn- ) , alt should be 10 uln and the total bilirubin should be < 2 uln;and ( 3 ) alt < 2 uln , but the liver biopsy shows a histological activity index ( knodell hai ) 4 , or degree of necroinflammation g2 , or degree of fibrosis s2 . for patients with persistently positive hbv dna who do not show the criteria mentioned above , the antiviral therapy should be applied if they are in the following conditions : patients older than 40 years and alt > uln.for patients older than 40 years and alt is sustained normal , a liver biopsy is recommended . if the knodell hai is 4 , or the degree of necroinflammation g2 , or the degree of fibrosis s2 , then antiviral therapy should be applied.if disease progression is evident , for example , spleen enlargement , liver biopsy is recommended , and antiviral therapy should be applied if necessary . patients older than 40 years and alt > uln . for patients older than if the knodell hai is 4 , or the degree of necroinflammation g2 , or the degree of fibrosis s2 , then antiviral therapy should be applied . if disease progression is evident , for example , spleen enlargement , liver biopsy is recommended , and antiviral therapy should be applied if necessary . as to the standards for determining the patients who need antiviral therapy , casld has very similar rules as aasld , easl , or apasl . all these associations recommend that the patients need to be evaluated based on the state of persistent hbsag , hbeag , the level of hbv dna , and the degree of liver inflammation and fibrosis , and clinicians may decide whether the patients are appropriate for antiviral therapy . histology of the liver biopsy provides direct evidence for grading the inflammation and staging the fibrosis in the liver ; however , liver biopsy is hard to perform in most hospitals because of the patients refraction . alt is routinely tested as a biochemical marker to reflect the degree of the liver inflammation , it is one of the important markers for identifying whether the patient in an immune activation stage or not . the uln for alt is defined as 40u / l in china based on the results of a national wide survey in the 1950s [ 6 , 7 ] . however , increasing evidence from china shows that this reference value can not dependably reflect the degree of the liver inflammation . for chronic hepatitis b patients with persistently normal alt , the liver biopsy results showed that 34.4 % of them had fibrosis with degree s2 or fibrosis degree s1 and inflammation degree g2 . < 0.5 uln , the proportion with significant liver inflammation and fibrosis was 16.6 % , whereas in patients with alt between 0.5 and 1 uln , the proportion increased to 40 % . for patients with alt between 1 uln and 2 uln these data indicate that the uln of alt may need to be revised for china . a large sample survey in italy showed the uln of alt should be 30 u / l for healthy men and 19 u / l for healthy women . the guidelines of aasld in 2009 recommended to use these values to evaluate the liver inflammation status of the patients . a study in korea showed that the uln of alt for koreans was 33 u / l for the healthy men and 25 u / l for healthy women , and these values had been applied in the guidelines for korea in 2012 . a large sample survey for uln of alt , including 28,642 healthy men and 24,413 healthy women was performed in china in 2011 and showed the uln of alt was 35 u / for men and 23 u / l for women in chinese han population . based on these new findings , casld should adjust the uln of alt reference value in the next version of the guidelines . otherwise , the patients with mildly elevated alt ( alt between 1 uln and 2 uln ) or persistently normal alt may not receive the opportunity of antiviral therapy . two kinds of antiviral agents have been approved for the therapy of chronic hepatitis b : ifn- and nucleot(s)ide analogs . the first includes conventional ifn- and peg - ifn- , and the second includes lamivudine ( lam ) , adefovir dipivoxil ( adv ) , telbivudine ( ldt ) , entecavir ( etv ) , and tenofovir ( tfv ) . the randomized clinical trials confirmed that peg - ifn- treatment could obtain a higher hbeag seroconversion rate in a limited period of treatment ; etv and tfv have a potent antiviral effect , low incidence of drug resistance , and good safety [ 11 , 1519 ] . these drugs are listed as the first - line antiviral drugs for chronic hepatitis b treatment by aasld and easl . the conventional ifn- , ldt , adv , and lam are the second - line antiviral drugs [ 11 , 20 ] . in china , compared with the average yearly incomes of the general population , the peg - ifn- and etv produced by foreign or sino - foreign joint venture pharmaceutical companies are very expensive and are only covered by a small proportion of medicare payments , or not covered at all . most of chinese patients with chronic hepatitis b can not afford peg - ifn- or etv , and consequently these drugs are rarely used for the initial antiviral treatment . the clinicians have to choose from the second - line drugs with affordable prices for the patients , especially in rural areas . by using the second - line drugs , the health system reduces the cost of treatment in a short term , but the medical expense will increase in the long term if the patients develop drug resistance or suboptimal response . with the development of local pharmaceutical companies , the generic drugs of conventional ifn- , lam , adv , and etv have been successfully produced and approved for the treatment of chronic hepatitis b in china . the application of domestic produced peg - ifn- in chronic hepatitis b is still in clinical trial . the multicenter , large sample clinical studies show that there are no significant differences in antiviral efficacy , bioavailability , and safety between the domestic ifn-/nucleot(s)ide analogs and the original drugs [ 2123 ] . the price of domestic generic drugs is only 5070 % of the original drugs . this provides more choices for the clinician in addition to reducing the economic pressure on the patients . traditional chinese medicines ( tcm ) have been used for treating chronic hepatitis b for a long time . over 90 % of chronic hepatitis preclinical and clinical trials , which were performed in china , indicated that some traditional chinese medicine preparations , like phyllanthus urinaria , decoction of small bupleurum ( a medicinal root found natively in east asia ) , matrine ( an alkaloid found in plants from the sophora genus with anticancer activity ) , or rhubarb , had a potential effect on inhibiting hbv replication , regulating the hosts immune function , and improving liver function [ 25 , 26 ] . a short - term clinical follow - up study found that matrine treatment could result in a similar curative effect as interferon in hbeag seroconversion and hbv dna reduction . the small bupleurum decoction and phyllanthus urinaria preparations are reported to have a similar therapeutic effect as conventional ifn [ 26 , 28 ] . although it seems that tcm can be used as alternative medicine for antiviral treatment , the antiviral effect needs to be evaluated in well - designed and randomized clinical trials . in addition , the composition of tcm is very complex , and it is difficult to explain the mechanism from the standpoint of modern medicine . this limits the application of tcm in the treatment of chronic hepatitis b [ 25 , 29 ] . due to the low cost , the second - line nucleot(s)ide analogs ( lam , ldt , and adv ) are widely used in china . when the second - line nas are chosen for long - term therapy regimens , drug resistance may occur and cause virologic breakthrough , hepatitis flare , and even death . therefore , rescue strategies need to be taken once the resistant strains of hbv are detected . for the lam - resistant strain , the therapy can switch to monotherapy with either adefovir or entecavir , add - on adv to lam or use a combination treatment of adv and etv . the experience from china is that etv + adv is superior to lam + adv , and lam + adv is better than monotherapy [ 30 , 31 ] . however , due to the risk of frequent resistance development against etv with lam - resistant strains , further research is needed to assess long - term cost - effectiveness of etv combination treatment to lam - resistant disease . for the adv - resistant strains , adding lam , or ldt , or etv to adv is recommended . current research shows that both ldt + adv combination therapy and etv monotherapy leads to significant decreases in serum hbv dna in the patients with resistance to adv , and ldt + adv combination therapy exhibits a significantly higher rate of hbeag seroconversion than etv monotherapy . for the patients with resistance to ldt or etv , adding adv a small sample survey showed that ldt + adv combination therapy led to significant decreases in serum hbv dna levels , normalization of alt , and increased the rate of hbeag seroconversion in patients with resistance to ldt . the serum hbv dna levels , serum alt levels , hbv genotype , hbeag status , and other factors in patients with initial antiviral treatment are important factors for predicting the response to antiviral therapy which has been called baseline guided therapy ( bgt ) . in recent years , the response of patients to treatment during antiviral treatment as the guidance ( response guided therapy , rgt ) has become the focus of attention . a multicenter study on optimization of telbivudine ( ldt ) treatment led by hou in china is based on the idea of rgt . if the serum level of hbv dna was 300 copies / ml , patients were switched to ldt plus adv combination therapy . after 105 weeks of treatment , the rates of hbv dna < 300 copies / ml in the ldt plus adv group and the ldt group were not significantly different ( 76.7 vs. 61.2 % ) . however , the rates of viral mutations and drug resistance were significantly different between these two groups ( 6.0 vs. 30.4 % , 2.7 vs. 25.8 % ) . some studies indicated that initial combination antiviral treatment can improve efficacy and reduce the rates of viral mutations in hbeag positive chb patients with high viral load ( 10 iu / ml ) and in decompensated cirrhosis patients who need long - term nas antiviral therapy [ 35 , 36 ] . the combination of ifn- and na or two nas are the mainstream strategy for combination treatment . although there are some reports from china showed that combination treatment was significantly superior to monotherapy ( see table 1 ) . however , the casld does not recommend the initial lam - adv combination therapy for chb patients.table 1results of initial combination antiviral therapy for chronic hepatitis bgroupsvirologic response % hbeag clearance % hbeag seroconversion % hbsag clearance % alt normalization % drug resistance % time ( weeks)number of patientsreferenceslam + adv1005110009650he et al . peg - ifn--2a + adv1007671249621 means that no data were reported results of initial combination antiviral therapy for chronic hepatitis b means that no data were reported tcm are also candidates for combination therapy . the combination of matrine and nucleot(s)ide analogs , such as lam , has synergistic effect and can achieve even better therapeutic effects for inhibiting hbv replication . they can restore liver function quickly and reduce the incidence of virus bounce after withdrawal of the nucleot(s)ide analogs [ 25 , 27 ] . the incidence of liver fibrosis , cirrhosis , hepatic decompensation , and hcc has been reduced . however , the clinicians have new challenges to face . in the us and european countries , primary non - response and suboptimal response is rare in patients who received tdf and etv treatment , but may occur due to pharmaceutical response of the host to the drugs . in china , poor adherence of patients to treatment , relative high costs of the first - line antiviral drugs and resistance development may be the key factors that cause the non - response , suboptimal response , and even treatment failure . since 2008 , several large cohort studies supported by the government have been carried out on the optimizing strategy for the treatment of chronic hepatitis b in china . this may serve as one of the important ways to solve this problem and to benefit most of the patients . in addition , recent studies show that the agonist of toll - like receptor 7 and hbsag / hbsab immune complex can induce prolonged suppression of hepatitis b virus in chronically infected individules by stimulating the innate and acquired immune response . therefore , the immune modulation therapy may be a new strategy for antiviral treatment on chronic hepatitis b. | the vaccination program against hepatitis b virus ( hbv ) has greatly reduced the incidence of hbv infection . however , almost one - fourth of the hbv infected patients worldwide are still located in china .
the healthcare burden from chronic hbv infection is a big challenge for the chinese government and clinicians .
antiviral therapy plays a central role in controlling chronic hbv infection and preventing the disease progression .
however , due to the specific economic and medical system issues , the first - line antiviral agents recommended by the aasld and easl have not been widely used for chinese patients . in this review
, we will discuss some key issues in the area of antiviral treatment for chronic hepatitis b in china . |
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pediatric trauma is a very significant cause of mortality and disability , being responsible for more deaths than all diseases combined . the burden of child injuries in india is not clearly known because our knowledge is inadequate about their epidemiology . as per national crime records bureau ( ncrb ) report of 2006 , there were 22,766 deaths ( < 14 years ) due to injuries among children . our study aims to determine the frequency of various types of childhood injuries in different sex and age groups and also to find out the various modes and place of trauma among study subjects and their distribution according to different age and sex groups . it also gives an idea about the relative mortality in various types of childhood injuries . this was a prospective study conducted at a tertiary care hospital over a 12-month period . a total of 791 patients ( age up to 12 years ) with trauma were admitted between august 2009 and july 2010 . isolated pediatric ophthalmic trauma , drowning as well as parental psychiatric disorders responsible for pediatric trauma , such as battered baby syndrome , were excluded from our study . a detailed history taking ( from parents / relatives / children ) and examination was done and all patients were assessed with regards to their age , sex , mode of trauma / injury , type of injury , site of trauma , place of trauma , and mortality . the children were classified according to age as : infants ( up to 1 year ) , toddlers ( 1 - 3 years ) , preschool ( 3 - 6 years ) and school - age children ( 6 - 12 years ) . modes of trauma were divided as : fall from height , road traffic accident ( rta ) , burn , sports related , assault ( sexual , sharp , blunt ) , poisoning , bites and stings . the types of injury were divided into subgroups : orthopedic , head , burns , abdomen , poisoning , bites and sting , chest , poly trauma and genital injuries . the places of trauma were divided into the following : home , road , farm , school / playground or park and others . the mortality data were shown according to different age groups as described earlier and according to the mode of injury . out of the total 6102 pediatric patients admitted , the cause of admission for 791 patients was trauma . school - going children were the most commonly injured ( 52.33% ) [ figure 1 ] . children mostly suffered from orthopedic injuries ( 37.80% ) . among the non orthopedic injuries , head and abdominal injury was the most common seen in the school - going children . while in burns , toddler group was the most commonly affected age group [ table 1 ] . fall from height ( 39.44% ) , rtas ( 27.83% ) and burns ( 15.18% ) were the most common mode of injury leading to pediatric trauma [ table 1 ] . 8 were injured by blunt object , 3 by sharp object and 2 cases were sexually assaulted . age distribution of trauma modes and type of injuries among children of different age groups in our study we found the home to be the most common place of injury [ figure 2 ] . the most common cause of injury among all traumatized children was a fall injury . , most of the falls occurred from stairs ( 31.73% ) , followed by the terrace ( 25.32% ) , whereas at the farm , the most common place of fall was from a tree ( 16.98% ) , followed by fall into well ( 2.88% ) [ table 2 ] . of 220 children involved in rta , 64.54% were pedestrians , 20.45% were two - wheeler passengers and 15% were four - wheeler passengers [ table 2 ] . characteristics of injuries due to fall , road traffic
accident , burn and poisoning out of total 120 burn patients , 45 ( 37.5% ) patients belonged to the toddler age group . burns due to scalds ( hot liquids ) accounted for 55.83% cases , while burns due to flames and electricity accounted for 23.33% and 10.83% of the cases , respectively . cracker and contact burns were the remaining cause of burn injury [ table 2 ] . most of these victims had ingested kerosene ( 67.39% ) followed by insecticide ( 13% ) , castor seed ( 10.86% ) and drugs ( 8.69% ) [ table 2 ] . out of 791 patients admitted , 51 died ( 6.44% ) . children of 1 - 3 years age group had the highest mortality ( 39.21% ) in their respective age group , followed by infants ( 15.38% ) . males ( 74.5% ) our study also revealed rta as one of the major causes of injury , causing the highest mortality ( 35.29% ) , followed by burns ( 27.45% ) and fall from height ( 15.68% ) [ table 3 ] . this was probably due to delayed presentation to our tertiary institute ( either via referrals or direct admission ) , or probably due to lack of knowledge and low literacy levels among the parents of these children . reported a prevalence of 14.2% , and another study done at naraingarh , india , reported a prevalence of 5.5% . many studies have been done from bangladesh , iran , nigeria , thailand , singapore and from major indian cities,[1215 ] and these studies have found boys to be more commonly injured then girls . home was found to be the most common place of injury , followed by road / street , with falls being the most common mechanism of pediatric trauma . the mean age of presentation in our study was 6.3 years which is in consonance with the above studies . in our study too , boys were more commonly hospitalized than girls , probably since in our country , boys are given more freedom as well as free hand to work or play outside their homes . male to female ratio was 1.9:1 in our study which is similar to the 1.5:1 to 3:1 ratio reported in the above studies . school - going children ( 6 - 12 years ) were the most common age group found to be affected in our study , which is also similar to that reported in other previous studies . majority of our injuries occurred at home , followed by road and school / playground . studies from trinidad and tobago , ethiopia , and nigeria all found the home environment to be the most common place for a childhood injury to occur . in our study , falls were the leading cause of trauma in all age groups , followed by rtas , except in the 6 - 12 year age group in which falls were the second most common etiology after rtas . stairs and terrace were the two most common causes of fall from height , while in a study from singapore , slipping and fall from bed were the most common causes of falls , once again signifying different epidemiological patterns in different parts of the world . in our study , most of the victims of rtas were pedestrians , followed by two - wheeler passengers . this finding is similar to that derived from studies done at maput and tehran . in our study , a vast majority of burn injuries occurred from hot liquids , followed by flame injuries . similar results were drawn from studies done in pakistan and south africa . in our study , followed by insecticides and drugs , whereas reports from other countries[2527 ] reveal kerosene to be the most common cause followed by drug ingestion and insecticides . rta was most common cause of death , followed by burn and fall from height . these results are similar to those of the studies done in developing countries , whereas studies from developed countries reveal rta to be the most common cause of death , followed by gunshot injuries . this study gives an idea about the epidemiology of pediatric trauma , with 6 - 12 years age group found to be the most affected and 1 - 3 years age group found to be the most vulnerable with regards to overall mortality . home was the most common place of injury , and fall and rta were the most common mechanisms of injuries . by knowing the epidemiology of pediatric trauma , we conclude that majority of pediatric injuries are preventable and pediatric epidemiological trends differ from those in adults . therefore , preventive strategies should be made in pediatric patients on the basis of these epidemiological trends . | aim : to assess the various epidemiological parameters that influence the causation of trauma as well as the consequent morbidity and mortality in the pediatric age group.materials and methods : a prospective study of 791 patients of less than 12 years age , was carried out over a period of 1 year ( august 2009 to july 2010 ) , and pediatric trauma trends , with regards to the following parameters were assessed : age group , sex , mode of trauma , type of injury , place where the trauma occurred and the overall mortality as well as mortality.results:overall trauma was most common in the school - going age group ( 6 - 12 years ) , with male children outnumbering females in the ratio of 1.9:1 .
it was observed that orthopedic injuries were the most frequent ( 37.8% ) type of injuries , whereas fall from height ( 39.4% ) , road traffic accident ( 27.8% ) and burns ( 15.2% ) were the next most common modes of trauma .
home was found out to be the place where maximum trauma occurred ( 51.8% ) .
maximum injuries happened unintentionally ( 98.4% ) .
overall mortality was found out to be 6.4% ( n = 51).conclusions : by knowing the epidemiology of pediatric trauma , we conclude that majority of pediatric injuries are preventable and pediatric epidemiological trends differ from those in adults .
therefore , preventive strategies should be made in pediatric patients on the basis of these epidemiological trends . |
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acromegaly is an uncommon disease ( estimated prevalence of 413 cases per 100,000 population ) [ 1 , 2 ] invariably due to a gh - secreting pituitary adenoma . excess gh production results in alterations in a person 's physical appearance , including enlargement of facial features ( nose , lips , ears , jaw , and forehead ) as well as enlargement of the hands and feet . less visible symptoms of acromegaly include obstructive sleep apnea , excessive sweating , premature osteoarthritis , hypertension , cardiomegaly , diabetes , and an increased risk of benign and malignant tumors [ 4 , 5 ] . given the symptoms associated with acromegaly , it is unsurprising that the disease has been reported to have negative effects on both self - image and quality of life ( qol ) [ 6 , 7 ] . despite the potential for acromegaly to affect a person 's qol in a number of ways , there is limited published research on this topic . the development of a questionnaire specifically designed to measure the effects of acromegaly on qol ( acroqol ) enabled dimensions of qol important in acromegaly to be measured , demonstrating reduced qol in those with acromegaly when compared to a normal population [ 9 , 10 ] . the effects of disease control on qol in acromegaly have proven to be mixed , with one published study showing an improvement in qol after successful disease control , while another reported a reduction in qol when radiotherapy was employed , even despite successful control of gh levels . these equivocal findings may be due to a lack of sensitivity of the acroqol measure , the influence of comorbidities , or cosmetic and orthopedic deformities . perhaps due to the relative rarity of acromegaly , there is a paucity of literature regarding the effects of acromegaly on body image ; one case report noted that body image was adversely affected in a person diagnosed with acromegaly . a more recent study examined the impact of exercise on body image in 11 patients with acromegaly , finding a positive impact on self - assessed body perception after three months of exercise . further , it is reported that biochemically controlled acromegaly patients typically show higher measurements of obesity , specifically visceral adiposity , than age- and gender - matched controls . several other conditions that affect one 's appearance have been reported to negatively impact body image ( bi ) , for example , obesity [ 16 , 17 ] , psoriasis , systemic lupus erythematous , cushing 's syndrome , and rheumatoid arthritis . in obese persons , identifying those who might respond to an intervention aimed at reducing body image disturbance or dissatisfaction is important when combining an operation with psychological interventions to assist the patient . given that acromegaly also negatively affects a person 's appearance , the condition might also lead to bi disturbance . if confirmed , then interventions aimed at helping patients to adjust their body image would potentially be beneficial when combined with medical control of acromegaly . however , to date , no studies have used the body image disturbance questionnaire ( bidq ) investigating people with acromegaly compared to other groups . gh deficits have been associated with reductions in qol and psychological functioning , particularly anxiety and depression , while excess gh has been linked with changes in personality , mood lability , and depression . even when gh excesses or deficiencies are treated , psychopathology and qol may remain problematic , although the literature is equivocal about this . however a recent study of nonfunctioning pituitary adenomas ( nfas ) found that qol was similar to those without endocrine disease once the disorder is sufficiently treated . these patients , in whom adenomas do not secrete excess gh , provide a useful comparison group for investigating the quality of life and body image disturbance effects of the two types of pituitary tumors on patients . existing literature suggests that patients with acromegaly have a greater impairment in qol than those with nfas and control participants , but that those with nfas will not have qol different to that of control participants . the current study aimed to test the hypothesis that body image disturbance would be more likely in acromegaly patients than the nfa patients and controls , because of the disfiguring nature of acromegaly . the impact of obesity on body image disturbance in patients with both conditions , as well as the controls , is also examined . endocrine clinic medical records were used to identify a convenience sample of participants diagnosed with acromegaly or nfas , who were invited to participate in the study face - to - face at routine clinic appointments , or by mail . control participants were recruited among family and associates of the patient groups using a snowball sampling technique to ensure that participant groups were closely matched with respect to socioeconomic status and ethnicity . this was a cross - sectional survey of three groups , patients with acromegaly , patients with nfas , and a control group . all participants signed an informed consent , and the study was conducted in accordance with the national health advisory committee 's ethical guidelines for observational studies and permission of the endocrine department . participants in all groups were mailed the questionnaire to complete at home , or they were given the questionnaire to complete while they were waiting to attend a clinic appointment . participants who agreed to a blood sample had these completed either prior to clinic appointments ( patient groups ) , or following completion of the questionnaire ( control group ) . gh and igf1 results were used to confirm diagnoses or treatment progress , and in nonacromegaly participants , to ensure there was no evidence of excess gh production . the questionnaire set consisted of demographic information ( including age , gender , ethnicity , height , weight , and relationship and employment status , as well as information about symptoms and comorbidities within the patient groups ) , together with the following measures : a single item self - esteem query ( i have high self - esteem ) with a likert response anchored by not at all true of me and very true of me ; this was chosen because of the known connection between body image problems and low self - esteem [ 2628].the hospital anxiety and depression scale ( hads ) . this was developed to detect signs of depression and anxiety in outpatients , has been used widely in medical contexts , and is reputed to have good reliability and validity [ 29 , 30].the body image disturbance questionnaire ( bidq ) contains seven scaled items that pertain to appearance - related concerns , mental preoccupation with these concerns , associated experiences of emotional distress , resultant impairment in social , occupational , or other important areas of functioning , interference with social life or with school , job , or role functioning , and consequential behavioral avoidance ; the measure yields qualitative and quantitative responses and the mean scale scores ( higher scores = more disturbance ) allow comparison between the present study groups.the duke health profile ( dhp ) is a 17-item scale that contains six health subscales ( physical , mental , social , general , perceived health , and self - esteem ) and four dysfunction scales ( anxiety , depression , pain , and disability ) . the scale 's authors report that the mean score for patients with painful health issues was 58.1 , compared to a mean of 83.9 for patients visiting their family practice for health maintenance . this general qol measure allows within - study comparisons between groups and scores can also be compared with those from that reference sample of general outpatients . a single item self - esteem query ( i have high self - esteem ) with a likert response anchored by not at all true of me and very true of me ; this was chosen because of the known connection between body image problems and low self - esteem [ 2628 ] . this was developed to detect signs of depression and anxiety in outpatients , has been used widely in medical contexts , and is reputed to have good reliability and validity [ 29 , 30 ] . the body image disturbance questionnaire ( bidq ) contains seven scaled items that pertain to appearance - related concerns , mental preoccupation with these concerns , associated experiences of emotional distress , resultant impairment in social , occupational , or other important areas of functioning , interference with social life or with school , job , or role functioning , and consequential behavioral avoidance ; the measure yields qualitative and quantitative responses and the mean scale scores ( higher scores = more disturbance ) allow comparison between the present study groups . the duke health profile ( dhp ) is a 17-item scale that contains six health subscales ( physical , mental , social , general , perceived health , and self - esteem ) and four dysfunction scales ( anxiety , depression , pain , and disability ) . the scale 's authors report that the mean score for patients with painful health issues was 58.1 , compared to a mean of 83.9 for patients visiting their family practice for health maintenance . this general qol measure allows within - study comparisons between groups and scores can also be compared with those from that reference sample of general outpatients . all completed questionnaires were scored and analyzed using statistica version 11 ( statsoft inc . , tulsa , ok , usa ) . one - way anova was used to confirm the age - matched nature of the patient groups . the scores on all measures recorded by the three groups within the study were compared using nonparametric analyses as appropriate ( mann - whitney u test or kruskall - wallis anova by ranks ) . kruskall - wallis analysis was used to examine between group differences among the obese participants within the study . a total of 82 participants took part in the study : 32 participants with diagnosed acromegaly , 29 participants with nfas , and 21 control participants . the majority of the acromegaly and nfa patients had been diagnosed between 1977 and 2012 by the endocrine service ; thus , time since onset varied across the sample . the demographic characteristics of the patient and control groups are shown in table 1 . from the patient symptom questionnaires the most frequently identified acromegaly features at diagnosis were enlarging of the hands and/or feet and visual acuity losses . current treatments for the acromegaly patients included slow - release octreotide ( sandostatin lar ) and cabergoline . acromegaly patients were taking from 0 to 13 medications at the time of study , depending on their comorbidities ( see table 2 for details ) . clinical characteristics of the nfa patients are included in table 1 . like the acromegaly patients , some were taking a range of medications depending on their comorbidities as shown in table 2 . igf-1 levels were ascertained for all patients and a volunteer sample within the control group ( 33% ) . the acromegaly patients were also categorized according to their igf-1 status ( high or controlled ) and analyses carried out for all measures in the study . patients with controlled igf-1 had lower pain levels as measured by the dhp than those whose igf-1 levels were high ; p = .03 . bmi data collected from all participants ranged between 18.9 and 61.73 , with a median of 29.46 . the distribution of the various bmi across the groups can be seen in table 1 . kruskall - wallis analysis showed no significant differences in bmi across the three groups , h(2 , n = 72 ) = .45 , p = .7985 . in addition , the numbers of overweight or obese participants were similar across the three groups . self - esteem responses were similar across each of the study groups , with the majority of participants reporting good self - esteem ( 58% ) ; the least positive responses were recorded by 2 acromegaly and 2 control group participants . when considering only obese participants , a similar spread across the diagnostic groups was found . using the caseness approach to scores on the hads , there were 12 acromegaly patients , 7 nfa patients , and 3 control participants with anxiety or depression . however , comparison between the study groups with respect to the hads mean scores showed no significant differences . examining the obese participants separately , anxiety and depression scores were worse in the patient groups ; anxiety was at the 64th percentile level in acromegalic males and 61st percentile in nfpa females . depression was at the 74th and 75th percentile for obese nfpa and acromegalic females , respectively , while in male patients nfpa men 's mean score was on the 68th percentile and the acromegalic males mean score reached the 91st percentile . the bidq asks respondents about their concern over the appearance of some parts of their body ; 63% of female and 27% of male participants expressed concerns about some aspect of their body . within each group , 54% of acromegalic women , 67% of women with nfpa , and 79% of control women expressed concerns , while 28% of acromegalic men , 22% of men with nfpa , and 43% of analysis of patient groups showed no significant differences between either of the patient groups , or the control group for the women , or the men . a question by group examination of each quantitative response in this measure showed that the acromegaly patients expressed similar concerns and distresses on this scale as did their study counterparts . overall a greater percentage of one or other of the two comparison groups recorded higher levels of distress or concern on every question within the bidq scale . however , among participants scoring 4 or more on the question as to which aspect of their body gave them concern , the nfpa and control group participants recorded a variety of bodily issues , while the acromegaly respondents were most distressed by facial changes . all participants completed the duke health profile to enable comparison of qol factors between groups . the three groups within the study were compared , and then further comparison was made with a reference group comprised of 683 primary care patients from us . the 17-item measure generates health scales and disability scales , scores from which are shown in table 5 . between - group analyses of the participants within the study found no significant differences on either the health scales or the disability scales . however when compared to the reference group of primary care patients , the acromegaly ( a ) and nfa patients recorded significantly lower physical health ( a , p = .0001 ; nfa , p = .009 ) and perceived health ( a , p = .0003 ; nfa , p = .04 ) indicating poorer health , but lower pain ( a , p = .0001 ; nfa , p = .0001 ) scores indicating less pain due to health issues . however as reported above , igf-1 levels in the acromegaly patients influenced pain scores on this measure . when the obese participants were examined separately , the acromegaly patients ' mean scores for physical health were significantly worse than the reference patients , p < .01 , and their perceived health was also significantly worse than the reference group , p < .05 ; the nfa patients also scored lower than the reference group for perceived health , p there were no mean score differences on other scales for obese participant groups within the study , or when study participants were compared with the external reference group . the obese acromegaly patients ' mean anxiety scores were significantly higher than the reference group , p < .05 , indicating more dysfunction . the obese nfa patients and the obese control group participants recorded less pain than the reference group , p < .0001 and p < .01 , respectively ; there was no significant difference between the pain scores for the obese acromegaly patients and the us reference group . since the study focus was body image disturbance in the three groups , the bidq average score and other characteristics among the study groups average bidq as well as bmi was related in both patient groups ( acromegaly , r = .44 , p < .05 ; nfa , r = .41 , p < average bidq was also related to hads anxiety in all three groups ( acromegaly , r = .57 , p < .01 ; nfa , r = .55 , p < .01 ; control , r = .69 , p < .01 ) and to hads depression in both patient groups ( acromegaly , r = .68 , p < .0001 ; nfa , r = .63 , p < .0001 ) . in acromegaly patients bidq scores were lower when dhp physical ( r = .63 , p < .01 ) , dhp mental ( r = .52 , p < .01 ) , and dhp general health ( r = .54 , p < bidq scores correlated positively with anxiety ( hads and dhp r = .48 , p < .05 ) , depression ( hads r = .69 , p < .0001 ; dhp r = .61 , p < .01 ) , and dhp pain ( r = .46 , p < .05 ) scores . among the nfa patients dhp mental health ( r = .38 , p < .05 ) and dhp esteem ( r = .61 , p < .01 ) were related to bidq scores ; and for control participants , dhp physical ( r = .622 , p < .05 ) and dhp pain ( r = .57 , p < .05 ) scales were correlated with bidq scores . because obesity appeared to significantly influence a number of the factors investigated in these groups , data from the obese participants ( n = 12 ) were examined separately . examination of the obese participants on a group by group basis showed that in the acromegaly patients body image problems were associated with age ( r = .58 , p < .05 ) and hads depression ( r = .59 , p < .05 ) . obese nfa patients ' bidq scores were related to hads anxiety ( r = .62 , p < .01 ) and hads depression ( r = .69 , p < .01 ) and higher where dhp mental health ( r = .48 , p < .05 ) , dhp perceived health ( r = .56 , p < .05 ) , and dhp esteem ( r = .69 , p < age and self - esteem were also positively linked ( r = .53 , p < .05 ) , and hads anxiety scores were correlated with dhp perceived health ( r = .51 , p < .05 ) and dhp pain ( r = .48 , p < .05 ) scores in this group ( n = 18 ) . in the obese control participants ( n = 7 ) there were no significant associations with the bidq average scores . hads anxiety was associated with age ( r = .77 , p < .05 ) , the dhp social scale ( r = .78 , p < .05 ) , and dhp pain ( r = .78 , p < .05 ) . this novel study examined body image disturbance in a sample comprising three groups , two patient groups with conditions that affect their endocrine systems , and a control group of friends and relatives of the patients . the first hypothesis tested here was that body image disturbance would be more likely in acromegaly patients because of the disfiguring nature of the condition . however this was not supported by the data , with no significant differences being found between groups with respect to body image disturbance . the second question as to how obesity affects quality of life , self - esteem , health profile , body image disturbance , and affect in these contexts was underpinned by the fact that almost 40% of study participants were obese ( bmi > 29 ) . in our study , the obese acromegaly patients reported poorer overall physical health and poorer perceived health than comparison reference groups . the nfa patients were similarly affected when obese , with anxiety , depression , and perceived health concerns being associated with body image disturbance , which accords with the associations noted in the development sample . our findings suggest that overweight and obesity contribute more to reduction in quality of life and the potential for body image concerns , than the acromegaly diagnosis . this may not have been so clear had there been more significant differences between the two study groups in other respects . the understanding that overweight and obesity relate strongly to quality of life links with a recent study of exercise , quality of life , and acromegaly that investigated whether participants reported qol after a period of regular exercise . while there were only 11 completers in that study , and the construct of body image was measured differently , those who did exercise regularly experienced a positive impact of the exercise on their self - assessed body perception . however the exercise regime did not improve the participants ' mood , or their quality of life . most participants in our study , whether overweight , obese , or otherwise , regarded themselves as having high self - esteem , with responses in this area being similar across the three groups . this may be part of the reason for lack of differentiation between patient groups with respect to other study parameters ; persons with high levels of self - esteem are less likely to experience body image disturbances regardless of other health diagnoses [ 2628 ] . concerning other aspects of mental health , while patient groups recorded more depression , the differences between groups were not statistically significant . across the range of quality of life issues assessed by the dhp , group differences were not significant again , although both patient groups experienced more physical problems and perceived health issues than the reference sample of us primary practice patients . pain as recorded by the dhp was worse in all three groups compared to the us patients . however it seems that combinations of these characteristics potentially contribute to differences in outcome ; interactions between the scales suggested that body image disturbance was more likely when patients were obese , and when anxiety , depression , and pain were present . the examination of correlations by group demonstrated age and depression are associated with body image disturbance in acromegaly , and anxiety and depression are related to body image issues in the nfpa group ; however , no such relationships were demonstrated in the control participants . this suggests that there are complex interactions among the factors we investigated that require further investigation . another endocrine related study recently published examined depression , pain , health , and body satisfaction in patients with cushing 's disease , compared with controls , but found no association between their groups with respect to depression , despite general health , pain , and physical function differences . however the authors also discussed the equivocal nature of the published evidence relating to depression and cushing 's disease and pointed out that adequate treatment of the condition does not always resolve these difficulties . these findings resonate with the equivocal nature of findings in other acromegaly studies and the current study this cross - sectional study within a relatively small sample demonstrated no significant differences among the three studied groups across these measures . this raises a question of the size of the study , and while this is almost as large as any prior study , the lack of clarity in our findings suggests a larger sample from a multicentre study may better be able to determine the scale of potential differences between these groups . however , the lack of significant differences may be simply explained by the fact that having a diagnosis of a condition that can cause disfigurement does not necessarily mean a person experiences that effect of the condition , or further , even if the disfigurement is present , the person may not be concerned by it . this accords with findings among obese people , not all of whom experience psychological difficulty associated with their obesity . one factor identified in the obese group is the age of onset of their obesity ; people who were obese as children more often reported greater body dissatisfaction when older , than those whose obesity onset was later in life . the average participant in the current study was aged in their fifties and this may have some explanatory value for our findings ; perhaps as the more general body image literature suggests , younger people would be more concerned about body image [ 26 , 28 ] . on the other hand , the condition is gradual in onset , and people may more readily adjust expectations when changes are gradual in contrast to situations such as surgery for breast cancer where body image is known to be a problem for patients particularly proximal to surgery . while the hypothesis that acromegaly might be associated with body image disturbance was not borne out , we have shown that obesity together with acromegaly can be associated with body image issues . equally both of these findings suggest that a patient 's bmi rather than their primary diagnosis will be more indicative of the likelihood that they might have body image problems . this understanding suggests the need for targeted interventions for those with either acromegaly or nonfunctioning pituitary disease when obesity is also a presenting factor . given the equivocal nature of research regarding endocrine conditions and quality of life , further research should seek an understanding of the benefits of such targeted interventions in terms of increased quality of life , decreased body image disturbance , and overall health benefits . | purpose .
excess growth hormone secretion in adults results in acromegaly , a condition in which multiple physical changes occur including bony and soft tissue overgrowth . over time
these changes can markedly alter a person 's appearance .
the aim of this study was to compare body image disturbance in patients with acromegaly to those with nonfunctioning pituitary adenomas ( nfas ) and controls and assess the impact of obesity in these groups .
methods . a cross - sectional survey including quality of life , body image disturbance , anxiety and depression measures , growth hormone , and bmi measurement was carried out . results .
the groups did not differ with respect to body image disturbance .
however separate analysis of obese participants demonstrated relationships between mood scales , body image disturbance , and pain issues , particularly for acromegaly patients . conclusions . while the primary hypothesis that acromegaly might be associated with body image disturbance was not borne out , we have shown that obesity together with acromegaly and nfa can be associated with body image issues , suggesting that bmi rather than primary diagnosis might better indicate whether patients might experience body image disturbance problems . |
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ulnar impaction syndrome ( uis ) was first presented by milch in 1941 , which was directed at the wrist ulnar sided pain caused by colles fracture - induced radial shortening and wrist ulnar impaction induced lunate necrosis.1 it is found by research and clinical experience that the wrist trauma is not the only risk factor of uis . the pathogenesis is mainly the wrist ulnar overload , which causes chronic impact of ulnar head , triangular fibrocartilage complex ( tfcc ) and lunate and triangular bone . this leads to nutrition disorder of the blood supply and synovial fluid of ulnar wrist structure , resulting in ulnar wrist joint degeneration . ultimately , a group of syndrome such as ulnar wrist pain and functional limitation occur.2 with the development of handicraft industry and increase of various works that need a large amount of repeated wrist ulnar deviation strength , the incidence of uis is increasing , but the traditional simple ulnar shortening osteotomy has more complications.3 in the western countries , uis is one of the main causes of carpal instability.4 in this study , the early diagnostic criteria of uis and its wrist arthroscopic treatment experience were explored . the objective is to provide a basis for further application of wrist arthroscopy to treatment of uis . 9 uis patients were enrolled in this study , including 7 males and 2 females . their ages were 1655 years old , with average age of 29 3.6 years . 4 patients were with traumatic tumble or sprain , and other 5 patients had long term repeated work with strength for grabbing or rotating with hand . the diagnosis criteria5 for uis were as follows : ( 1 ) wrist trauma history and long term repeated hand work history ; ( 2 ) the history of wrist ulnar - side rotation , compression and ulnar wrist pain , with or without clicking ; ( 3 ) x ray : most of the ulnar positive variation ( ulna is over 2 mm greater than radius ) is associated with osteosclerosis or / and cystic degeneration under proximal articular surface of ulna head , lunate bone and triangular bone , which may also be associated with kienbck 's disease,6 while minority of ulnar neutral variation only need fist wrist pronation x - ray ( dynamic ulnar positive variance);7 ( 4 ) magnetic resonance imaging ( mri ) performance : the signal changes of the lunate bone , triangular bone , as well as tfcc ; ( 5 ) wrist arthroscopic changes : chondromalacia of lunate bone , triangular bone and various types of tfcc injury . all 9 patients were associated with different degree of tfcc injury . they had wrist chronic pain and wrist weakness symptoms , which seriously affected the daily life and work . the conservative treatments such as the use of nonsteroidal anti - inflammatory drugs , wrist immobilization , and cold compression could not obtain a good result . physical examination x - ray examination revealed that , 8 cases had ulnar positive variance including 1 case with positive variance more than 4 mm [ figure 1 ] , with 1 case of negative variance . mr joint imaging showed different degree of signal change in the wrist bone in 9 patients [ table 1 ] . ulnar impaction syndrome with positive variance > 4 mm demographics and surgical methods for 9 cases under brachial plexus anesthesia or general anesthesia , the patient was in the supine position and the tourniquet was used . the patient was fixed at shoulder abduction 90 , elbow flexion 90 and wrist flexion 15 , with finger implementing the traction . 34 portals were established at the dorsal carpal , and the arthroscopy sheath was inserted as the water inlet . after checking the radiocarpal joint ( rcj ) , the pinhead was inserted through the 6-r approach as a water outlet , and a cannula was inserted . a 2.9 mm full radius shaver was inserted to remove degenerative joint capsule tissue and synovial tissue , making the vision more clear.8 after confirming the scapholunate ligament , the carpometacarpal ligaments including radial head ligament , the radioscapholunate ligament and radial triangular ligament were in turn confirmed . after full inspection on dorsal lunate bone and tfcc , the arthroscopy was transformed to the 6-r approach to examine the rcj ulnar part . the degree of tfcc damage was detected by the probe to determine the injury condition of each ligament . the stability degree of lunotriquetral joints ( ltj ) was observed . in 9 patients , 8 cases with positive variance had tfcc ii c damage that is , central damage ( tfcc central perforation ) , and 5 cases were complicated with lunar and ulna osteomalacia . for patients with ulnar positive variance less than 4 mm , the arthroscopy was placed in the 34 approach ( if necessary , arthroscopy was placed on the 45 or 6-r approach to expand the observation scope ) . after resection of tear or degeneration of the tfcc central part and reserve of the intact peripheral part , the arthroscopic wafer resection was performed through the tfcc central perforation area . a 1/2 or 1/3 of the radial side was firstly removed , using the shaver as contrast . the shaver device with a diameter was about 3 mm and the resection distal ulna depth did not exceed 4 mm . if the ltj stability was poor , or the ulnar positive variance was greater than 4 mm , the routine ulnar shortening osteotomy was conducted . at the same time , the ligament reconstruction was performed to restore the stability of ltj . almost , 1 patient with ulnar positive variance greater than 4 mm was treated with ulnar shortening osteotomy and arthroscopic ligament reconstruction , with tfcc exploration and removal of the wear . the junction of distal and middle 1/3 ulna were chosen for the ulnar shortening osteotomy , as this site was the intersection of the diaphysis nutrient artery and the epiphysis - metaphysis arteries . the amputation end was rich in blood supply and was good for healing , which could be performed with plate fixation , laying a good foundation for early functional exercise.9 after operation , cefmetazole sodium ( 2 g ) was intravenously injected for preventing infection ( once per day , 7 days ) . the patients with the ulna osteotomy were treated with using plaster for 2 months and the patients with only the arthroscopic treatment were treated with plaster external fixation for 1-week all the patients were evaluated clinically for pain , functional status , range of motion and grip strength after a followup . the wrist function scoring system of green and obrien10 modified by cooney et al.11 [ table 2 ] was used for the final clinical assessment of patients . clinical scoring system of green and obrien modified by cooney data were expressed as mean standard deviation . a t - test was used to analyze the differences between two groups , and p < 0.05 was considered as statistically significant . under brachial plexus anesthesia or general anesthesia , the patient was in the supine position and the tourniquet was used . the patient was fixed at shoulder abduction 90 , elbow flexion 90 and wrist flexion 15 , with finger implementing the traction . 34 portals were established at the dorsal carpal , and the arthroscopy sheath was inserted as the water inlet . after checking the radiocarpal joint ( rcj ) , the pinhead was inserted through the 6-r approach as a water outlet , and a cannula was inserted . a 2.9 mm full radius shaver was inserted to remove degenerative joint capsule tissue and synovial tissue , making the vision more clear.8 after confirming the scapholunate ligament , the carpometacarpal ligaments including radial head ligament , the radioscapholunate ligament and radial triangular ligament were in turn confirmed . after full inspection on dorsal lunate bone and tfcc , the arthroscopy was transformed to the 6-r approach to examine the rcj ulnar part . the degree of tfcc damage was detected by the probe to determine the injury condition of each ligament . the stability degree of lunotriquetral joints ( ltj ) was observed . in 9 patients , 8 cases with positive variance had tfcc ii c damage that is , central damage ( tfcc central perforation ) , and 5 cases were complicated with lunar and ulna osteomalacia . for patients with ulnar positive variance less than 4 mm , the arthroscopy was placed in the 34 approach ( if necessary , arthroscopy was placed on the 45 or 6-r approach to expand the observation scope ) . after resection of tear or degeneration of the tfcc central part and reserve of the intact peripheral part , the arthroscopic wafer resection was performed through the tfcc central perforation area . a 1/2 or 1/3 of the radial side was firstly removed , using the shaver as contrast . the shaver device with a diameter was about 3 mm and the resection distal ulna depth did not exceed 4 mm . if the ltj stability was poor , or the ulnar positive variance was greater than 4 mm , the routine ulnar shortening osteotomy was conducted . at the same time , the ligament reconstruction was performed to restore the stability of ltj . almost , 1 patient with ulnar positive variance greater than 4 mm was treated with ulnar shortening osteotomy and arthroscopic ligament reconstruction , with tfcc exploration and removal of the wear . the junction of distal and middle 1/3 ulna were chosen for the ulnar shortening osteotomy , as this site was the intersection of the diaphysis nutrient artery and the epiphysis - metaphysis arteries . the amputation end was rich in blood supply and was good for healing , which could be performed with plate fixation , laying a good foundation for early functional exercise.9 after operation , cefmetazole sodium ( 2 g ) was intravenously injected for preventing infection ( once per day , 7 days ) . the patients with the ulna osteotomy were treated with using plaster for 2 months and the patients with only the arthroscopic treatment were treated with plaster external fixation for 1-week all the patients were evaluated clinically for pain , functional status , range of motion and grip strength after a followup . the wrist function scoring system of green and obrien10 modified by cooney et al.11 [ table 2 ] was used for the final clinical assessment of patients . a t - test was used to analyze the differences between two groups , and p < 0.05 was considered as statistically significant . 9 patients obtained stage - i healing of incision , with no joint infection , vascular or nerve damage , or other complication . they were followed up for 2 - 18 months , with average 13 2.6 months . 9 patients recovered normal life and work , with no ulnar wrist pain again . almost , 1 patient remained wrist weakness . there was a significant difference of the wrist activity between the last followup and before operation ( p < 0.05 ) [ table 4 ] . for one case ( female ) with left wrist pain and myasthenia , x - ray and mri examination showed existence of free body in left wrist joint , positive ulnar variance and tfcc damage [ figure 2 ] , which confirmed the uis . under brachial plexus anesthesia , the free body in left wrist joint was removed by wrist arthroscopy [ figure 3a ] . the tfcc central damage was visible after resection of free body [ figure 3b ] . the arthroscopic wafer resection was performed via tfcc central damaged zones , reserving the intact peripheral parts [ figure 4 ] . the patient was kept in plaster for 2 weeks and obtained good recovery through function exercise after removal of the plaster [ figure 5 ] . indications for surgery comparison of wrist activity before operation and last followup x - ray and magnetic resonance imaging ( mri ) of wrist with forearm showing ( a ) the ulnar positive variance and free body in ulnar carpal joints ; ( b ) ulnar positive variance , triangular fibrocartilage complex central damage , lunate abnormal signals arthroscopic views showing ( a ) the free body , visible during arthroscopic operation ( b ) triangular fibrocartilage complex central damage was visible after resection of free body arthroscopic view showing ( a ) after arthroscopic removal of free body , the arthroscopic wafer resection was performed was performed via triangular fibrocartilage complex ( tfcc ) central damaged zones , reserving the intact peripheral parts ( b ) free bodies from articular cavity clinical photographs showing ( a ) physical examination before operation ( b ) physical examination after operation in 9 patients , the average scores of pain , functional status , range of motion and grip strength were 21.93 0.80 , 23.80 1.50 , 20.42 0.60 and 18.60 0.10 points respectively . according to cooney 's modification of green and obrien 's score for clinical outcome , 6 cases were graded excellent , with 2 cases of good and 1 case of fair . normal wrist joint is composed of rcj , distal radioulnar joint and radiolunate joint , which are not communicated . the triangular fibrocartilage locates in the ulnar - side of ulnar distal articular surface and originates from the ulnar margin of the distal radius articular surface and the ulnar notch , passing through distal radioulnar joint . the edge is divided into distal and the proximal ligaments , which respectively terminates at the tip and the basilar part of the styloid process of ulna . when the positive ulnar variance occurs , the ulnar bone repeatedly impacts the lunate bone , triangular bone and tfcc , causing damage to them . the tfcc injury is very common and occurs earlier.1213 due to anatomical position , the lunate bone has higher morbidity than the triangular bone . ulnar impaction syndrome is a group of syndrome , including ulnar wrist pain and functional limitation . the ulnar wrist pain and activity limitation can occur in the tfcc damage and necrosis of lunate bone and triangular bone , which influence mutually . in this study , x - ray examination shows that , uis is associated with osteosclerosis or / and cystic degeneration under proximal articular surface of ulnar head , lunate bone and triangular bone . at the same time , mri more clearly displays the occurrence of positive variance , tfcc is irregular - shaped , thinner or even disappear , which shows the positive correlation.14 the proximal ulnar side of lunate bone and proximal radial side of triangle bone have different degree of chondromalacia . according to the five level classification method from recht et al.,15 all of these can be reflected in mri . ulnar impaction syndrome has low universal rate , low diagnosis rate and high rate of missed diagnosis . although it is definitely diagnosed , the conservative treatment and simple ulnar shortening osteotomy are often applied . as the arthroscopic treatment has not been universalized , after uis occurrence , the wrist joint tfcc injury , wrist joint instability , various types of ligament injury and other complications are not treated properly . it has the advantages of less invasive , lower complication rate and rapid postoperative recovery . in view of the positive correlation between uis and tfcc degeneration and chondromalacia of lunate bone and triangular bone , we believe that the wrist arthroscopy should be the dominant method in the treatment of uis . according to the damage degree of wrist injury , the operation modes are different , which are as follows : ( 1 ) if the ulnar positive variance is less than 4 mm and is associated with tfcc central damage , it is suitable for arthroscopic wafer resection via tfcc central damaged zones , reserving the intact peripheral part , with resection thickness less than 4 mm . ( 2 ) if the ulnar positive variance is greater than 4 mm , the osteotomy should be firstly performed , followed by wrist arthroscopic tfcc debridement . ( 3 ) if it is associated with chondromalacia of lunate bone and triangular bone and instability of wrist joint , which mostly belong to the kienbck 's disease , the debridement should be performed , with repair for ligament injury . however , the therapy of kienbck 's disease should be further explored , which looks forward to multidisciplinary treatment.8 ( 4 ) for tfcc damage and even the main uis damage , the arthroscopy is the preferred treatment method , which has a good future.1617181920 along with the popularization of mri and gradually raised awareness of uis among majority of clinicians , the diagnosis level of uis has obtained a substantial progress than before . in the treatment of uis , the wrist arthroscopy can improve the diagnosis rate , optimize the treatment plan , shorten the treatment cycle , with good treatment results . however , at present the wrist arthroscopy is mainly applied only for shortening osteotomy , and the operation is rough and unable to effectively deal with all complications . with the improvement in cognition and application of wrist arthroscopy , the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . | background : the development of handicraft industry and increase of various such works that need a large amount of repeated wrist ulnar deviation strength , the incidence of ulnar impaction syndrome ( uis ) is increasing , but the traditional simple ulnar shortening osteotomy has more complications .
this study aimed to explore the early diagnostic criteria of uis and its wrist arthroscopic treatment experience.materials and methods:9 uis patients were enrolled in this study . according to magnetic resonance imaging ,
x - ray and endoscopic features , the diagnostic criteria of uis were summarized and the individualized treatment schedule was made .
if the ulnar positive variance was less than 4 mm , the arthroscopic wafer resection was performed .
if the ulnar positive variance was more than 4 mm , the arthroscopic resection of injury and degenerative triangular fibrocartilage complex and ulnar osteotomy were conducted.results:in all patients , the wound healed without any complications .
all patients returned to normal life and work , with no ulnar wrist pain again .
one patient had wrist weakness .
there was a significant difference of the wrist activity between the last followup and before operation ( p < 0.05 ) . according to the modified wrist function scoring system of green and obrien ,
there were 6 cases of excellent , 2 cases of good and 1 case of appropriate and the overall excellent and good rate was 92.3%.conclusion : in the treatment of uis , the arthroscopy can improve the diagnosis rate , optimize the treatment plan , shorten the treatment cycle , with good treatment results . |
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olivine - type lifepo4 has
been extensively studied as a promising cathode material for li - ion
batteries , due to its good reversibility , safe operating voltage ( 3.45
v vs li / li ) and high reversible capacity of 160 mah / g ( compared to the practical capacity of 140
mah / g for licoo2 , the commonly
used cathode in the portable communications industry ) . however , the
low operating voltage of lifepo4 leads to an energy density
that is considered low for use in electric transportation . substituting
fe with a transition metal that has a higher redox potential
( e.g. , mn , co , or ni with redox
potentials of 4.1 , 4.8 , and 5.1 v , respectively , vs li / li ) increases the theoretical energy density of the olivine cathode
material . however , limnpo4 , licopo4 , and linipo4 have more sluggish kinetics than isostructural
lifepo4 , and are more difficult to fully lithiate and delithiate . licopo4 has the lowest hole polaron migration barrier of
the high voltage olivines and subsequently has the highest electronic
conductivity among limnpo4 , licopo4 and linipo4 . furthermore , the high redox potential of the co / co couple means that licopo4 has a high theoretical
energy density of 800 wh / kg , as compared with 580 wh / kg for lifepo4 , but is not as affected as linipo4 by the electrolyte
decomposition that occurs at high voltages . bramnik et al . in 2007 studied the electrochemical reaction
of
licopo4 using in situ x - ray diffraction ( xrd ) and observed
an intermediate li0.7copo4 phase , where the
composition was determined based on vegard s law ( i.e. , assuming
a linear relationship of the unit cell parameters to li content ) . however , a later study combining x - ray and neutron
diffraction determined an intermediate li0.6copo4 stoichiometry . thus , the li content
of this phase likely lies in the range 0.6 and 0.7 , but it is not
clear whether the difference in li content arises from the ability
of the intermediate phase to tolerate some degree of solid solution ,
or whether it reflects differences in the approaches used to estimate
the li content . loss of long - range order was also observed , which
agrees with the chemical delithiation results of wolfenstine et al . the observation of intermediates in the olivine system is
not unique
to licopo4 . intermediates of lixfepo4 ( 0.5 < x < 0.7 ) have been
observed on cooling the solid solution phases formed at elevated temperatures , from chemical oxidation and more recently ,
during electrochemical cycling of micron - sized particles . the isolated intermediates generally have a
li content of between 0.6 and 0.7 , the li content again being typically
inferred using vegard s law . first - principles density functional
theory ( dft ) calculations performed on the li0.6fepo4 phase found that it is energetically favorable for li vacancies
to be ordered in pairs . an a2/3 intermediate ( a = na , li ) has also been observed during charging
of the polyanionic materials nafepo4 and livpo4f . the crystal structure of the na phase , na2/3fepo4 ( space group pnma ) , was
originally proposed to have a ( 3a 3b c ) supercell ( relative to the parent olivine unit
cell ) , in which the fe cations are
aligned in the ( 110 ) and ( 220 ) planes and the na vacancies
reside between the fe ions for charge balance . boucher et al . , on the basis of tem and synchrotron data showed ,
however , that this cell could be redrawn using a smaller ( ab 3b c ) monoclinic p21/n superstructure , which
is only 3 times larger in volume than the original cell . the licopo4copo4 system
exhibits
complex magnetic properties , the magnetic data providing insight into
the electronic structures of the end and intermediate members , and
of direct relevance to the present study , often aiding the interpretation
of the nuclear magnetic resonance ( nmr ) spectroscopy . ehrenberg et
al . carried out an experimental magnetic study on the end member licopo4 and copo4 phases , and on their intermediate lixcopo4 ( x = 0.6 )
phase , and showed that the electronic ground state of all three compositions
is high spin . high - spin licopo4 and copo4 ground states had also previously been predicted
using dft plus hubbard u ( dft+u ) calculations , and self - consistently
calculated effective hubbard u parameters for co and
co. the result for the fully
delithiated co - only phase is not necessarily expected
given that the electronic configuration of co is d , for which a low spin configuration ( corresponding to a fully
occupied t2 g orbital set in octahedral coordination ) is
typically lower in energy than the high spin ( t2g)(eg ) configuration . additionally , an
intermediate spin co has been reported to exist at the
surface of licoo2 , owing to changes in the co o
coordination near and at particle surfaces , and in li - excess licoo2 materials . a magnetic and neutron diffraction study on a licopo4 polycrystalline sample reported an antiferromagnetic alignment
of the co spins , along the vector in the pnma unit cell . ehrenberg et al.s
neutron diffraction study confirmed this alignment of spins , and found
a similar alignment of the co spins in their intermediate lixcopo4 ( x = 0.6 ) phase . the neutron diffraction data acquired on a sample
with average composition of li0.2copo4 was interpreted
in terms of an additive contribution from all three stable phases
( licopo4 , li0.6copo4 , and copo4 ) , and the magnetic structure of copo4 was found
to comprise of co spins aligned antiferromagnetically along the
direction of the copo4 unit cell ( i.e. , perpendicular to
the spin alignment in the licopo4 cell ) . in situ
x - ray ( powder ) diffraction is a valuable tool for monitoring
the evolution of the long - range order of crystalline battery materials
during electrochemical cycling . solid - state nmr , on the other
hand , yields insight into the local coordination of the nucleus being
studied and is also proven to be a fundamental technique for the study
of battery materials . the paramagnetic co and co ions in the olivine structure result in hyperfine interactions between
the unpaired electrons and the nucleus under study ( li
and p in this case ) , and these interactions dominate
the nmr response . the strongly covalent p o bonds in the olivine
structure lead to a large transfer of electron density onto the p
atom via co o p pathways , the resulting through - bond
or supertransferred fermi contact interaction leading to very large p isotropic shifts ( e.g. , iso
3000 ppm for licopo4 ) . the
through - space nuclear - electron interaction ( i.e. , the dipolar interaction )
in these systems results in broad spinning sideband manifolds , even
when fast magic angle spinning ( mas ) is used . the presence of multiple
chemical environments invariably complicates the spectra , as the isotropic
resonance due to one environment may overlap with spinning sidebands
from another , and so pulse sequences that separate sidebands from
isotropic resonances are particularly useful in such cases . for example ,
both the magic angle turning phase adjusted sideband separation ( matpass )
and the adiabatic magic angle turning ( amat ) experiments have been
used to study related paramagnetic cathode materials ; the former method is used in this work . this paper builds
on our preliminary report of the p spectra and xrd patterns
of lixcopo4 and on the work recently published
by kaus et al . we combine the complementary
experimental techniques of nmr and xrd with first - principles dft calculations
to study the electrochemical ( de)lithiation of licopo4 . we obtain invaluable insights into not only how licopo4 delithiates , but also into the co / co ordering
and the li composition of the lixcopo4 intermediate phase . because only limited information is obtained
experimentally on li / vacancy ordering in the intermediate
phase , we use dft calculations to evaluate the lowest energy configurations ,
within the co / co ordering determined from
the experimental nmr and xrd data on the intermediate phase . the effect
of the magnetic ordering and spin states of the co and
co cations on the phase energetics is explored by dft ,
first for the end member phases , and then for the intermediate lixcopo4 structure . carbon - coated licopo4 ( c - licopo4 ) was synthesized
via the solid - state
method using cobalt oxalate ( sigma - aldrich ) , lithium carbonate ( sigma - aldrich ,
99.997% ) , ammonium dihydrogen phosphate ( sigma - aldrich , 99.999% ) and
10 wt % ketjen black ( azkonobel ) in a stoichiometric mixture . after
high - energy ball milling for 20 min , the reaction mixture was pelletized
and heated to 600 c under flowing argon . the precursors were
heated for 6 h , cooled and reheated for 11 h twice , before a final
24 h heating step . the additional heating
and cooling steps were carried out to decrease the amount of impurities
present in the final product . phase purity was confirmed using
powder x - ray diffraction with a panalytical empyrean x - ray diffractometer
with a cu k source . the total scan time was 9 h and 52 min ,
using a step size of 0.017 over a 2 range from 5 to 140.
the topas software was used to perform the rietveld refinement . the electrode was prepared by ball milling
80 wt % c - licopo4 , 10 wt % carbon super p li ( timcal ) and
10 wt % polyvinylidene fluoride ( kynar ) for 1 h. dry n - methyl pyrolidene ( sigma - aldrich ) was added dropwise to the mixture
to make a slurry . a 150 m spreader was used to cast the film
onto aluminum foil , which was dried in an oven at 60 c overnight .
a circular punch of 7/16 in . diameter was used to cut the cathode
( 2 mg ) . for the ex situ nmr studies , the as - synthesized c - licopo4 powder ( 1020 mg ) a coin cell - type battery was assembled in an argon - filled glovebox ,
using li metal ( sigma - aldrich ) as the counter electrode , a whatman
gf / b borosilicate microfiber filter as the separator and 1 m lipf6 solution in a 1:1 mixture of ethylene carbonate / dimethyl
carbonate as the electrolyte . the electrode was prepared by mixing
85 wt % c - licopo4 , 5 wt % super p carbon ( alfa aesar ) ,
5 wt % carbon black ( vulcan
xc-72 , cabot corporation ) and 5 wt % polytetrafluorethylene ( sigma - aldrich ) . the powder was pressed into a 13 mm - diameter pellet of 150
m thickness and weighing 22 mg . the pellet was assembled
into the ampix in situ cell in an argon - filled
glovebox , using li metal as the counter electrode , a whatman gf / b
borosilicate microfiber filter as the separator , and 1 m lipf6 solution in a 1:1 mixture of ethylene carbonate / dimethyl
carbonate as the electrolyte ( tomiyama pure chemical industries ) . in situ xrd experiments were performed
at the powder diffraction beamline , 11-bm , at the advanced photon
source at argonne national laboratory . high angular resolution x - ray
diffraction data were collected using a 12 channel analyzer detector
array ( = 0.413609 , beam size 1.5 0.5 mm ) . data spanning a 026 2 range
were collected using a step size of 0.002. each measurement
took 7 min 40 s. rietveld refinements were undertaken within the topas
academic software . hahn echo and matpass spectra were collected
on a bruker 200 avance iii spectrometer ( 4.7 t field strength ) at
a larmor frequency of 81 and 78 mhz for the p and li experiments , respectively , using a 1.8 mm mas samoson probe .
for the p matpass experiments a series of five 90
pulses with a pulse width of 1.6 s were employed , rotor synchronized
at a mas frequency of 3839 khz . the recycle delay was 0.015 s. the decay time constant of the hahn
echo / matpass sequence , t2 , was
obtained at 4.7 t for lixcopo4 using a series of hahn echoes where , the delay separating
the centers of the 90 and 180 pulses , was varied between
250 s and 16 ms in 8 increments . the rotors were packed by mixing
kbr and the partially cycled c - licopo4 composite electrode
in a 2:1 ratio , in order to reduce paramagnetic interactions with
the field that prevented the rotors from spinning . the p and li data were referenced to an 85 wt % h3po4 and 1 m licl aqueous solution , respectively , at 0
ppm . configurations with different spin
states and spin alignments were generated for licopo4 ,
copo4 , and for the intermediate phase ( assumed to be li2/3copo4 as discussed below ) , and their energetics
were computed in a series of solid - state dft simulations . the vienna
ab initio simulation package ( vasp5.2 ) was implemented within spin - polarized
dft , and the projector - augmented wave ( paw ) approach was used to describe the electron . full
relaxation of the atomic positions and cell parameters , and total
energy calculations , were carried out in the absence of symmetry constraints . ernzerhof ( pbe ) exchange - correlation
functional was used throughout , applying
the hubbard u model within the rotationally invariant
formalism proposed by liechstenstein et al . to correct for the known deficiencies of pure functionals for highly
localized 3d states , as explained in more detail below . after testing for convergence of the total energy
of the licopo4 and copo4 end members with respect
to the plane wave cutoff energy and k - point mesh density , a cutoff
energy of 500 ev and a 24 k - point grid were selected for calculations
within the 4 formula unit cells of the end member compounds . a smaller
k - point grid ( 16 k - points ) was used for the ( a
3b c ) li2/3copo4 supercells comprising 12 formula units . the threshold difference
for self - consistent field ( scf ) convergence in the total free energy
was set to 1 10 ev , and a gaussian - type
smearing of the fermi level , equivalent to a temperature of 1.2 k ,
was applied . starting structures for the end member phases were taken
from a previous study by ehrenberg et al . , whereas the optimization of the intermediate phase proceeded from
the licopo4 structure in which a third of the li ions were removed . the effect of spin polarization on the
total energy of the different lixcopo4 phases ( x = 0 , 2/3 , 1 ) was investigated
by initializing each co spin in a particular spin configuration ( low
spin t2geg or high spin
t2geg for d co ions ; and low spin t2geg , high spin t2geg , or intermediate spin t2geg for co ions ) in the scf process . the intermediate spin configuration was explored since it has been
found in other co systems . the total
magnetization of the cell was fixed to the value corresponding to
the initial magnetic spin configuration ( equal to the sum of all individual
co spins in ferromagnetic cells , or to zero in antiferromagnetic cells )
in the first structural optimization run , but the total cell magnetization
constraint was released in a subsequent structural optimization run . the charge density was recalculated at the start of each new optimization
and single point energy run , from the wave function obtained at the
end of the previous run . the final energy , and spin and charge distributions
of the different configurations were obtained from a single point
energy run in the absence of magnetic constraints . values of
5.05 and 6.34 ev were chosen for the effective hubbard
interaction parameter ueff = u j for co ( in licopo4 ) and co ( in copo4 ) , respectively , where u is the intraband coulomb term and j is the intraatomic
exchange term . the ueff values were obtained
self - consistently and used successfully in a previous study on the
limpo4 compounds ( m = mn , co , ni , fe ) . to compare the total energies obtained for the various
li configurations with stoichiometry li2/3copo4 to the total energies obtained for the licopo4 , and copo4 end members , this value is the weighted
average of the ueff values optimized for
co in licopo4 and co in copo4 , assuming co and co are present
in a 2:1 ratio . the effect of using a single ueff value
to calculate the energies of the end member licopo4 and
copo4 compounds is further discussed in the supporting information ( si ) . moreover , the effect
of electronic localization , through the use of a u hubbard correction
term , on the energies of the different spin and magnetic states of
the end member licopo4 and copo4 phases , and
on the formation energies of the different intermediate configurations ,
was further explored by repeating all calculations in the ferromagnetic
state within the pure gga approach ( u = 0 ev ) . table
s2 in the supporting information summarizes
the outcome of all the calculations carried out in this study . carbon coated licopo4 ( c - licopo4 ) , pnma , was synthesized via the solid - state method . additional heating steps beyond that used in
the lifepo4 synthesis were
required to decrease the amount of impurities present in the product
( including li3po4 and co2p ; see the supporting information ) . xrd and solid - state p and li nmr experiments were carried out on
c - licopo4 and there were no detectable impurities ( see
the supporting information ) . the unit - cell
parameters evaluated from a rietveld refinement on the xrd pattern
agree well with literature values ( a = 10.202(48 )
, b = 5.922(28 ) , c =
4.700(23 ) and volume = 283.9(24 ) ) . a single isotropic resonance is observed in
both the p and li nmr spectra ( iso of 2985 ppm and 104 ppm , respectively ) as expected
from single distinct p and li environments in licopo4 . in situ xrd
was carried out on the c - licopo4-composite film in the
ampix cell for 1.5 galvanostatic cycles
at a charge rate of c/20 ( see the supporting information ) . a two - dimensional representation
of the xrd patterns is shown in figure 1a . the region 2 = 7.88.3 ( at = 0.413609
) shows the ( 211 ) and the ( 020 ) reflections for licopo4 and copo4 , and most clearly reveals the changes in the
unit cell parameters upon cycling . upon charging , the characteristic
two - phase behavior between licopo4 = > lixcopo4 ( at 4.82 v ) and lixcopo4 = > copo4 ( at 4.89 v ) is observed
via
the simultaneous disappearance and appearance of the reflections of
the starting material and product , respectively . after the first charge ,
the bragg reflections are broadened to the point where they are difficult
to distinguish from the background . however , a small peak at 2
8.1 , corresponding to the lixcopo4 phase , appears on discharge after the disappearance
of the copo4 ( 020 ) reflection , suggesting that both two - phase
reactions involving the intermediate are reversible . , as we see both a significant loss of long - range order after the
first charge ( shown by the decrease in intensity of the bragg reflections )
and the formation of an intermediate , lixcopo4 . so - called amorphization has also
been observed in ex situ xrd after chemical delithiation , which indicates
that this behavior is intrinsic to the material and is not a consequence
of interaction with the x - ray beam . more recently ,
a slight loss of long - range order has also been seen for lifepo4 , but the phenomenon is less pronounced than for licopo4 . xrd data for the first
1.5 galvanostatic cycles of licopo4 cycled at a rate of c/20 . ( a ) two - dimensional image
of the patterns at a 2 range of 7.88.3 , shows
the transformation of the ( 211 ) and ( 020 ) reflections in licopo4 and copo4 . the y - axis is scan
number ( time ) and x - axis is 2. the results
from the rietveld refinements using the topas academic software show changes in ( b ) the scale factor ( the sum
of the scale factors is plotted in yellow ) and ( c ) the unit cell volume
in the three phases ( licopo4 , lixcopo4 and copo4 shown in red , purple
and blue , respectively ) . ( d ) gitt experiment ( in green ) for licopo4 ( 4th charge ) is overlaid onto the unit - cell volume data obtained
for the 1st charge ; the ocv is indicated with a black dotted line . rietveld refinements were carried
out using the diffraction patterns
acquired during the first charge ; the scale factor of the refined
phases and the unit - cell volume are shown in panels b and c in figure 1 , respectively . the analyses confirm the qualitative
observation of two distinct two - phase reaction processes ( indicated
by a block of green crosses above the figure ) . however , it is surprising
that there are regions , denoted by diagonal red lines above the figure ,
within which there is very little evidence of any reaction , i.e. ,
there is neither the formation of a new phase , nor a significant change
in the lattice parameters ( a solid solution mechanism would manifest
itself in the xrd patterns as a gradual change in the unit - cell parameters ) . very small changes do , however , occur with respect to the peak position ,
which are difficult to observe owing to the accompanying broadening
of the peaks ; these changes are more clearly seen in figure s3c in
the supporting information . this suggests
that small structural changes ( including some partial delithiation )
are required to trigger the onset of the next process . the electrochemical
activity in these regions is likely dominated by side reactions , i.e.
oxidation of the electrolyte , formation of the solid
electrolyte interface , and/or metal dissolution . we tentatively suggest that the growth of the copo4 phase from the intermediate phase is associated with a large
activation energy , and the side - reactions compete with this structural
transformation . the areas indicated with green crosses in panels b
and c in figure 1 sum to 160 mah / g ,
suggesting that the main electrochemical reaction occurs within these
regions ( the theoretical capacity of licopo4 is 167 mah / g ) . the crystalline phase fraction continues to diminish , even in regions
where side reactions dominate , suggesting that the reduction in long - range
order is not directly correlated with the delithiation reaction , but
instead depends on the time that the cell remains at high potentials . overall , a very large capacity of 250 mah / g is measured
on the first charge . however , the discharge capacity for the first
cycle is 149 mah / g ( 89% of the theoretical capacity ) , suggesting that ,
while side reactions are significant ( with 40% of the charge capacity
being irreversible on the first cycle ) , li intercalation and deintercalation
in licopo4 is largely unaffected by them . this is the highest
reversible capacity documented in the literature for this material ,
and is evidence that the loss of long - range order does not impact
the reversibility of the reaction , at least for the first few cycles . the additional capacity associated with the side reactions at the
beginning of charge ( i.e. , below 4.75 v ) drops noticeably in subsequent
cycles , while the additional capacity at higher voltages is still
observed ( see the supporting information ) . no attempt was made here to minimize the side reactions through
the introduction of additives or by surface coating of electrode particles ,
since our focus here is on the structural transformations that occur
in this system . we note that a recent xrd and nmr study of this system
has shown these side reactions can be partially reduced by the use
of electrolyte additives like hfip . interestingly ,
the volume of the licopo4 primitive olivine
cell decreases slightly from 284.1 to 282.1 ( decrease
of 0.7% ) as its phase fraction decreases to zero , approaching that
of the lixcopo4 phase when
it first appears , at 281.4 . similarly , the cell
volume of lixcopo4 varies noticeably
from 281.4 to 278.2 ( decrease of 1.1% ) across its
two - phase coexistence with licopo4 , and appears to sustain
a larger solid solution range than the two end members . of particular
note , the phase that forms during each of the two - phase reactions
( lixcopo4 followed by copo4 ) has a larger solid solution regime ( i.e. , a larger range
in cell volume ) than the phase being consumed ( licopo4 and
lixcopo4 , respectively ) . one
explanation for this is that the new phase , which nucleates and grows
within the parent phase , is distorted to compensate for the difference
in cell parameters and to reduce the coherency strain at the grain
boundary . the greater deviation in li composition in the nucleating
phase is consistent with the work carried out by ravnsbk et
al . on the lifexmn1xpo4 materials , which they attribute to
a small amount of a coherently nucleating phase . there appears to be a lag between the two major plateaus
observed
in the galvanostatic cycling and the two - phase reaction regions emerging
from the refinements , a voltage increase from 4.82 to 4.89 v being
observed before the licopo4 phase has fully converted to
lixcopo4 . however , the agreement
is significantly better if we compare the electrochemically active
regions determined by xrd with the plateaus obtained from the open
circuit voltage ( ocv ) within galvanostatic intermittent titration
technique ( gitt ) experiments , as shown by black dashed lines in figure 1d . note that the gitt data was taken from the fourth
cycle , where the latent ( side reaction ) period observed
at the beginning of the charge is significantly shorter . thus , the
gitt data has been offset by 25 mah / g in order to align the electrochemically
active regions with the xrd volume changes . the overpotential increases
noticeably before the end of the first plateau ( with an ocv of 4.75
v ) , resulting in an increase in the potential measured under galvanostatic
conditions from 4.82 to 4.89 v. the overpotential appears to correlate
with the concentration and cell volume of the minority phase within
the two - phase reaction . during the 4.75 v plateau , the overpotential
is at a minimum when lixcopo4 nucleates and is the minority phase within the licopo4 particle . as lixcopo4 grows
and licopo4 becomes the minority phase , the overpotential
increases . the overpotential increases at the same point in the electrochemistry
as the decrease in the volume of licopo4 from its initial
value of 284.1 , the volume presumably contracting
so that it can be accommodated within the lixcopo4 majority particle . the voltage associated
with li extraction will be increased by the overpotential required
to drive the energetically unfavorable decrease in cell volume in
licopo4 . to estimate the li content in the intermediate
phase , we compared
the lattice parameters of the intermediate structure with those of
the end members , and the li content was obtained using vegard s
law . on the basis of the volume of lixcopo4 , a value of x = 0.72 is obtained .
however , the a , b , and c lattice parameters considered separately would indicate a li stoichiometry
of x = 0.80 , 0.47 and 0.66 , respectively ( the end
member cell parameters are shown in the supporting
information ) . therefore , due to the anisotropic changes in
the lattice parameters of lixcopo4 , compared to those of copo4 and licopo4 , we are only able to provide , based on the xrd results , a range
( 0.47 x 0.80 ) within which we expect
to find the li composition of the intermediate . ex situ p and li nmr spectroscopy was
carried out on licopo4 at different
states of charge and discharge , obtained by galvanostatically cycling
the battery at a rate of c/20 ( figure 2 ) . the lixcopo4 intermediate
is observed in the spectra of both nuclei , and is therefore deemed
stable in the absence of a current . ex situ ( a ) p matpass and
( b ) li hahn
echo mas nmr spectra recorded at a magnetic field strength of 4.7
t and at a spinning speed , r , between 38 and 39
khz , at different points in the first galvanostatic cycle of licopo4 , cycled at a rate of c/20 . ch115 and ch185 refer to the spectra
of the licopo4-composite electrode , which has been charged
up to 50 , 115 , and 185 mah / g , respectively . the spectra labeled discharged
and dis25 , were both fully charged and subsequently
either fully discharged or discharged for 3 h , respectively . all of
the spectra have been normalized so that the maximum intensity is
equal to 1 . there is again a significant delay
between the start of the electrochemistry
and the onset of delithiation in licopo4 , which agrees
with the xrd data and strongly indicates the existence of side reactions
in this region , the peaks from lixcopo4 only being observed after li extraction corresponding to
a capacity of 50 mah / g . however , once commenced , the reaction apparently
occurs steadily . after 185 mah / g , the lixcopo4 peaks decrease with the growth of the copo4 peak ( this is only observable in the matpass p nmr
spectra , as there is a negligible contribution from li in a both two - phase
mechanisms are reversible on discharge : the p copo4 peak ( iso = 3201 ppm ) decreases with the
growth of the lixcopo4 peaks ,
which then decrease with the growth of the licopo4 peak .
the shift and line width of the licopo4p
resonance ( at iso = 2989 ppm ) after one cycle is
similar to that observed initially , indicating that the amorphization
observed with in situ xrd corresponds only to a loss of long - range
structure , the short - range structure being unchanged ; this is consistent
with the reversible electrochemistry of the licopo4 phase . the nmr results do not imply much compositional variation , as observed
in the xrd data , specifically for the intermediate lixcopo4 phase . this must in part be due
to the limited number of samples examined by using nmr spectroscopy ,
but may also be a result of the absence of a current in the ex situ
nmr experiments , permitting solid solutions to relax into more structurally
homogeneous end member phases of the reactions , so that only resonances
associated with the licopo4 , lixcopo4 and copo4 phases are observed . hahn - echo p nmr spectra were also acquired for the same samples ( see
the supporting information ) ; yielding similar
shifts and intensities for the various resonances across the electrochemical
range . we chose to analyze the matpass spectra in greater detail since
these spectra do not suffer the additional complications of the overlap
between the isotropic resonance of one environment and the sidebands
of another . our li and p nmr spectra are
essentially identical to those obtained recently in an nmr and xrd
study of this material . from the
nmr spectra , we are able to extract information about
the local structure of the intermediate phase : two p
resonances are observed at iso = 2610 and 2210 ppm ,
and two li resonances at iso = 69 and
125 ppm . the li resonance at 125 ppm in
the intermediate overlaps with the isotropic shift observed for the
fully lithiated phase , suggesting that it arises from a li nucleus
predominantly surrounded by co ions . from the charged li spectrum ( i.e. , the licopo4 electrode that has
been fully charged to 5.0 v ) , where the features of the intermediate
phase are most prevalent , the integrated intensity indicates that
the two environments are present in an approximately 1:1 ratio ( the
measured ratio is 1:1.23 , the higher intensity of the 125
ppm resonance being ascribed to minor contributions from the overlapping
licopo4 resonance ) . the p matpass ch185
spectrum ( i.e. , the point at which the licopo4 electrode
has been charged to a capacity of 185 mah / g ) shows the clearest resolution
of the p intermediate peaks and suggests that the two
p environments are present in an approximately 2:1 ratio ( a value
of 2:0.87 was calculated for resonances iso = 2610
and 2210 ppm , respectively ) . note that the different spin spin
( t2 ) relaxation times for the two p and two li environments have been accounted
for in the intensity analysis ( see the supporting
information ) . although it is possible to have two p environments
that have similar isotropic shifts and that give rise to overlapping
peaks in the nmr spectrum , our former studies of the lifexmn1xpo4 and lifexco1xpo4 materials have shown that the
hyperfine shift is highly sensitive to the geometry of the transition
metals around p and to the oxidation state . it is therefore unlikely
for two p environments to have the exact same shift ,
and , as there is no significant additional peak broadening or distortion
of the more intense resonance , this suggests that the observed resonances
correspond to two distinct environments occurring in a 2:1 ratio .
it would be helpful to perform li or p 2d
homonuclear correlation experiments on the intermediate phase to obtain
insight into the spatial proximities of the environments giving rise
to the resonances . however , nmr simulations ( see the supporting information ) show that such experiments are extremely
challenging because of the large hyperfine dipolar interactions which
are much larger than the dipolar coupling between the nuclei , and
the short spin lattice ( t1 ) relaxation
times relative to the time required to establish significant correlations
between the different nuclei using a dipolar recoupling scheme . although it is surprising at first sight to observe p nmr resonances associated with lixcopo4 lying beyond the range demarked by the end member licopo4 and copo4 shifts , a similar phenomenon was observed
for the p environments in lifexco1xpo4 , where many of the
observed resonances did not fall between those of pure lifepo4 and licopo4 . this
is attributed to the high degree of sensitivity of the p hyperfine shift to the individual geometries and d - orbital occupancies
of the m o - p ( m = fe , co , and co ) bond pathways . we now use the constraints set by
the nmr spectra
( in particular , the number of different p and li local environments
and their spectral weights ) , in combination with the lattice parameters
extracted from the xrd refinements , to propose a composition and structure
for the intermediate phase . the anisotropic changes of the lattice
parameters of the intermediate with respect to the unit - cell parameters
of the end members led us to consider a range of intermediate phase
compositions , i.e. , li0.470.80copo4 . it is not possible to devise a li and co / co ordering scheme within the original olivine unit - cell that gives
rise to two different p resonances with a 2:1 intensity
ratio , since this cell contains four p atoms . therefore , a reasonable
first assumption is that the intermediate structure is represented
by an expansion of the original olivine primitive cell . in order to
analyze the different possible structures in a systematic way , we
proceed by considering the following supercell expansions : ( 2a b c ) , ( a 2b c ) , ( a b 2c ) , ( 3a b c ) , ( a 3b c ) , and ( a b 3c )
( i.e. , three cells are doubled and three cells are tripled along either
the a , b , and c axes ) to create six supercells . we also consider a seventh expansion ,
the na2/3fepo4 superstructure , which formally involves cell tripling in two directions , ( either
( 3a 3b c ) or ( ab 3b c ) ) . because the p fermi contact shifts are much
more sensitive to the nature and/or oxidation state of the transition
metals in the first coordination shell than the li nmr
spectra , we first analyze the p spectra
before proceeding to examine the li environments . there are five co
positions ( labeled m1 , m2 , m2 , m3 , and m4 in figure 3a ) in the first cation coordination shell of p in
the olivine structure , of which two are equivalent ( m2 and m2 )
because of the presence of a mirror plane passing through the p atoms
in the pure limpo4 and mpo4 phases . seven tables were created ( see the supporting information ) with the 8 , 12 , and 12
p local sites ( from cell doubling , tripling , and the ( ab 3b c ) supercell ,
respectively ) and the five different ( labeled ) co atoms in the first
coordination shell around the p sites . in these supercells , the periodicity
imposes further limits on the possible range of p environments ; e.g. ,
in the ( 2a b c ) supercell , p1 is surrounded
by two co1 atoms from two different cells on the m1 and m3 positions ,
two co5 atoms from two cells on m2 and m2 and a co6 atom on
m4 . first coordination shell of co atoms around ( a ) p and ( b ) li in
the olivine structure . tables showing ( c ) the 12 p and ( d ) 12 li sites
generated in the ( a 3b c ) supercell of the olivine structure , and their nearest co atoms . the co atoms shaded in red and white represent co and
co , respectively , generating three p and two li local
environments in the co / co arrangement shown
here . all configurations obtained when
the cell is doubled were immediately
dismissed because there are 8 p atoms in the supercell , so it is never
possible to obtain two p sites in a 2:1 ratio . we then
considered the different cells ( and different p local environments )
generated by assuming that the different co sites were either co or co , in the appropriate ratios to form li
compositions falling within the range li0.470.80copo4 . when the unit cell is tripled along either
the a , b , or c axis ,
every arrangement
of co and co ions generates more than two p sites , in disagreement with the p nmr experimental
data ( illustrated in figure s7d i in the supporting information ) . after a thorough analysis in which
all possible combinations were explored , the closest agreement with
the nmr data was achieved with a co to co ratio of 2:1 , resulting in three different p environments in a 1:1:1
ratio ( figure s7d , f in the supporting information ) . when the cell is tripled along the b - direction ,
however , two of these three p environments consist of one co on either the m2 or the m2 site , and four co on the remaining sites ( p1 , p2 , p4 , p5 , p8 , p9 , p11 , and p12 in
figure 3c ) . because the mirror plane on p is
retained in the cosubstituted materials , lifexmn1xpo4 and
lifexco1xpo4 , it strongly suggests that in lixcopo4 , m2 and m2 are also equivalent . the bond lengths and bond angles generated by our calculations support
this assumption , as discussed in the dft study . therefore , even though
three p environments arise , two of them are expected to give rise
to identical shifts in the presence of the mirror plane , resulting
in two peaks occurring in a 2:1 ratio . there are three symmetrically
equivalent possible combinations
of co / co ordering for the ( a 3b c ) superstructure . they can be superimposed
onto each other by a translation in the direction perpendicular to
the co chains ( see the supporting information ) , which , as observed in figure 3d , results
in a total of two distinct li environments : four li sites surrounded
by six co ( denoted li , figure 4d ) and eight li sites surrounded by three co and
three co ( denoted li , figure 4e ) . li is assigned to the li resonance
at 125 ppm , as it overlaps with the pristine li
resonance ( i.e. , li surrounded by 6 co ions ) . the composition
li2/3copo4 implies that four li ions must be
removed from the supercell . given the experimentally observed intensities ,
the li ions must be removed from the li sublattice , yielding
two li environments in a 1:1 ratio , in agreement with the li nmr results . note that this differs from the ordering scheme
proposed in the recent li and p nmr study . ( a c ) three p environments ( present in a 1:1:1
ratio ) and
( d , e ) two li environments ( which are present in a 1:1 ratio ) in the
li2/3copo4 structure . because m2 = m2 ,
a and b are equivalent . ( f ) there are 8 li sites ( shown in
green ) , 4 of which must be removed for charge balancing ( co , co and li are shown in blue , magenta , and orange ,
respectively ) . the ( ab 3b c ) supercell
present in the na2/3fepo4 intermediate structure was explored , and
the same fe / fe ordering observed in both
these studies was implemented for co / co ( figure
s8 in the supporting information ) . however ,
this ordering results in five different p sites . when m2 and m2
are assumed to be equivalent , three distinct p environments
are predicted in a 1:1:1 ratio , which still disagrees with the p nmr data . the ( a b 3c ) supercell , which was recently suggested by kaus et al . for the li2/3copo4 intermediate , is presented in figure s7d in the supporting information and results in the presence of five inequivalent p
environments in a 2:1:1:1:1 ratio , so it is not considered further . energy
diagrams of ( a ) licopo4 and ( b ) copo4 in their
experimental ( exp ) and dft
optimized ( opt ) structures , within the gga+u ( ueff = 5.48
ev ) approach . total energies were calculated for cells in which all
co spins were ferromagnetically ( fo ) aligned ( in either their low
( ls ) or high ( hs ) spin state ) , and on cells in which the hs co spins
were antiferromagnetically ( af ) aligned , as reported in a previous
magnetic study on these phases . first - principles dft
calculations were performed on the end member phases and on the proposed
li2/3copo4 intermediate structure to determine
the lowest - energy spin state and magnetic alignment at all three stages
of charge of the licopo4 electrode . the calculations considered
both structures with fixed experimental cell parameters and atomic
positions , as obtained from rietveld refinement of neutron and xrd
data , and fully optimized structures .
the relative stability of different li / vacancy ordering
schemes in the intermediate was also explored . the calculations on licopo4 and copo4 were
performed using an average ueff value
of 5.48 ev , as derived for the mixed valence li2/3copo4 system . a high spin ground state was predicted for both end
member phases , in agreement with previous experimental and theoretical studies . the previous dft study reported that different magnetic
ordering schemes of the transition metal ions lead to energy differences
of 1060 mev per formula unit . our results on the licopo4 and copo4 end member
phases , presented in the two diagrams in panels a and b in figure 5 , show that these energy differences can be orders
of magnitude larger when the different spin states of the co and co atoms are also considered , a result in line
with the fact that intraatomic exchange interactions are typically
a few electron volts , whereas interatomic exchange interactions are
in the millielectron volt range . only one antiferromagnetic
state is reported here for licopo4 , as the antiferromagnetic
low - spin cells converged to the high - spin state upon release of magnetic
constraints . the total energies of the high spin ferromagnetic and
antiferromagnetic licopo4 states differ by 8 mev per formula
unit in both the experimental and optimized structures . as expected ,
the low - spin state is highly unfavorable and lies more than 300 mev
per formula unit above the high - spin ground state . in the dft
calculations performed on the copo4 structure ,
all cells with the co spins initialized in the intermediate - spin
state converged to high - spin states and are therefore not represented
in figure 5b . again , the low - spin state is much higher in energy ,
by more than 1 ev per formula unit . in summary , our calculations
confirm the antiferromagnetic ground
state and spin alignment deduced experimentally , for both licopo4 and copo4 structures , by ehrenberg et al . this study on the end member compounds yields
results in good agreement with previous experimental and theoretical studies , and can now
be used to determine the relative stabilities of the different ( li
and magnetic ) configurations considered here as potential li2/3copo4 intermediate phases . as previously described , the p nmr data for the intermediate phase li2/3copo4 suggest a tripling of the licopo4 unit cell along
the b - direction . the site occupation disorder ( sod ) code , devised to determine a minimal
set of symmetry - unique configurations for site - disorder problems in
solids , was used with the pnma licopo4 unit cell as the input structure . the ( a 3b c ) supercell ( figure 4f ) was created . it contains 12 li atoms , four of which were
systematically removed by the sod code in order to generate all possible
structures with a li2/3 stoichiometry . the code generated
thirty - two symmetry inequivalent supercells , in which all the co sites
were assumed to be equivalent . co / co ordering
along the b - direction , identified on the basis of
the p experimental nmr data , was implemented for each
of the thirty - two possible li configurations . nineteen of these structures
were discarded , as they did not agree with the li nmr
data ( i.e. , they involved the removal of one or more li ion(s ) from the li sublattice ) . close examination of the
13 remaining structures lead us to reject a further seven of them
that had either triple or higher aggregates of li vacancies close
to each other , and/or a highly anisotropic charge distribution . the
final six configurations ( a f ) are shown in figure 6 . six different li / vacancy ordering schemes
on which
dft calculations were performed , which are all consistent with the
experimental li and p nmr and xrd data . co and co are shown in blue and magenta , respectively ,
and li and li are shown in orange and green , respectively . the energies of the most stable
( antiferromagnetic high spin ) configurations
for the end member structures optimized ab initio were used to calculate
the formation energies of the six different li / vacancy
configurations . a lixcopo4 convex
hull was generated ( presented in figure s10 in the supporting information ) , which includes the energy of all
the ( intermediate and high ) spin states , and magnetic configurations
( ferromagnetic , antiferromagnetic ) which reached electronic convergence
without spin constraints in the last single - point energy dft calculation . additionally , the formation energies of all of the licopo4 , li2/3copo4 , and copo4 spin and
magnetic configurations investigated in this study are summarized
in table s2 in the supporting information . the lixcopo4 convex hull
presented in figure 7 only shows the lowest
energy spin and magnetic state , for each of the six different li configurations
considered within the pure gga and gga+u ( ueff = 5.48 ev ) approaches . we will analyze the gga+u results in this
section , then comment on the insight into the mechanisms stabilizing
the li2/3copo4 intermediate obtained from the
gga results in the discussion section . within
gga+u , apart from configuration b , none of the li arrangements considered
for the li2/3copo4 structure are stable in the
intermediate - spin state . the ( ferromagnetically and antiferromagnetically
coupled ) intermediate - spin state either does not reach electronic
convergence in the absence of magnetic constraints , or converges to
the high - spin state when the magnetic constraints are released . configuration
b is the only exception to the rule : its ferromagnetic high - spin state
is not stable and converges to the ferromagnetic intermediate - spin
state in the absence of constraints on the co spins . moreover , this
ferromagnetic intermediate - spin state is found to be the lowest - energy
spin state for this configuration , and the second lowest energy configuration
out of all the li configurations investigated in this study . figure 7 and figure s10 and table s2 in
the supporting information clearly demonstrate
that all of the li2/3copo4 configurations tested
in this study are thermodynamically stable with respect to the two
end member phases . the lowest energy configuration observed in figure 7 ( configuration a in the antiferromagnetic high
spin state ) has a negative formation energy of 309 mev per formula
unit . a map of the spin density of this configuration is presented
in table s2 in the supporting information . the formation
energies of the three most stable configurations ( figure 6a c ) differ by less than 5 mev per formula
unit , and we may expect more than one structure to be present at room
temperature . these three low energy configurations ( a , b and c ) have
minimal differences in the arrangement of the li vacancies , and can
be interconverted by li ion hops to adjacent li sites . it is
likely that the intermediate structure contains a disordered li sublattice
comprising all of these different li local environments . configurations
d f contain vacancies arranged in pairs , which were shown in
calculations on li0.6fepo4 materials to be the
most energetically favorable configurations . convex
hull for lixcopo4 , calculated
within the gga ( ueff = 0
ev ) and gga+u ( ueff = 5.48 ev ) approaches . the formation
energies of the different lixcopo4 ( x = 0 , 2/3 , 1 ) phases are plotted against
li composition , in ev per formula unit ( ev / f.u . ) . the insets show
the 0.32 to 0.22 ev / f.u . and the 0.12 to 0.05
ev / f.u . regions of the convex hull , corresponding to the range of
formation energies of the li2/3copo4 intermediate
structures in gga+u and in gga , respectively . although gga calculations
were only performed in the ferromagnetically aligned state , gga+u
calculations were performed in the ferromagnetically and antiferromagnetically
aligned states . only the lowest energy spin and magnetic state , for
each of the different li2/3copo4 li arrangements
investigated , is plotted . table s2 in the supporting
information summarizes the outcome of all calculations performed
on the li2/3copo4 cells , in gga and in gga+u . is , hs , fo and af denote the intermediate spin state , the high spin
state and the ferromagnetically and the antiferromagnetically aligned
phases , respectively . as previously discussed , the experimental observation of
only two p resonances implies an equivalence of the m2
and m2
metal positions in the first p coordination shell . to determine whether
this equivalence is observed computationally , we compared the co ( m2)o p and co(m2)o p
bond pathway geometries , in the lowest - energy li2/3copo4 supercell optimized ab initio ( configuration a in the high - spin
antiferromagnetic state ) . p
bond angles involving either m2 or m2 ( i.e. , the mirror plane
is preserved ) , helping to validate our assertion that only two p local environments are present . the excellent agreement between the number of distinct li and p
local environments and their relative populations , predicted using
the model outlined above and the experimental li and p nmr data obtained for the intermediate structure , led us
to propose an intermediate phase with stoichiometry li2/3(co)2/3(co)1/3po4 , and with a superstructure obtained by tripling the unit
cell in the b - direction . this result should be contrasted
to previous structural reports for other a2/3 polyanionic
intermediates ( a = li , na ) , li2/3vpo4f and a2/3fepo4 . although a li2/3vpo4f intermediate has been identified when livpo4f is electrochemically charged , the crystal structure is still
unknown . the a2/3fepo4 and li2/3copo4 intermediates adopt different superstructures with the former
consisting of a tripling in both the b- and a - directions ( figure s8 ) . three inequivalent sites are generated
from this superstructure , in disagreement with the experimental observations
for li2/3copo4 . interestingly , we observed two p resonances in our previous studies of li2/3fepo4 obtained by cooling micron - sized particles of chemically
delithiated lifepo4 , suggesting that more than one superstructure
may exist for li2/3fepo4 . the absence of superlattice reflections in the in situ
xrd data shown here was accounted for by performing simulations of
the li2/3copo4 diffraction pattern . the simulations
show that the intensity of the superlattice reflections are 1/100th
of that of the main bragg peaks ( see the supporting
information ) , and are not observable because of the significant
broadening of the peaks ; further tem analyses are ongoing . our
calculations predict that all six li2/3copo4 intermediate configurations investigated here lie below the
licopo4copo4 convex hull , conversely
to the li2/3fepo4 case , for which the intermediate
is only metastable and lies above the lifepo4fepo4 convex hull . therefore , in the co system ,
the thermodynamics will drive the formation of the intermediate phase . the activation energy barrier associated with the structural transformations
will also be lowered over that involving the direct delithiation of
licopo4 to form copo4 , since the intermediate
phase is able to buffer the volume changes associated with delithiation . the relative changes in the sizes of the a , b , and c unit - cell parameters , and subsequent
changes in the areas of the ab , ac and bc faces of the unit cell ( table 1 ) , allow us to speculate on a possible orientation
of any interface between licopo4 and li2/3copo4 and between li2/3copo4 and copo4 within a reacting particle . for the licopo4 = >
li2/3copo4 and li2/3copo4 = > copo4 reactions , the changes in the area of the
bc
cell faces are 0.7% and 0.1% , respectively . these changes are minor
in comparison to the changes of 2.4% and 6.1% in the area of the ac
cell faces for the licopo4 = > li2/3copo4 and li2/3copo4 = > copo4 reactions ,
respectively . the latter being largely a result of the large change
in the a - parameter during this reaction . these results
strongly suggest that any interface between licopo4 and
li2/3copo4 , and between li2/3copo4 and copo4 , that forms during the electrochemical
reaction , is in the bc - plane , in agreement with the
work by ravnsbk et al . on lifexmn1xpo4 . although coherency strain energy calculations have been
carried out by van der ven and cogswell on the lifepo4 system , it is not
trivial to rationalize the additional stabilization of the intermediate
phase in the licopo4 and copo4 particles . ab initio calculations on the li2/3copo4 intermediate
predict its thermodynamic stability with respect to the disproportionation
reaction leading to the formation of licopo4 and copo4 , in good agreement with experimental observations of a stable
li2/3copo4 intermediate phase in the absence
of a current . these observations should be contrasted with mechanisms
proposed for lifepo4 nanoparticles , where the lifepo4fepo4 interface was suggested to be unstable
and the delithiation reaction to occur particle - by - particle , as proposed
by the domino - cascade model . our recent
results and those of zhang et al . have suggested , in agreement with prior theoretical
predictions , that nanoparticles of lifepo4 react via a solid - solution mechanism rather than by forming
an abrupt interface between the two end - member phases , at least at
very high rates . the changes are calculated relative
to the licopo4 and li2/3copo4 structures ,
respectively . licopo4 , li2/3copo4 , and copo4 also
show considerable variations in cell parameters from
their equilibrium values during the in situ xrd measurement . this
phenomenon is most pronounced when the phases exist as minority phases
within the electrode ( see figure s13 in the supporting
information ) . for example , the b - parameter
of li2/3copo4 approaches that of licopo4 in the initial stages of delithiation . this indicates that
a substantial fraction of the particles within the electrode undergo
electrochemical reaction simultaneously , in contrast to results obtained
at low cycling rates for lifepo4 . thus , this system does
not react particle - by - particle ( the domino - cascade model ) . the mechanism
observed here for licopo4 is related to the solid - solution
mechanism , but is subtly different . , the li2/3copo4 phase must be able to accommodate a wide range of
nonstoichiometry during the reaction ( resulting in structural flexibility / elasticity )
so that when it first forms within the licopo4 phase it
contains excess lithium ( i.e. , li2/3+copo4 ) . because little evidence for nonstoichiometry is observed in the
ex situ p nmr experiments , we suggest that these are
also metastable solid solutions , but further calculations to determine
the energetics of different compositions within this system are required
to investigate this phenomenon in greater depth . nevertheless ,
the dft calculations performed in this study provide
insight into the mechanisms stabilizing the li2/3copo4 phase . the u hubbard term in gga+u allows charges to be localized
onto particular ions of mixed - valence systems , while pure gga results
in delocalization of charge over all metal atoms . the ab initio study
by zhou et al . on the lixfepo4 system showed that the electronic configurational entropy
needed to be taken into account to produce an accurate model for the
lifepo4-fepo4 phase diagram and to reproduce
the eutectic point at approximately x = 0.5 . this
electronic entropy , arising due to the tendency to form distinct fe and fe oxidation states in the gga+u calculations
and their subsequent arrangements , was found to be larger than the
configurational entropy associated with the distribution of li ions and vacancies in the lattice . in our work , the li2/3copo4 formation energies obtained within the
gga approach are not as large as those calculated in the gga+u case ,
the additional stability of the intermediate configurations observed
in gga+u arising from an electronic term related to localizing the
charges in the structure . however , we note that the failure of pure
dft is not as severe in the case of li2/3copo4 , where the method still predicts the intermediate configurations
to be below the convex hull . in contrast , in lixfepo4 , lda and gga do not predict the experimentally
observed phase separation at low t. this result is in agreement with previous gga studies on
the licopo4/copo4 system . finally , the formation energies obtained within the pure gga approach
on the different li2/3copo4 structures reveal
that configurations a , b , and c , are lower in energy than configurations
d , e and f , whether or not a u correction is applied . this suggests
that the greater stabilization of configurations with single li vacancies
( a , b , and c ) , compared to configurations containing pairs of li vacancies
( d , e , and f ) , is due to more favorable electrostatics , in contrast
to what has been observed for li0.6fepo4 . both xrd and nmr studies
show that the electrochemical delithiation
reaction of licopo4 involves two distinct two - phase mechanisms ,
namely licopo4 = > li2/3copo4 and
li2/3copo4 = > copo4 , both of which
are reversible upon discharge . li2/3copo4 is
stable in the absence of a current , a result confirmed by dft calculations
of the energetics of this phase . the ex situ nmr spectra of a range
of delithiated samples provide direct insight into the p and li local
environments occurring in the intermediate phase : two li environments
are present in a 1:1 ratio ( one environment containing a first coordination
shell of only co cations ) , along with two p environments
in a 2:1 ratio . a thorough analysis of these nmr spectra led to the
conclusion that the intermediate has the composition li2/3copo4 , with co / co ordering along
the b - axis . the dft calculations indicate that the
lowest energy li / vacancy ordering also occurs along the b - axis , with the vacancies residing next to the co ions . their total energies lie within a range
of 5 mev per formula unit , and the three configurations only differ
by single li displacements between neighboring li
sites in the b - direction , in keeping with the fact
that there is only a 50% occupancy of the sites neighboring both co and co ions . thus , it is very likely that the
intermediate phase contains a degree of disorder on the li sublattice . a comparison of the formation energies obtained in gga+u and gga on
the different li2/3copo4 intermediate configurations
reveals that an electronic term contributes to the stability of the
intermediate phase . | in situ synchrotron diffraction measurements
and subsequent rietveld
refinements are used to show that the high energy density cathode
material licopo4 ( space group pnma ) undergoes
two distinct two - phase reactions upon charge and discharge , both occurring
via an intermediate li2/3(co2+)2/3(co3+)1/3po4 phase .
two resonances
are observed for li2/3copo4 with intensity ratios
of 2:1 and 1:1 in the 31p and 7li nmr spectra ,
respectively .
an ordering of co2+/co3 + oxidation
states is proposed within a ( a 3b c ) supercell , and li+/vacancy
ordering is investigated using experimental nmr data in combination
with first - principles solid - state dft calculations . in the lowest
energy configuration , both the co3 + ions and li vacancies
are found to order along the b - axis .
two other low
energy li+/vacancy ordering schemes are found only 5 mev
per formula unit higher in energy .
all three configurations lie below
the licopo4copo4 convex hull and they
may be readily interconverted by li+ hops along the b - direction . |
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an ideal twin - spot marcm would permit differential expression of markers in paired sister clones and restrict marker expression to the cells homozygous for a particular chromosome arm and its homologous counterpart ( fig . akin to marcm , we reasoned it should utilize independent suppressors that reside on opposing homologous chromosome arms and distal to the recombination site whereby loss of heterozygosity would trigger expression of the two distinct markers ( fig . for this new marcm technique , we exploited rna interference ( rnai ) to suppress gene expression in a transcript - dependent manner rather than by targeting promoters or proteins 13 - 16 . based on our recent success silencing other transcripts with microrna ( mirna)-encoding transgenes 17 , 18 , we developed non - interfering uas - mirna transgenes that silence uas - driven mcd8::gfp and uas - driven rcd2::rfp , respectively ( supplemental fig . co - induction with uas - gfp - mirna potently silenced uas - mcd8::gfp but minimally affected the induction of uas - rcd2::rfp ( supplemental fig . 1b , e ) . consistently , uas - rcd2-mirna only suppressed uas - rcd2::rfp without compromising the expression of uas - mcd8::gfp ( supplemental fig . we can , thus , selectively silence uas - mcd8::gfp and uas - rcd2::rfp using uas - gfp - mirna and uas - rcd2-mirna , respectively . to test the efficacy of the twin - spot marcm technique , we assembled a pilot system on the 2l chromosome arm , induced clones , and assessed the results in wing discs . using the ubiquitous tubp - gal4 , we labeled paired clones in different colors in the otherwise unstained wing discs ( fig . 1d ) , the first evidence that twin - spot marcm works as we intended . we next applied twin - spot marcm to the mbs to establish its validity for studying the nervous system . neurogenesis of the mbs has been well studied with the existing marcm technology and independent methods 5 , 19 . past experiments using marcm , which labels only one side of a twin spot , indicate mutual exclusion of nb and two - cell clones . however , we can not ensure the pairing relationship between nb clones and two - cell clones , tell a two - cell clone from two nearby single - cell clones , or deduce the order of birth from randomly generated clones . to highlight clones in the mb lineage of 93 mb nb clones induced in nhl , we found 91 of them accompanied by exactly two mb neurons ( fig . the two exceptions were each paired with a group of three neurons ( data not shown ) . the presence of one extra neuron on the gmc side of nb progeny could derive from mitotic recombination within a coexisting gmc of the same or a neighboring lineage . moreover , the later we induce mitotic recombination , the smaller the size of the resulting nb clones ( fig . irrespective of nb clone size , we consistently detect a two - cell clone paired with it due to differential labeling by twin - spot marcm . notably , mb nb clones of different sizes are accompanied by two - cell clones of different types , perfectly reflecting the order of birth from , / , to / neurons ( fig . these results reinforce the current model of mb neurogenesis and show our twin - spot marcm system effectively labels paired sister clones in the brain . we anticipated pairing of a gfp - labeled neuron with one rfp - labeled neuron in clones produced by mitotic recombination in gmcs . in brains that have multiple gmc - induced clones , it may not be possible to tell which gfp neuron pairs with a particular rfp neuron since sister neurons can reside far apart as shown in our two - cell clones ( fig . however , the numbers of gfp- and rfp - labeled single - cell clones in each mosaic mb should be constantly equal . while we frequently saw the expected pairing of / or / neurons ( fig . 2m - r ; n>20 for each ) , this was not the case in clones of neurons . first , we seldom obtained neurons labeled without nb clones ( only 5 out of 200 mosaic brains derived from recombination in the early 1 instar larval stage ) . second , equal numbers of gfp and rfp neurons do not occur in most groups of neurons without nb clones ( 3 were not paired ) . since we never observe a mb nb clone either alone or accompanied by only one mb neuron , it is unlikely that a significant number of mb neurons are negative for gal4-ok107 or die prematurely . instead , it is more likely our technique does not faithfully label clones induced in gmcs due to stage - specific differential perdurance of rnai leading to delayed or incomplete derepression of the uas - reporters after the loss of their respective uas - mirna transgenes . this can also explain why the gmc - induced clones of neurons were weakly marked as compared to clones of / or / neurons ( data not shown ) . any perdurance of repression in marcm derives from expression of repressors prior to mitotic recombination and thus inheritance of repressors directly from heterozygous precursors . in the mirna - based twin - spot marcm system , gal4 drives expression of both the repressors and the reporters ; we labeled mb sister clones with mb247-gal4 , a mb driver expressed strongly in post - mitotic neurons but not in precursors 12 . we did not detect any delay in the induction of uas - reporter and we identified neuron pairs not only in adult brains ( fig . 2j - l ; n=46/48 ; two cases were one neuron paired with two neurons ) but also in larval brains ( fig . 2s - u ; n=12 ) , in which the original marcm technique can not label single - cell clones due to tubp - gal80 perdurance ( data not shown ) . visualizing both sides of a twin - spot is essential for depicting a complete proliferation pattern for any progenitor . for example , consistent pairing of a multi - cellular nb clone and a two - cell clone through development of a lineage would confirm the models that self - renewing nbs serially produce gmcs and that gmcs produce two neurons . it was long thought that all drosophila nbs undergo this simple pattern of neurogenesis until recent identification of pan ( posterior asense - negative ) lineages and their transit - amplifying precursors 20 , 21 . each transit - amplifying precursor , derived from self - renewing pan nbs , may give rise to multiple gmcs through a limited run of self - renewing asymmetric division . in addition , gmcs from many neuronal lineages of the drosophila ventral nerve cord ( vnc ) initially produce two daughter cells during larval neurogenesis , but only one of them survives to mature into an adult neuron ( williams d.w . and truman j.w . personal communication ) . we performed a study of neurogenesis patterns in the drosophila central brain and vnc using twin - spot marcm with the pan - neuronal gal4-c155 . for mosaic clones in the central brain , 43 of the 108 ( 40% ) nb clones , including 10 mb nb clones , are paired with two neurons ( fig . 3a , b , d , e , g , h ) ; 53 of the 108 ( 49% ) nb clones are accompanied by only one neuron ( fig . 3c , f , i ) ; and 10 of the 108 ( 9% ) nb clones , located in the dorsomedial region where pan lineages reside , associate with a cluster of 8 to 12 cells ( fig.3l , o , r ) . there were two nb clones without counter pair association ( 2% ; data not shown ) . in contrast , for mosaic clones in the vnc , only 26 of the 128 ( 20% ) nb clones are paired with two neurons ( fig . 3j , m , p ) while 100 of the 128 ( 78% ) nb clones are paired with a single neuron ( fig . 3k , n , q ) and two nb clones were not associated with any post - mitotic neurons ( 2% ; data not shown ) . gal4-ok107 labels , in addition to mb neurons , a cluster of six neurons in each brain lobe whose projections finally converge in various midline neuropils , collectively called the central complex ( fig . , determining the birth order of each neuron requires technology that provides at least the resolution of twin - spot marcm . by labeling gmc progeny in one color and the remaining lineage in another , we can determine which gmc has given rise to a particular neuron in any protracted neuronal lineage . to show this capability we found that all central complex nb clones have sister clones that contain only one cell , which suggests we are observing half of the gmc progeny ( fig . 4r - v ) , and that these six gal4-ok107-positive central complex neurons acquire different trajectories based on birth order . for example , the first - born neuron , as indicated by five cell bodies in the associated nb clone , consistently extends into a dorsal sub - compartment of the noduli and extends few processes ventrally ( fig . in contrast , the third - born neuron always innervates a ventral sub - compartment of the noduli ( fig . in addition , each neuron has distinct stereotyped branches off the primary neurite ( e.g. triple arrows in fig . , we ultimately identified the unique characteristics and birth order of each gal4-ok107-positive central complex neuron . the first four neurons each innervate a distinct sub - compartment of the noduli ( fig . 4d - g ) and the last two neurons project into the ellipsoid body ( fig . individual neurons also acquire stereotyped patterns of neurite elaboration in the lateral accessory lobe ( fig . it is extremely challenging to detect temporal cell fate transformation in a fast - tempo neuronal lineage where neuron fate may change with each self - renewing nb division . to exemplify how twin - spot marcm permits better determination of temporal cell fates , we examined whether and how chinmo , which perturbs temporal identity in mb neurons 12 , affects temporal fates in the sublineage of six central complex neurons . knocking out chinmo from a gmc of the central complex sublineage causes the 3 sibling to exhibit a temporal cell fate like that of the 4 neuron in the wild - type lineage . neurons derived from chinmo mutant 3 gmcs , as determined by associated wild - type nb clones containing three cells , acquired projections characteristic of wild - type 4 siblings ( fig . they innervate the middle sub - compartment of the noduli with the wild - type sibling of the nb clone ( arrow and arrowhead in fig . in addition , mutant 3 siblings , like wild - type 4-born neurons , acquire a much more diffuse pattern of neurite elaboration in the lateral accessory lobe than wild - type 3-born neurons ( triple arrows in fig . 4f , g , i ) . as to removal of chinmo from the central complex nb , we consistently fail to detect cells with an appropriate 3 temporal fate in chinmo mutant clones produced prior to birth of the 3 offspring ( n=3 ) . for example , in a chinmo mutant nb clone induced during production of the second gmc , which leads to a normal 2 sibling , we do not detect axons of mutant cells in the ventral sub - compartment of the noduli ( arrow in fig . we conclude chinmo is required in only one of the six fates of the gal4-ok107-positive central complex neurons . an ideal twin - spot marcm would permit differential expression of markers in paired sister clones and restrict marker expression to the cells homozygous for a particular chromosome arm and its homologous counterpart ( fig . akin to marcm , we reasoned it should utilize independent suppressors that reside on opposing homologous chromosome arms and distal to the recombination site whereby loss of heterozygosity would trigger expression of the two distinct markers ( fig . for this new marcm technique , we exploited rna interference ( rnai ) to suppress gene expression in a transcript - dependent manner rather than by targeting promoters or proteins 13 - 16 . based on our recent success silencing other transcripts with microrna ( mirna)-encoding transgenes 17 , 18 , we developed non - interfering uas - mirna transgenes that silence uas - driven mcd8::gfp and uas - driven rcd2::rfp , respectively ( supplemental fig . co - induction with uas - gfp - mirna potently silenced uas - mcd8::gfp but minimally affected the induction of uas - rcd2::rfp ( supplemental fig . 1b , e ) . consistently , uas - rcd2-mirna only suppressed uas - rcd2::rfp without compromising the expression of uas - mcd8::gfp ( supplemental fig . we can , thus , selectively silence uas - mcd8::gfp and uas - rcd2::rfp using uas - gfp - mirna and uas - rcd2-mirna , respectively . to test the efficacy of the twin - spot marcm technique , we assembled a pilot system on the 2l chromosome arm , induced clones , and assessed the results in wing discs . using the ubiquitous tubp - gal4 , we labeled paired clones in different colors in the otherwise unstained wing discs ( fig . 1d ) , the first evidence that twin - spot marcm works as we intended . we next applied twin - spot marcm to the mbs to establish its validity for studying the nervous system . neurogenesis of the mbs has been well studied with the existing marcm technology and independent methods 5 , 19 . past experiments using marcm , which labels only one side of a twin spot , indicate mutual exclusion of nb and two - cell clones . however , we can not ensure the pairing relationship between nb clones and two - cell clones , tell a two - cell clone from two nearby single - cell clones , or deduce the order of birth from randomly generated clones . to highlight clones in the mb lineage of 93 mb nb clones induced in nhl , we found 91 of them accompanied by exactly two mb neurons ( fig . the two exceptions were each paired with a group of three neurons ( data not shown ) . the presence of one extra neuron on the gmc side of nb progeny could derive from mitotic recombination within a coexisting gmc of the same or a neighboring lineage . moreover , the later we induce mitotic recombination , the smaller the size of the resulting nb clones ( fig . irrespective of nb clone size , we consistently detect a two - cell clone paired with it due to differential labeling by twin - spot marcm . notably , mb nb clones of different sizes are accompanied by two - cell clones of different types , perfectly reflecting the order of birth from , / , to / neurons ( fig . these results reinforce the current model of mb neurogenesis and show our twin - spot marcm system effectively labels paired sister clones in the brain . we anticipated pairing of a gfp - labeled neuron with one rfp - labeled neuron in clones produced by mitotic recombination in gmcs . in brains that have multiple gmc - induced clones , it may not be possible to tell which gfp neuron pairs with a particular rfp neuron since sister neurons can reside far apart as shown in our two - cell clones ( fig . however , the numbers of gfp- and rfp - labeled single - cell clones in each mosaic mb should be constantly equal . while we frequently saw the expected pairing of / or / neurons ( fig . 2m - r ; n>20 for each ) , this was not the case in clones of neurons . first , we seldom obtained neurons labeled without nb clones ( only 5 out of 200 mosaic brains derived from recombination in the early 1 instar larval stage ) . second , equal numbers of gfp and rfp neurons do not occur in most groups of neurons without nb clones ( 3 were not paired ) . since we never observe a mb nb clone either alone or accompanied by only one mb neuron , it is unlikely that a significant number of mb neurons are negative for gal4-ok107 or die prematurely . instead , it is more likely our technique does not faithfully label clones induced in gmcs due to stage - specific differential perdurance of rnai leading to delayed or incomplete derepression of the uas - reporters after the loss of their respective uas - mirna transgenes . this can also explain why the gmc - induced clones of neurons were weakly marked as compared to clones of / or / neurons ( data not shown ) . any perdurance of repression in marcm derives from expression of repressors prior to mitotic recombination and thus inheritance of repressors directly from heterozygous precursors . in the mirna - based twin - spot marcm system , gal4 drives expression of both the repressors and the reporters ; we labeled mb sister clones with mb247-gal4 , a mb driver expressed strongly in post - mitotic neurons but not in precursors 12 . we did not detect any delay in the induction of uas - reporter and we identified neuron pairs not only in adult brains ( fig . 2j - l ; n=46/48 ; two cases were one neuron paired with two neurons ) but also in larval brains ( fig . 2s - u ; n=12 ) , in which the original marcm technique can not label single - cell clones due to tubp - gal80 perdurance ( data not shown ) . visualizing both sides of a twin - spot is essential for depicting a complete proliferation pattern for any progenitor . for example , consistent pairing of a multi - cellular nb clone and a two - cell clone through development of a lineage would confirm the models that self - renewing nbs serially produce gmcs and that gmcs produce two neurons . it was long thought that all drosophila nbs undergo this simple pattern of neurogenesis until recent identification of pan ( posterior asense - negative ) lineages and their transit - amplifying precursors 20 , 21 . each transit - amplifying precursor , derived from self - renewing pan nbs , may give rise to multiple gmcs through a limited run of self - renewing asymmetric division . in addition , gmcs from many neuronal lineages of the drosophila ventral nerve cord ( vnc ) initially produce two daughter cells during larval neurogenesis , but only one of them survives to mature into an adult neuron ( williams d.w . we performed a study of neurogenesis patterns in the drosophila central brain and vnc using twin - spot marcm with the pan - neuronal gal4-c155 . for mosaic clones in the central brain , 43 of the 108 ( 40% ) nb clones , including 10 mb nb clones , are paired with two neurons ( fig . 3a , b , d , e , g , h ) ; 53 of the 108 ( 49% ) nb clones are accompanied by only one neuron ( fig . 3c , f , i ) ; and 10 of the 108 ( 9% ) nb clones , located in the dorsomedial region where pan lineages reside , associate with a cluster of 8 to 12 cells ( fig.3l , o , r ) . there were two nb clones without counter pair association ( 2% ; data not shown ) . in contrast , for mosaic clones in the vnc , only 26 of the 128 ( 20% ) nb clones are paired with two neurons ( fig . 3j , m , p ) while 100 of the 128 ( 78% ) nb clones are paired with a single neuron ( fig . 3k , n , q ) and two nb clones were not associated with any post - mitotic neurons ( 2% ; data not shown ) . gal4-ok107 labels , in addition to mb neurons , a cluster of six neurons in each brain lobe whose projections finally converge in various midline neuropils , collectively called the central complex ( fig . , determining the birth order of each neuron requires technology that provides at least the resolution of twin - spot marcm . by labeling gmc progeny in one color and the remaining lineage in another , we can determine which gmc has given rise to a particular neuron in any protracted neuronal lineage . to show this capability we found that all central complex nb clones have sister clones that contain only one cell , which suggests we are observing half of the gmc progeny ( fig . 4r - v ) , and that these six gal4-ok107-positive central complex neurons acquire different trajectories based on birth order . for example , the first - born neuron , as indicated by five cell bodies in the associated nb clone , consistently extends into a dorsal sub - compartment of the noduli and extends few processes ventrally ( fig . in contrast , the third - born neuron always innervates a ventral sub - compartment of the noduli ( fig . in addition , each neuron has distinct stereotyped branches off the primary neurite ( e.g. triple arrows in fig . , we ultimately identified the unique characteristics and birth order of each gal4-ok107-positive central complex neuron . the first four neurons each innervate a distinct sub - compartment of the noduli ( fig . 4d - g ) and the last two neurons project into the ellipsoid body ( fig . individual neurons also acquire stereotyped patterns of neurite elaboration in the lateral accessory lobe ( fig . it is extremely challenging to detect temporal cell fate transformation in a fast - tempo neuronal lineage where neuron fate may change with each self - renewing nb division . to exemplify how twin - spot marcm permits better determination of temporal cell fates , we examined whether and how chinmo , which perturbs temporal identity in mb neurons 12 , affects temporal fates in the sublineage of six central complex neurons . knocking out chinmo from a gmc of the central complex sublineage causes the 3 sibling to exhibit a temporal cell fate like that of the 4 neuron in the wild - type lineage . neurons derived from chinmo mutant 3 gmcs , as determined by associated wild - type nb clones containing three cells , acquired projections characteristic of wild - type 4 siblings ( fig . they innervate the middle sub - compartment of the noduli with the wild - type sibling of the nb clone ( arrow and arrowhead in fig . in addition , mutant 3 siblings , like wild - type 4-born neurons , acquire a much more diffuse pattern of neurite elaboration in the lateral accessory lobe than wild - type 3-born neurons ( triple arrows in fig . 4f , g , i ) . as to removal of chinmo from the central complex nb , we consistently fail to detect cells with an appropriate 3 temporal fate in chinmo mutant clones produced prior to birth of the 3 offspring ( n=3 ) . for example , in a chinmo mutant nb clone induced during production of the second gmc , which leads to a normal 2 sibling , we do not detect axons of mutant cells in the ventral sub - compartment of the noduli ( arrow in fig . we conclude chinmo is required in only one of the six fates of the gal4-ok107-positive central complex neurons . twin - spot marcm permits the visualization of both sides of a twin spot , enhancing the power of marcm and supporting creative and informative mosaic studies in the complex nervous system . in particular , labeling gmc clones and their paired nb clones reveals the developmental origin of neurons . given the fundamental influence that developmental origin seems to exert on neuron - type specification , single - neuron analysis with twin - spot marcm promises to identify individual neuron types , resolve their connectivity , and elucidate underlying mechanisms . as in the original marcm system , twin - spot marcm is built on top of gal4/uas . use of a ubiquitous gal4 driver is mandatory for revealing mosaic patterns in their entirety . by contrast , one can utilize subtype - specific gal4 drivers to restrict labeling to subsets of repressor - negative cells for more focused mosaic analysis . systematic efforts are underway to generate diverse gal4 drivers that together cover the entire brain 22 . clonal analysis with such gal4 drivers in twin - spot marcm should allow us to ultimately identify all neuron types and unambiguously determine their developmental origins . however , most gal4 drivers label neurons in complex patterns ; they mostly label multiple sublineages . we propose combining those subtype - specific gal4s for comprehensive lineage analysis by twin - spot marcm . a power of marcm is that we can drive additional uas - transgenes specifically in the marcm - labeled clones for cell - autonomous rescue of mutant clones or targeted expression of a gain - of - function transgenes . this sort of application is not impossible with the mirna - based twin - spot marcm . in theory , and as already demonstrated in other model organisms 23 , one can artificially make any gene repressible by a specific mirna by introducing the mirna 's target sequences into the untranslated regions of the gene of interest . this way , we may express multiple uas - transgenes differentially between sister clones in mosaic tissues . twin - spot marcm clearly preserves all the powers of marcm and further extends the targeted labeling as well as gene manipulation to both sides of twin spots . labeling both sides of a twin spot should allow us to determine a complete proliferation pattern for any neural progenitor which we demonstrated by showing major patterns of post - embryonic neurogenesis with gal4-c155 in twin - spot marcm . first , we hypothesize that gal4-c155 labels every neuron in the larval central brain and vnc . indeed our observation that 98% of nb clones have sister clones reinforces this notion . second , neurons of the same lineage exist as clusters in the drosophila central brain and vnc , a fact shown without exception in clonal analyses of drosophila brain . third , nb clones generated during early larval life may be accompanied by smaller sister clones of three different sizes . one nb clone may pair with a single - cell clone , a two - cell clone , or a small multi - cellular clone . the small multi - cellular clones were consistently found in the dorsomedial region of the brain , while no spatial correlation could be detected as to the pairing with single - cell or two - cell clones . given that neurons and glial cells in the fly central brain derive from distinct post - embryonic precursors 24 , 25 and that cell bodies of central neurons of the same lineage are tightly packed together 26 , these phenomena indicate unit production of one , two , or a small cluster of mature neurons depending on the lineages . in addition , the recently identified transit - amplifying precursors in pan lineages probably underlie the production of multiple neurons from some nb self - renewal 20 , 21 . multiple modes of neurogenesis demonstrate the complexity of assembly of the drosophila cns , and the use of twin - spot marcm in lineage and phenotypic analyses should facilitate our understanding on this process . one major challenge with single - neuron analysis is how difficult it is to determine if we are comparing the same neuron as we look at brains from different organisms . with twin - spot marcm , we can at least ensure the comparison of neurons with the same developmental origin . intriguingly , neurons of the same developmental origin consistently acquire the same fate in different organisms . analysis of single neurons classified based on their developmental origins should permit description of neural circuitry with single - cell resolution . one can further study the genetics of origin - dependent cell fate specification as a step toward elucidating underlying mechanisms . notably , twin - spot marcm permits independent assessment of mutant clones ' prospective cell fates and has allowed us to demonstrate that chinmo , a btb - zinc finger protein , is specifically required for the specification of the third temporal fate in a sublineage of six central complex neurons . in sum , with twin - spot marcm , we may realistically begin the endeavor to identify every single neuron in the drosophila brain . these together will allow us to build a complete cellular and developmental brain map and lay the foundation for a comprehensive understanding of brain development and function . | a comprehensive understanding of the brain requires analysis of individual neurons . here
we describe twin - spot marcm for high - resolution lineage tracing by independent labeling of paired sister clones .
we determine patterns of neurogenesis and the influences of lineage on neuron - type specification .
notably , neural progenitors may yield intermediate precursors that make one , two , or more neurons .
furthermore , neurons acquire stereotyped projections according to their temporal position within various brain sublineages .
twin - spot marcm also permits birth dating of mutant clones , enabling the detection of a single temporal fate that requires chinmo in a sublineage of six drosophila central complex neurons . in sum
, twin - spot marcm can reveal the developmental origins of neurons and the mechanisms that underlie cell fate . |
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bacterial vaginosis ( bv ) , the most common bacterial genital infection in women of reproductive age , has been linked to considerable gynecologic and obstetric morbidity . bv may be a cofactor in male - to - female and female - to - male human immunodeficiency virus type ( hiv ) transmission and other sexually transmitted infections ( stis ) including herpes simplex virus type-2 ( hsv-2 ) [ 14 ] . the highest prevalence of bv has been found in sub - saharan africa where hiv infection is also common [ 3 , 5 ] . it has been postulated that bv may increase susceptibility to hiv infection through a variety of mechanisms including altering the host 's defense system , absence of the normal h2o2-producing vaginal lactobacilli ( which maintain the natural acidic ph of the vagina ) , loss of the protective myeloperoxidase halide - hydrogen peroxide system , and by production of metabolic by - products that may increase hiv replication [ 7 , 8 ] or activate target cells for hiv infection . if any of these hypotheses are correct , reduction of bv prevalence could significantly reduce female hiv acquisition . furthermore , the high prevalence of bv in sub - saharan africa remains unexplained . though considerable evidence supports sexual transmission of bv , factors like clothing , climate , and hygiene may also influence vaginal flora . of the various risk factors associated with bv , the contribution of each to causing bv may vary depending on hygienic , sexual , and other practices . bv treatment with metronidazole although effective in the initial clearing of symptoms and signs of bv has since proven disappointing even in industrialized countries with recurrence rates reaching 40% within 3 to 6 months after treatment with oral or intravaginal preparations [ 1113 ] . in uganda , administration of metronidazole , azithromycin , and ciprofloxacin to large populations of adults of reproductive age every ten months did not reduce the prevalence of bv below 50% . barrier contraceptive devices , such as the diaphragm , lubricants , and microbicides have been associated with changes in vaginal flora [ 1417 ] . specifically , contraceptive use of the diaphragm with nonoxynol-9 ( n-9- ) based spermicide has been associated with altered vaginal bacterial microflora including decreased rates of lactobacillus colonization and urinary tract infections ( utis ) [ 15 , 16 ] . however , most have regarded alteration of vaginal flora and increased risk of uti as a direct result of the n-9 spermicide and not related to the diaphragm alone . importantly , buffergel a candidate microbicidal gel with the ability to maintain a low vaginal ph was associated with profound reductions in bv after a short period of use among participants in a phase 1 safety trial . in several small trials , miphil , a gel with acid - buffering properties , demonstrated effectiveness in treating women with bv . here , we studied the effect of the diaphragm used with replens , a commercially available vaginal lubricant with weak ph buffering capacity , on the presence of bv for up to 24 months of followup among women enrolled in a large randomized controlled hiv prevention trial , the methods for improving reproductive health in africa ( mira ) study . the mira trial recruited sexually active ( an average of at least four sex acts per month ) , hiv - seronegative , women aged 1849 years who were free of chlamydia and gonorrhea between september , 2003 and september , 2005 . women were recruited from family planning , well - baby , and general health clinics , and from community - based organizations , through printed media and radio . two thousand four hundred and ninety - nine women were enrolled at the zimbabwe site , where the mira - bv ancillary study was conducted ( the main mira trial was also conducted in johannesburg and durban , south africa ) . participants who met protocol inclusion and exclusion criteria were then randomized into one of two groups : the intervention group , who received a clinician - fitted diaphragm ( all - flex arcing spring diaphragm ; ortho - mcneil pharmaceutical , raritan , nj , usa ) , a supply of lubricant gel ( replens , lil ' drug store products , cedar rapids , ia , usa ) , and male condoms and the control group , who received male condoms only . all participants received a comprehensive hiv prevention package consisting of hiv / sti pretest and posttest counseling , treatment of curable , laboratory - diagnosed stis , and intensive risk reduction counseling , which emphasized condom negotiation and was tailored to each participant 's individual circumstances . the follow - up period for the study was designed to be staggered , with the first enrolled participants were followed up for 24 months , the last enrolled were followed up for 12 months , and an overall expected average of 18 woman - months of followup per participant . the study protocol was reviewed and approved by the university of california san francisco institutional review board committee on human research and by the medical research council of zimbabwe ethical review committee , the medicines control authority of zimbabwe and western institutional review board . an independent external audit , sponsored by ibis reproductive health , was done by the quintiles corporation in november 2005 , after study accrual was completed . the study was conducted as an ancillary study of the mira trial in two clinics within 30 km of harare : chitungwiza , a periurban municipality and epworth , a slightly poorer and less developed suburb . bv study procedures followed almost identically procedures for the main mira trial , which have been published in detail . briefly , at screening , we obtained verbal consent to assess initial eligibility , followed by written informed consent for screening procedures , including diagnostic hiv and sti testing and answering an interviewer - administered questionnaire on demographics and sexual behavior . the enrolment visit was scheduled within 2 weeks and not more than 30 days after screening , for participants who met the initial eligibility criteria . by design , approximately 500 participants from the two zimbabwe clinics were enrolled serially into the bv - ancillary study . at their enrolment visit , participants provided written informed consent for the main trial and for the bv - ancillary study , women had to give consent to have an additional vaginal swab obtained at each visit . women were reevaluated for study eligibility and had a pelvic examination that included collection of a vaginal swab for gram stain evaluation of bv using nugent criteria . after randomization in to the main mira trial women in the intervention group were counseled to insert the diaphragm into their vagina at any time that was convenient to them before coitus and to leave it in place for at least 6 hours after sex . they were given detailed instructions on maintenance , cleaning , and storage of their diaphragm . women were asked to empty an applicator of gel ( about 2.5 g ) into the dome of the diaphragm at the time of insertion , to spread gel onto the rim to facilitate insertion , and to insert another applicator of gel into the vagina before each act of vaginal sex . at each visit , women received a 3-month supply of gel and were counseled that the effectiveness of the diaphragm and lubricant gel for the prevention of hiv infection was not known . to prevent hiv , they were asked to use condoms regardless of whether or not they used the diaphragm and gel . participants were also told that they should not use the diaphragm and gel as a method of contraception . women were advised to use other contraceptive methods and were provided with hormonal contraceptives through the clinic . they were encouraged to return to the clinic if they experienced any problems or needed more study products . at all visits , participants in both study groups received counseling on risk reduction and as many male condoms as desired . counselors emphasized that condoms are the only known method to prevent hiv and stis and that condoms should be used for every act of sex . participants returned 2 weeks after enrolment for resupply of products , for counseling , and to have any problems assessed . thereafter , follow - up visits were scheduled quarterly to assess hiv and sti status and to obtain a vaginal swab for gram stain evaluation of bv . recent medical history , use of study products , and sexual behavior were ascertained through a face - to - face clinician - administered questionnaire and audio computer - assisted self - interview ( acasi ) . ( e.g. , soap , commercial douche , and natural products ) during the past two - weeks was assessed through a face - to - face - administered questionnaire . women were counted as having attended their quarterly visit if they visited during the period from 14 days before , to 73 days after , the scheduled date . clinicians addressed any medical problems ; a pelvic examination and urinalysis or wet mount were done when clinically indicated , and treatment was provided when appropriate . data were managed at the center for international data evaluation and analysis at the university of california san francisco . the primary outcome was bv detection by nugent 's criteria during followup . to minimize recall bias , per - protocol analyses were based on a priori product use at last sex as one of our two measures of diaphragm and condom use in all analyses . we also assessed a measure of cumulative use of the methods since last study visit ( always , sometimes , or never ) . for calculation of sample size we assumed that bv detection during followup would be compared between arms in an intent - to - treat ( itt ) analysis using generalized estimating equation ( gee ) logistic regression model controlling for a within - participant correlation between repeated outcomes of 0.2 , with an annual loss to followup of 5% . based on these assumptions , 250 women per arm insured 80% power to detect a 25% difference in prevalence between the two study arms as significant at the 5% level ( based on a two - sided test ) . this corresponded to a relative risk ( odds ratio ) of 0.75 ( 0.70 ) . the itt analysis compared serial bv prevalence during followup between groups using a logistic regression model , including a binary indicator of group assignment as the primary predictor variable and accounting for possible within - participant correlation between repeated outcomes using generalized estimating equations ( gees ) methods . results of the primary analysis were summarized by the estimated odds ratios comparing bv serial prevalence during followup in women in the intervention group to that in the control arm , with associated 95% confidence intervals ( cis ) . we used the same criteria for conducting the per - protocol analyses as were performed for the determination of the effect of the diaphragm on hiv acquisition . in the per - protocol analyses the between - group comparison of the primary outcome , excluding follow - up periods where participants in the intervention group ( diaphragm , replens , and condom promotion ) ( 1 ) did not report diaphragm use at last sex and ( 2 ) did not report use of the diaphragm consistently since the last follow - up visit , generally three months . person time in the control group was included even if no condom use was reported but was excluded if diaphragm use was reported . the sponsor , the bill & melinda gates foundation maintained oversight of the trial through regular progress reports and meetings with investigators , and its program officer had input into key scientific decisions as a member of the study technical advisory committee . the sponsor had no other role in the data collection , data analysis , data interpretation , or writing of the report . the authors had access to all the data and shared final responsibility for the decision to submit for publication . five hundred and forty - three women were enrolled in the bv ancillary study between february and october 2004 ; 9 subjects did not have baseline and 6 subjects did not have follow - up bv results leaving 528 ( 97% ) evaluable subjects ( figure 1 ) . baseline sociodemographic characteristics , sexual behavior , and stis were similar between the intervention and control arms ( table 1 ) . at enrolment , 102 ( 39% ) in the intervention arm and 111 ( 42% ) in the control arm had bv . since the prevalence of bv during followup differed predictably between women with and without bv at enrollment , we analyzed both groups separately . over the course of the study , participants randomized to the intervention arm reported consistent ( always ) diaphragm use during the previous three months at 904 ( 60.1% ) out of 1505 visits . women also reported always using gel at 908 ( 60.3% ) visits indicating that in most instances when the diaphragm was used , gel was used as well . at enrollment , 29% of women reported always use a condom during the previous three months while 74% of women reported condom use at last sex with no significant differences found between study arms ( table 1 ) . during followup a greater proportion of women enrolled in the control arm reported condom use at last sex ( range per visit : 78%88% ) than in the intervention arm ( range per visit : 45%60% ) . for participants with bv at baseline , the odds of prevalent bv decreased an average of 12% per visit with a 60% overall decline from baseline during followup ( or = 0.40 , 95% ci 0.250.65 ) ; this decline was similar between arms ( p = 0.99 ; figure 2 ) . for those without bv at enrollment , the odds of prevalent bv increased nonsignificantly during the study in comparison to baseline an average of 6% per visit with a 50% overall increase for participants in the intervention arm ( or = 1.5 , 95% ci 0.992.25 ) in comparison to a 3% per visit and a 24% overall increase from baseline for women in the control arm ( or = 1.24 , 95% ci 0.771.99 ) ; this increase was not significantly different between arms ( p = 0.55 ; figure 2 ) . only 2.1% of participants were treated for symptomatic bv , and antibiotic use during the last four weeks for any indication was reported infrequently during the course of the study ( range per visit : 0.2%0.7% ) . use of water to clean the vagina in the past two weeks was common during followup and did not significantly differ by study arm ; intervention arm range per visit : 67%77% ; control arm range per visit : 64%76% . use of other products to clean the vagina in the past two weeks was less commonly reported overall and did not differ by study arm during the course of the study either ; intervention arm range per visit : 6%12% ; control arm range per visit : 5%12% . in the itt analysis , bv prevalence over time did not differ between the intervention and control groups for women who had bv at enrollment ( or = 1.01 , 95% ci 0.521.94 , p = 1.0 ) and did not have bv at enrollment ( or = 1.21 , 95% ci 0.652.27 , p = 0.5 ) ( figure 2 , table 2 ) . in the per - protocol analysis , limited to women reporting diaphragm use at last sex , women in the intervention arm with and without bv at enrollment did not have an altered odds of bv ( or = 0.90 , 95% ci 0.461.76 ; and or = 1.34 , 95% ci 0.682.62 , resp . ) in comparison to the control arm ( table 2 ) . we performed a second per - protocol analysis limited to women reporting consistent diaphragm use since the last visit in the intervention arm ; women without bv at enrollment had a nonsignificant increased odds of bv ( or = 1.83 , 95% ci 0.903.71 ) compared to those in the control arm ; the intervention was not associated with an altered odds of bv in women with bv at enrollment ( or = 1.17 , 95% ci 0.562.45 ) ( table 2 ) . first , we compared the prevalence of bv between groups at followup , restricted to women with normal vaginal flora ( nugent 's score 03 ) at enrollment . participants in the intervention group with normal vaginal flora at enrollment did not have an altered odds of bv during followup in comparison to the control group in itt ( or = 1.37 , 95% ci 0.623.07 ) and in per - protocol analysis limited to women who reported diaphragm use at last sex ( or = 1.64 , 95% ci 0.713.82 ) . however , women with normal vaginal flora at enrollment reporting consistent diaphragm use since their last visit had an increased odds of bv during followup ( or = 2.52 , 95% ci 1.026.22 ) in comparison to the control arm ( table 2 ) . next , we compared the prevalence of bv among participants with normal lactobacillus ( score : 0 - 1 ) found on gram stain at enrollment . among women with normal lactobacillus on gram stain at enrollment , being in the intervention arm was not associated with an altered odds of bv during followup in itt or in the two per - protocol analyses ( table 2 ) in comparison to the control arm . our study demonstrated no difference in the risk of bv ( as measured by nugent score ) for women provided with the diaphragm plus replens gel in addition to male condoms compared to those provided male condoms alone . however , in the analysis limited to women with normal vaginal flora at enrollment , participants who reported consistent diaphragm use over the preceding three months had a significantly increased odds of bv during followup compared to the control arm . this result did not seem to stem from differential vaginal cleansing practices or antibiotic use including treatment for symptomatic bv between study arms . it is worth noting the statistical power was reduced following stratification of the results by presence versus absence of bv at enrollment . thus , we can not rule out smaller differences between arms in regards to bv prevalence at followup as was our original intention . furthermore , while we believe that our data on use of the diaphragm and gel are valid , we could not confirm their use in this trial . as has previously been reported , the study attained a high rate of condom use that was maintained over time . however , as in the multisite mira hiv prevention trial , women enrolled in this ancillary study and randomized to the control arm reported higher condom use than those in the intervention arm , suggesting that diaphragm use may have been compensatory , that is , that women provided with the diaphragm were less likely to negotiate condom use with their partners . sexual intercourse without a condom has been associated with prevalent and recurrent bv [ 23 , 24 ] , providing further evidence that a sexually transmitted factor plays a role in bv pathogenesis . thus , we hypothesize that increased condom use in both arms led to the significant decline in bv over time among those with bv at enrollment and that differential condom use between arms and/or diaphragm use in the intervention arm may have contributed at least partially to the lower prevalence of bv among those with normal vaginal flora at enrollment randomized to the control arm in our per - protocol analysis . alternatively , we can not rule out that the device itself could predispose to abnormal vaginal flora . studied the safety of the diaphragm used with ky personal lubricant and cellulose sulfate ( cs ) gel and cs gel alone over a six - month period in a similar population . although they did not report any statistically significant difference between treatment groups , bv appeared to be most common among women in the diaphragm and cs arm ( 34.2% by amsel 's and 39.5% by nugent 's score ) and least common among those in the cs - alone group ( 15.0% by amsel 's and 22.5% by nugent 's score ; p = 0.07 and p = 0.14 , resp . ) . it is possible that even intermittent use of the diaphragm could physically affect the vaginal distribution of immune factors such as immunoglobulin ( ig ) a , secretory leukocyte protease inhibitor ( slpi ) , cytokines , and chemokines originating from the endocervix and endometrium ; evidence suggests that these factors inhibit growth of abnormal vaginal flora while promoting colonization of the vagina by lactobacilli [ 26 , 27 ] . a prior investigation of young women including university students in the usa demonstrated an increased risk of bv in women using the diaphragm for contraception , an association that had previously been thought to be due to the use of n-9 [ 15 , 16 ] . among women initiating new contraceptive methods , the diaphragm used with n-9 was associated with increased vaginal colonization by escherichia coli , enterococcus species , anaerobic gram - negative rods , and bv . while data related to diaphragm use without a spermicide is not available , recent studies have investigated the effects of replens on vaginal microflora . replens contains carbopol and polycarbophil , negatively charged polymers that maintain the vaginal ph in the physiologic range , that may have microbicidal properties and have some activity against bv - associated bacteria [ 18 , 28 ] . although we did not directly assess use of replens , we believe there was very little diaphragm use reported without gel . thus , when used with a diaphragm , replens gel did not appear to protect against bv . following the hiv results of the mira trial , wider - scale use of the diaphragm for hiv prevention is unlikely at this time . however , women who choose to use the diaphragm for pregnancy prevention should be counseled about the potential increased risk of bv . with the development of multipurpose technologies to prevent hiv and pregnancy underway , this investigation provides additional insight regarding bv pathogenesis with important implications for microbicide , cervical barrier , and combination product research . | background . bacterial vaginosis ( bv ) has been linked to female hiv acquisition and transmission .
we investigated the effect of providing a latex diaphragm with replens and condoms compared to condom only on bv prevalence among participants enrolled in an hiv prevention trial .
methods .
we enrolled hiv - seronegative women and obtained a vaginal swab for diagnosis of bv using nugent 's criteria ; women with bv ( score 710 ) were compared to those with intermediate ( score 46 ) and normal flora ( score 03 ) . during quarterly follow - up visits over 1224 months a vaginal gram stain was obtained .
the primary outcome was serial point prevalence of bv during followup .
results .
528 participants were enrolled ; 213 ( 40% ) had bv at enrollment .
overall , bv prevalence declined after enrollment in women with bv at baseline ( or = 0.4 , 95% ci 0.29.56 ) but did not differ by intervention group . in the intention - to - treat analysis
bv prevalence did not differ between the intervention and control groups for women who had bv ( or = 1.01 , 95% ci 0.521.94 ) or for those who did not have bv ( or = 1.21 , 95% ci 0.652.27 ) at enrollment .
only 2.1% of participants were treated for symptomatic bv and few women ( 516% ) were reported using anything else but water to cleanse the vagina over the course of the trial .
conclusions .
provision of the diaphragm , replens , and condoms did not change the risk of bv in comparison to the provision of condoms alone . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
mode of delivery method is defined as choosing either the vaginal or caesarean section ( c - section ) delivery . vaginal delivery is the natural method of birth , though about 10% of normal deliveries may be complicated , caesarean section delivery is suggested to prevent either maternal or fetal morbidities and mortality . however , nowadays , many c - sections are performed upon maternal request with no medical cause . world health organization ( who ) recommended that no more than 1015% of pregnancies should be terminated by c - section . the term elective caesarean section delivery refers to those c - section deliveries which are performed with no medical cause . it has been well documented that mortality and morbidity for c - section deliveries are greater than normal vaginal delivery . protecting mothers from unnecessary medical technologies is one of the who strategies to promote maternal health . international confederation of midwives has announced that performing caesarean section deliveries with no medical indication is immoral . although reducing the rate of elective c - section delivery has been considered by health professionals authorities , this rate is increasing in some countries . in the usa , caesarean delivery rate increased from 20/7% in 1996 to 31/1% in 2006 and to 32/9% in 2009 . in arab countries , according to the results of a study this rate , in iran , is about 50% showing iran is far from the who c - section advocated rate ; therefore , it seems to be crucial to conduct studies to focus on the reasons of such increases and to promote programs to reduce this health issue . considering c - section as a behavior ; before any intervention to reduce the rate of c - sections deliveries , it is essential to understand the reasons for this behavior . because of the importance of the values and beliefs in directing behavior , understanding underlying elements of behavior are necessary to promote any health promotion program . as such , a valid and reliable tool is needed to extract personal values and beliefs . taking the previous studies and researches into consideration , there is no exact measure on maternal beliefs . only two studies which are mostly focused on the cognitive aspects of behavior are exist . considering the fact that the nature of human behavior is very complex as many psychosocial factors are affecting it , the available tools are not provide enough reasons to extract maternal influential factors on mode of delivery . therefore , designing a reliable and valid questionnaire to extract the psychological factors related to the women 's preferences for mode of delivery seems to be more essential . fear and anxiety are one of the most frequent reasons to choose c - section women might consider themselves at risk of probable morbidities . many studies confirmed that negative beliefs are the main reasons for choosing any mode of delivery . such beliefs , as perceived threat , as well as evaluating the benefits or risks , are the key constructs of health belief model ( hbm ) . this model which is based on the behavioral sciences theory is an interpersonal health education model which is composed of theoretical constructs as perceived susceptibility , perceived benefit , perceived barriers , and self - efficacy . in addition to these factors , some researchers believe that pain intolerance also is another effective factor on choosing the delivery mode . that is to say that this factor is inconsistent with the self - efficacy constructs in hbm . based on above - mentioned results , hbm can be an appropriate model to design the materials . since it is unlikely that one specific model can predict the behaviors appropriately , it is recommended that for having more comprehensive understanding , other components and beliefs might be taken into consideration , too . in this regard , some studies demonstrated that physicians , midwifes , and relatives ideas , as well as following the fashion are very significant factors in choosing c - section delivery . this concept is consistent with the construct of normative beliefs which is found within the theory of planned behavior . hence , the aim of this study was to develop a questionnaire to access pregnant women 's preferences for mode of delivery . it was hoped this might help to fill the gaps and perhaps contribute to the existing literature on the topic . this was a cross - sectional study carried out in 2011 in ahvaz the south west of iran public and private health care centers . combining the previous theory - based questionnaires , as well as studying the related text books ; the researchers made a questionnaire which was piloted in a small sample of pregnant women . several methods were used so as to verify the validity and the reliability of the questionnaire as : ( 1 ) extracting items from the related texts and questionnaire and interviewing with the women . ( 2 ) estimating the content validity based on the experts viewpoints . ( 3 ) evaluating face validity based on the pregnant women 's ideas . ( 4 ) using exploratory factor analysis ( efa ) to assess the construct validity . ( 5 ) measuring discriminate validity . ( 6 ) evaluating the reliability using cronbach alpha . in the first stage , having used the published texts , also , based on the viewpoints of the professional faculty members , the researchers designed a questionnaire consisting of 62-item questionnaire . these questions which were based on some constructs of hbm and normative beliefs of the theory of planned behavior were designed to evaluate factors affecting the mode of delivery . in order to , qualitatively measure content validity , in the second stage , the questionnaire was given to 10 experts and their corrective ideas were applied . then , content validity ratio ( cvr ) and content validity index ( cvi ) were calculated to assess the validity quantitatively . the results , then , were used to ensure researchers from the best selection of the items . in so doing , 10 experts including 4 health education experts , 4 midwives , and 2 health experts were asked to answer the questions arranged in three levels ( necessary , useful but not necessary , and unnecessary ) . based on their answers , cvr was calculated . for each question , the impact score for each item was calculated as multiplying the importance of an item with its frequency . the impact scores of > 1.5 were considered suitable . in order to measure cvi , the questions were reviewed by a panel of experts and rated on simplicity , relevance , and clarity on a four - point likert - type scale the cvi of each statement was calculated and as recommended values of 0.80 were considered acceptable . at the end , 49 questions remained . each item is rated on a five - point likert scales ranging from strongly agree to strongly disagree giving a possible score of 15 for each item . in the third step , to measure the construct validity and , also , to determine the factor structure of the questionnaire , at first , the questionnaires were given to 342 nonrandom pregnant women referred to public and private health care centers . the inclusion criteria were : being aged 1835-year - old , having the history of pregnancy without adverse outcomes , not suffering from chronic diseases during the present pregnancy and not having the history of fertility problems . demographic characteristics of the pregnant women included recording of age , education of pregnant women and their husbands , gestational age , and family monthly income . data were analyzed applying descriptive and inferential tests using spss 15 ( spss , inc . , a set of observed variables was used to identify a set of latent constructs . to determine the adequacy of the sample size , kaiser meyer a threshold of > 0.4 for corrected item - total - correlation was chosen sufficient . known groups comparison was performed to test how well the questionnaire discriminates between pregnant women with the different intention for their mode of delivery with no medical reason ( either c - section or vaginal delivery ) . 112 women ( 31/6% ) chose c - section delivery , and 120 participants ( 33/9% ) chose vaginal delivery as their definite preference . t - test , also , was used to verify the discriminated validity between these two groups . however , item correlation with intended factors was assessed to calculate reliability ( p < 0.05 ) . data were analyzed applying descriptive and inferential tests using spss 15 ( spss , inc . , a set of observed variables was used to identify a set of latent constructs . to determine the adequacy of the sample size , kaiser meyer a threshold of > 0.4 for corrected item - total - correlation was chosen sufficient . known groups comparison was performed to test how well the questionnaire discriminates between pregnant women with the different intention for their mode of delivery with no medical reason ( either c - section or vaginal delivery ) . 112 women ( 31/6% ) chose c - section delivery , and 120 participants ( 33/9% ) chose vaginal delivery as their definite preference . t - test , also , was used to verify the discriminated validity between these two groups . however , item correlation with intended factors was assessed to calculate reliability ( p < 0.05 ) . the mean age of women was 23.9 ( 4.07 ) years , and the mean gestational age was 32.1 ( 4.3 ) weeks . demographic characteristics of the study population the results obtained from validity analysis showed good levels of the cvr ( 0.86 ) , cvi ( 0.84 ) , and impact score ( is = 5 ) for items . in the qualitative face validity , all participants acknowledged that they had no problems in reading and understanding the items . after content validity phase , 42 items were remained for the next stage of validation process . olkin measure of sampling adequacy test was 0.738 , showing the adequacy of the sample size for factor analysis . the principal component analysis with varimax rotation was performed for the items resulting in a seven factor solution . table 2 shows the rotated factor matrix of these seven factors and indicates the factor loading of each of 21 items . the results of the factor analysis for exploring the key factors with rotated varimax from a questionnaire , which evaluates the related factors with the mode of delivery , and internal consistency ( cronbach alpha ) among pregnant women factors were named so that they could reflect underlying measured variables . accordingly , these seven factors were named as : ( 1 ) self - efficacy ( 5 items ) , ( 2 ) false impression of the benefits of c - section delivery ( 4 items ) , ( 3 ) exaggerating the risks of vaginal delivery ( 3 items ) , ( 4 ) perceived susceptibility ( 3 items ) , ( 5 ) normative beliefs ( 2 items ) , ( 6 ) desire for acceptance ( 2 items ) and ( 7 ) health professionals idea ( 2 items ) . 63.619% of the total variance was explained by these seven factors . to measure the discriminate validity , in table 3 , the scores of each factor were compared between different groups based on their intention to either vaginal or c - section . the results of the t - test indicated that these factors showed a significant difference between the pregnant women who chose c - section delivery and those whose selection was vaginal birth in self - efficacy , false impression of the benefits of c - section delivery , exaggerating the risks of vaginal delivery , perceived susceptibility , normative beliefs , desire for acceptance , and even in total score ( p = 0.001 ) . discriminate validity of the explored factors in two groups based on their intention for mode of delivery however , such difference was not found concerning the health professionals idea factor ( p = 0.19 ) . the mean scores of the second group in all these dimensions were higher . generally , the mean score of the total factors in a group choosing vaginal delivery were more than those of the group that will undergo c - section delivery , 68.92 9.78 and 55.38 8.58 , respectively [ table 3 ] . cronbach alpha ( internal consistency ) for all 21 items in the questionnaire was 0.747 . this number for self - efficacy , false impression of the benefits of c - section delivery , exaggerating the risks of vaginal delivery , and normative beliefs were more than 0.7 , indicating that all these dimensions had high internal reliability . other dimensions as perceived susceptibility , desire for acceptance , and health professionals idea showed other results ( 0.649 , 0.534 , and 0.332 , respectively ) [ table 2 ] . the aim of this study was to evaluate the overall psychometric properties of pregnant women 's preferences for mode of the delivery questionnaire . so far no specific research has been found focusing exclusively on the behavioral beliefs related to the mode of delivery . in fact , this is a key construct within many health education theories and seems to be the most fundamental behavioral constructs related with the choice of delivery method . self - efficacy refers to an individual 's perception of his or her competence to successfully perform a specific behavior . given any sort of behavior , it can motivate individuals to engage in the behavior or even to change that behavior . therefore , recognizing this construct would help to better explain individual differences in health behaviors . the second and the third extracted factors were false perception of the benefits of c - section delivery and exaggerating the risks of vaginal birth , respectively . in spite of the fact that , in many studies , it is demonstrated that both the mother and the baby are more at risk in c - section delivery than vaginal birth , many people still perceive c - section delivery as having less risks than vaginal delivery . penna et al . in their studies showed that high socioeconomic class and awareness of delivery time were the other important reasons for women choosing c - section delivery . therefore , it seems that providing appropriate educational program can help pregnant women to perceive the advantages and disadvantages of vaginal delivery and c - section . therefore , it seems necessary that pregnant women be taught truly about the advantages and disadvantages of c - section delivery . with colleagues , penna and soltani and sandall in their studies showed that social welfare and controlling the exact time of delivery and hospital release were the main reasons for women 's tendency toward c - section delivery . it refers to one 's perception of the risk or the chances of contracting a health disease or condition . individuals who perceive that they are susceptible to a particular health problem will engage in behaviors to reduce their risk of developing a health problem . hajian et al . found that if individuals know about the risks of c - section delivery , it is more probable that they , when having no medical indications , choose vaginal delivery . as consistent with the findings of this research , in another study by penna and arulkumaran , liu et al . , and angeja et al . normative belief was found as an important factor impacting on the pregnant women 's decision making of the mode of delivery . the ideas of the women 's spouse , family , friends , and close others are very influential . although this factor is not mentioned in any behavioral model , the item of distorted body image is a variable mentioned in other studies . although results of discriminate validity showed no significant differences between the women choosing c - section delivery and those who chose vaginal delivery , this factor was found to be influential in the studies of turner et al . and guittier et al . in their studies , turner et al . showed that the ideas of midwives were very effective in choosing the mode of delivery . one possible explanation for nonsignificant discriminate validity for this subscale might be due to the fact that while other studies considered midwives idea , this study investigated all health professional 's idea . since the personnel 's ideas can change the pregnant women 's preferences for the mode of delivery , it is very necessary that some educational classes , focusing on the morals and social skills , be held for the health professionals too . in spite of the fact that the cronbach alpha and reliability of all factors were in high levels , desire for acceptance showed a weak reliability . moreover , the individual 's perception of the health professionals was unacceptable in terms of reliability . taking cramines 's and zeller 's ideas into consideration , the number of items are one of the important factors constructing alpha cronbach 's level , such result can be justified since these two factors had only 2 questions . they , also , believe that mean correlation of the items is another way to evaluate reliability . in their cross table , they suggested expected alpha between 0.333 and 0.572 . having only 2 items , acceptability orientation with the mean correlation of 0.355 showed a higher alpha , 0.524 . according to this theory , if the number of questions become double , alpha will be between 0.5 and 0.727 . based on the cromines 's idea , expected alpha for the personnel 's idea , having 2 items and mean correlation of 0.247 , should be about 0.333 . in this study , it was measured as 0.332 . if the numbers of the items change to 4 or 6 , alpha levels will increase up to 0.500 or 0.600 . many experts believe that with the increase in the sample size , the alpha coefficient would increase too . it is recommended , then , that in the further studies the effect of the above - mentioned factors be examined . not to conduct confirmatory factor analysis is the study 's limitation . in general , the findings suggest that this newly generated scale is a reliable and valid specific questionnaire for assessing pregnant women 's preferences for mode of delivery . however , further studies are needed to establish stronger psychometric properties for the questionnaire . | introduction : the rate of caesarean delivery is increasing worldwide .
maternal beliefs may be influential on the mode of delivery .
this study aimed to validate pregnant women 's preferences for mode of delivery questionnaire among pregnant women.materials and methods : this was a cross - sectional study which was done in ahvaz public and private health care centers .
a total of 342 low - risk pregnant women were included in a study conducted in spring 2011 in ahvaz , iran . after careful consideration and performing content and face validity , a 62-item measure was developed and subjects completed the questionnaire .
reliability was estimated using internal consistency and validity was assessed by performing face , content and structure and discriminate validity .
data were analyzed using explanatory factor analysis , t - test , and correlations in spss 16.results:the findings of content and face validity showed almost perfect results for both content validity ratio = 1 and content validity index = 1 .
the explanatory factor analysis indicated a 7-subscale measure ( eigenvalue > 1 , factor loading > 0.4 ) , and discriminate validity revealed satisfying results p < 0.05 for 6 out of 7 subscales . internal consistency as measured by the cronbach 's alpha coefficient was acceptable for subscales.conclusions:in general , the findings suggest that this newly generated scale is a reliable and valid specific questionnaire for assessing pregnant women 's preferences for mode of delivery .
however , further studies are needed to establish stronger psychometric properties for the questionnaire . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
vascular surgery needs the temporary interruption of arterial blood flow with occluding clamps isolating the diseased vessel tract and resulting in physiologic changes often superimposed on advanced pathologic conditions . in particular during aortic aneurism repair an increased afterload follows aortic clamp placement which can itself induce , firstly , hypertension and , afterwards , a sudden decrease in afterload with a potentially severe ischemia reperfusion injury can follow clamp removal . this phenomenon can be associated to severe hypotension , lactic acidemia , myocardial ischemia and cardiovascular collapse . perioperative management should be aimed at providing hemodynamic stability , adequate oxygenation and preserving organ function . methylene blue ( mb ) has been used as a vasopressor agent in various settings ( such as sepsis , liver failure and transplantation ) and its role has been well described in cardiac surgery to treat vasoplegic syndrome [ 2 , 3 ] . the physiological response to aortic cross clamp release is in part characterized by the production of no , an endothelial derived vasodilator which acts on smooth cells in the vessel wall resulting in hypotension . the rationale for mb use in hypotensive syndromes is represented by the fact that mb directly inhibits nitric oxide ( no ) synthases , both constitutive and inducible . however , at our best knowledge there are no report in literature regarding the use of mb in vascular surgery . the recently introduced co monitoring flotrac / vigileo allows co determination from the arterial pressure waveform of any peripheral arterial line in conjunction with patients anthropometric data , with less potential complications compared to more invasive devices . we describe the use of mb during an intervention of aortic aneurism repair with infrarenal cross clamp . the patient is 61-year old , male , with a mitral valve prolapse and dilated cardiomyopathy ( ejection fraction 35% ) . standard monitoring was used throughout the operation , including continuous electrocardiography , pulse oximetry , capnography , bispectral index ( bis ) , end tidal gas analysis and urine output . a 20 gauge radial artery catheter was used to measure arterial pressure , a flotrac / vigileo was used to measure cardiac index ( ci ) and stroke volume variation ( svv ) , an oesophageal temperature probe monitored core temperature . fluid and drug infusion were administrated via two venous catheters and one double lumen central venous catheter . propofol ( 1 mg / kg ) , sufentanyl ( 0.5 mcg / kg ) and atracurium ( 0.5 mg / kg ) . anesthesia was maintained with sevoflurane titrated on a bis value of 40 - 50 , atracurium ( 0.2 mg / kg / h ) and sufentanyl i.v . boluses of 5 mcg as required . colloids and cristalloids were administrated to achieve a diuresis of 1 ml / kg / h , central venous pressure ( cvp ) of 12 - 15 mmhg and svv < 12% . bolus ) was administered to maintain a mean arterial pressure ( map ) above 65 mmhg . before aortic clamping , blood pressure ( bp ) was maintained at mean values of 90/60 mmhg with a cvp of 14 mmhg , svv = 10 and a ci = 2.6 l / min per square meter . after infrarenal cross clamp bp increased to 110/60 mmhg , svv = 7% and ci decreased to 1.9 l / min / m . the operation was complex : aortic clamp was maintained for 135 min . and blood losses were estimated about 4500 ml ( 1300 ml of blood were re - infused by a blood cell saver system , red blood cells transfusions were administered to maintain hb value above 10 g / dl ) . nevertheless bp decreased to 65/45 mmhg with a cvp of 6 mmhg , svv = 14% and ci of 2.5 l / min / m.thus we administered 10 mg of ephedrine . after 10 minutes the total amount of fluids required was 5500 ml of crystalloids , 1350 ml of colloids and 1250 ml of red blood cells . the operation was concluded and the patient was moved to the intensive care unit ( icu ) . the classic investigation of gelman represents a cornerstone of our knowledge of the pathophysiology of hemodynamic changes during aortic cross clamping and unclamping . aortic clamping increases mean arterial pressure ( map ) and systemic vascular resistances ( svr ) up to 50% , due to mechanical afterload increase as well as a physiologic increase because of the activation of renin and release of cathecolamines , prostaglandins , and other active vasoconstrictors . unclamping of the aorta can result in severe reduction in map , svr and even co unless aggressive therapy is undertaken prior to unclamping . most anesthesiologists use fluid administration together with vasoconstrictors ( i.e norepinephrine ) or other drugs as calcium chloride to offset the negative inotropic and dromotropic effects caused by the reperfusion - induced release of factors such as e potassium , acids and other mediators . although the main factor contributing to hypotension is volume redistribution to the lower body after clamp release , many humoral mediators are released from the underperfused areas and exacerbate the hemodynamic changes . such factors are as follows : renin , angiotensin , epinephrine , norepinephrine , pgi 2 , endothelin , tromboxane , lactate , potassium , oxygen free radicals , platelet activator , cytokines , activated complement and neutrophil sequestration [ 8 , 9 ] . another mechanisms contributing to the reduction in svr occurring after reperfusion is represented by abnormalities in no synthesis and vascular smooth muscle cell guanylate cyclase activation . no is produced by two types of nitric oxide synthase , a constitutive type and an inducible one . no activates soluble guanylate cyclase to produce cyclic guanosine monophosphate ( cgmp ) which causes vasodilation and may also decrease myocardial contractility . mb directly inhibits both types of no synthases band it is also believed to act in competition with no in binding guanylate cyclase , thus reduction cgmp production , and indirectly counterbalancing reperfusion - induced . mb has been studied in different clinical situations such as sepsis , cardiopulmonary bypass , liver failure an transplantation [ 11 , 12 , 13 , 14 , 15 ] . these studies used different doses and modalities of drug administration , but most of them reported an improvement of hemodynamics in the patients treated with mb . in fact some investigations found an increase in svr with no changes in ci , while others supposed that this drug mainly exerted its effects on a action on myocardial function [ 2 , 10 , 11 , 12 , 13 ] . similarly , some experimental and in vitro studies support this hypothesis reporting a direct effect of mb in increasing myocardial contractility [ 3 , 16 ] . adverse effects of mb include cardiac arrhythmias , coronary vasoconstriction , angina , decreased co , renal blood flow and increased pulmonary vascular pressure . other side effects are headache , fever , hemolytic anemia , nausea , vomiting and abdominal pain . a dangerous interaction with mao inhibitors is described and it can result into a postoperative serotonin syndrome . nevertheless the most common side effect noted with mb is the self limiting discoloration of skin and urine [ 13 , 17 ] . in regard to our patient our findings might be explained through a first effect of mb on myocardial function which sustained hemodynamic immediately after reperfusion , this effect is mostly mediated by guanylate cyclase inhibition ; the effects on no induced vasodilatation are likely to be gradual and slower , but unfortunately we did not measure no products in our investigation . as far as mb dose is concerned , we arbitrarily chose the dose and modalities previously described in liver transplantation with a single bolus of mb . we can not exclude that could be better a bolus followed by a continuous infusion , in order to obtain the immediate improvement of myocardial function and the effect on endothelial no . limitations : a potential limitations of this case report is the use of flotrac / vigileo during aortic cross clamp , since a randomized controlled trial on this concern has never been performed . however flotrac / vigileo is validated in many surgical settings , and there are no reasonable motivations against its reliability in this setting . the whole intraoperative management including fluid and vasopressors administration have contributed to the hemodynamic finding and the good outcome and they can not be ascribed only to mb use , thus we can not draw any definitive conclusion regarding mb use is this setting with a case report . randomized controlled trials are needed to assess the clinical effects of mb in vascular surgery as well the most efficacious doses and administration modalities . | the open abdominal aortic surgery includes a well - known phase in which arterial blood flow is stopped by occluding clamps , resulting in peculiar physiologic changes usually superimposed on advanced pathologic conditions .
an anesthetic plan should aim at providing hemodynamic stability and preserving organ function .
clamp removal leads to an acute fall in blood pressure following a decrease in systemic vascular resistance , caused by reactive hyperemia due to opening of the previously minimally perfused vascular beds .
several different mediators , including the nitrous oxide ( no ) pathway , have been thought to be responsible for this hemodynamic effect .
the massive production of no by the inducible isoform of no synthase could be partially responsible for the profound vasodilatation and myocardial dysfunction .
the dye methylene blue ( mb ) has been used as to prevent vasodilatation in other clinical situations like sepsis , cardiopulmonary bypass and liver transplantation .
we describe its use in a patient with poor hemodynamic status , who was submitted to aortic aneurism repair with infrarenal cross clamp .
the intervention was also associated with a severe bleeding . in this case
mb allowed us to control hypotension with relatively low doses of vasopressors . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the postpartum care ( ppc ) visit is an important opportunity to assess the physical and psychosocial health of the mother . the american academy of pediatrics ( aap ) and the american college of obstetricians and gynecologists ( acog ) recommend that women , regardless of age , seek postpartum care between 4 and 6 weeks after childbirth . the postpartum care visit may be utilized to counsel mothers on infant care and family planning , encourage breastfeeding , identify and treat medical conditions common to the postpartum period , and manage preexisting or emerging chronic conditions . despite the known benefits of the ppc visit , there are many access and utilization barriers to care . as a result , healthy people 2020 aims to increase the proportion of women , across demographic and socioeconomic boundaries , who attend a ppc visit after giving birth , thereby highlighting postpartum care as a national priority to promote the health of women and children . data on the utilization , content , and quality of the ppc visit are limited and often lack a comprehensive preventive care component . according to the centers for disease control and prevention 's ( cdc ) pregnancy risk assessment monitoring system ( prams ) 2004 , based on data from 11 states and new york city , 89% of us women who deliver attend a ppc visit . however , the prevalence of women who attend a postpartum care visit is lower among certain subgroups . approximately 71.2% of women who had received late prenatal care and 71% of women with 8 years of education attend a postpartum care visit . in addition , only 66% of women who did not receive prenatal care and 59.5% of women whose infants did not have newborn checkups obtain ppc . among the women who do attend the ppc visit , many factors influence the decision to seek ppc . a study by kogan et al . reported that the strongest indicator of whether a woman would obtain ppc was her use of prenatal care . other studies have reported that women with inadequate or no prenatal care are less likely to adhere to recommendations for continuity of care after birth , which include newborn visits , childhood immunizations , and postpartum visits . among a nationally represented sample of women who had a live birth in the 1988 national maternal and infant health survey ( n = 9953 ) , women who had no prenatal care had a 3.39 greater odds of not receiving ppc compared to women who had prenatal care . a commitment to preventive health during pregnancy ( i.e. , prenatal care ) may be a strong indicator of maternal health care utilization after birth . new mothers are often sleep deprived , overwhelmed , and have limited time to tend to their personal health care needs . women with unintended pregnancies may find it difficult to come to terms with their pregnancy . after birth , postpartum depression and other mental health conditions may result , impacting the woman 's willingness to take initiative and follow through with medical appointments . alcohol and drug use can further hinder a woman 's ability to attend to her health care needs . in addition , women who had a baby born preterm or low birth weight may be less likely to seek ppc , as their attention and time are devoted to the care of the newborn [ 5 , 11 ] . furthermore , enduring traumatic events , such as a still birth or infant death have a major impact on mental health status , which may result in low ppc utilization rates . some studies report that women with less than a high school education , less than 26 years of age , a household income of less than $ 20,000 , and high parity are associated with a low acceptance rate of the ppc visit [ 2 , 5 , 7 ] . the disparities in ppc utilization require attention to improve access to quality care for women across socioeconomic and demographic boundaries . access barriers further decrease the likelihood that a new mother will schedule a ppc visit . medicaid programs serve a higher proportion of pregnant women of low socioeconomic status with special needs than do private insurers . although medicaid recipients receive hospital coverage , after birth outpatient visits such as ppc visits , are not always covered expenses . as a result , these women tend to seek acute , reactive care and do not have a long - term health care plan . recent legal immigrants experience additional barriers to care as they are ineligible for medicaid services and language barriers can be a deterrent to seeking care as well . lack of comprehensive health insurance coverage is a considerable challenge to meeting maternal healthcare needs . furthermore , cultural and religious beliefs influence an individual 's regard for the postpartum visit . social support , including partner involvement , can have a bearing on perceived feasibility of scheduling a ppc visit while caring for a newborn . logistical barriers such as inaccessible transportation , long waits during appointments , and lack of child care further limit the likelihood of a postpartum visit . in light of the expanding hispanic population in the us , our study is focused on a predominantly hispanic multiethnic sample of low - income women . to address gaps in the literature , our study used data from a recent probability sample of mothers from los angeles county to study the determinants of and barriers to ppc utilization . to our knowledge , this is the first study to date to highlight actual and perceived barriers to ppc in this subpopulation . data from the 2007 los angeles mommy and baby ( lamb ) study ( n = 4 , 075 ) were used to examine the extent to which postpartum care utilization is related to sociodemographic characteristics and to identify factors that reduce the likelihood of postpartum care utilization among women living in los angeles ( la ) , california . the lamb study was a cross - sectional , population - based study that examined maternal and child health outcomes during the preconception , prenatal , and postpartum periods . eligible participants were la residents who had a live - birth in la county in 2007 . this study was a collaborative effort between the university of california , los angeles ( ucla ) and the los angeles county department of maternal , child , and adolescent health . a total of 10,000 surveys were mailed to eligible women within 6 months after delivery . the response rate was 56% , after adjusting for faulty addresses , language issues , maternal deaths , and loss of follow - up due to inability to locate the respondent . the recruitment process consisted of a ( 1 ) notification letter 4 months after child birth , ( 2 ) mailed questionnaires , ( 3 ) mailed reminder letters , ( 4 ) and telephone follow - up to nonresponders . the questionnaires were translated into spanish and chinese and a telephone translation service provided access in 88 languages . data from completed surveys were linked to birth certificate data resulting in a final analytic sample of 4,075 . this study was approved by both the los angeles county department of public health and the ucla institutional review boards ( irbs ) in 2007 . the current analysis focused on the determinants of , and barriers to , postpartum care . respondents were asked whether or not they obtained a postpartum care visit ( yes / no ) . a range of maternal sociodemographic factors including race ( non - hispanic white , hispanic , non - hispanic black , asian / pacific islander ( api ) , and native american ) , age in years ( 016 , 17 - 18 , 1929 , 3039 , 4049 ) , marital status ( married , separated / divorced , widowed , never married but living together , never married but living apart ) , income ( < $ 20,000 , $ 20,00039,000 , $ 40,00059,000 , $ 60,00099,000 , > $ 100,000 ) , and education ( less than high school , high school graduate , some college or more , and not stated ) were included in the analyses ( table 1 ) . when two races / ethnicities were provided , the first listed race / ethnicity was used . additional variables of interest included insurance type ( no insurance , medi - cal / medicaid , private insurance , other ) , prenatal care utilization ( yes / no ) , care received prior to pregnancy ( reasons for seeking care : to have a healthy pregnancy , chronic medical problem , problem with previous pregnancy , expected to get pregnant , encouraged by doctor or nurse ) , intendedness of pregnancy ( yes , yes but not trying very hard , trying hard to keep from getting pregnant , or neither trying nor preventing pregnancy ) , preterm birth , low birth weight ( yes / no ) , and whether or not the infant attended a newborn visit ( yes / no ) . we analyzed the data using sas 9.3 ( sas institute inc . , cary , nc ) . weights were added to account for the sampling design and survey nonresponse . to test differences between women who did and did not obtain postpartum care on select socio - demographic factors and other possible determinants , analyses were performed ( table 2 ) . multivariate logistic regression analyses were used to identify predictors of postpartum care utilization adjusting for selected covariates ( table 3 ) . adjusted odds ratios ( ors ) and 95% confidence intervals ( cis ) were computed using the beta coefficients ( ) and standard errors ( ses ) from the multivariable logistic analyses . the sample was predominantly hispanic , between 19 and 39 years old , married , had an annual income < $ 20,000 , and obtained a high school - level education or less . table 2 displays the bivariate analyses of select maternal demographic and socioeconomic characteristics by postpartum visit status . in the bivariate analyses , maternal race / ethnicity , age , income , marital status , education , pregnancy intendedness , prenatal care , care received prior to pregnancy , and preterm table 3 shows results of the multivariable analysis on the association between predictors and the lack of postpartum care utilization while controlling for confounders . in model 1 , hispanic mothers were significantly less likely to lack a ppc visit compared with non - hispanic white mothers ( or = 0.57 , 95% ci 0.38 , 0.87 ) . mothers aged 30 to 39 ( or = 0.68 , 95% ci 0.51 , 0.91 ) were less likely to lack a ppc visit compared to mothers aged 19 to 29 . mothers from families with income less than $ 20,000 were almost three times as likely to lack a ppc visit ( or = 2.89 , 95% ci 1.43 , 5.82 ) compared to mothers with households of > $ 100,000 . separated or divorced and never married mothers were more likely to lack a ppc visit compared to married mothers . mothers who reported not trying hard to get pregnant or trying to prevent pregnancy were also more likely to lack a ppc visit , compared to those who were trying . mothers who did not attend prenatal care were more than 3 times as likely to lack a ppc visit ( or = 3.08 , 95% ci 1.68 , 5.63 ) . when insurance status replaced family income in model 2 , mothers who had medi - cal insurance were twice as likely to lack a ppc visit compared to those on private insurance ( or = 2.19 , 95% ci 1.54 , 3.11 ) . hispanic ethnicity , maternal age , marital status , pregnancy intendedness , and prenatal care utilization remained significant predictors . figure 1 shows the most commonly reported reasons for not receiving a postpartum check - up . women reported that they felt fine , were too busy with the baby , had other things going on , and felt there was no need for ppc . our paper highlights the many factors influencing a woman 's decision to seek postpartum care . our study population was multiethnic , predominantly hispanic and low - income , based on the demographics of los angeles county . in the multivariable analyses , postpartum care utilization was lowest among women who were low income , separated hispanic women were more likely to obtain ppc compared to non - hispanic white women . consistent with other studies , prenatal care was the strongest predictor of postpartum care utilization [ 5 , 7 , 8 ] . women trying to get pregnant may be more inclined to seek care prior to , during , and after pregnancy . one would expect that a continuum of maternal care may reflect a women 's commitment to life - long health . however , on the contrary to our expectations , care received prior to pregnancy was not a significant determinant of ppc utilization . the perinatal period represents a window of opportunity to engage and educate women about the importance of postpartum care . the top 5 reasons for not seeking ppc indicate that women who lacked ppc did not consider the ppc visit a high priority ( figure 1 ) . interestingly , access to care was not perceived as a top reason for not obtaining ppc . interventions aimed at changing attitudes and perceptions about the importance of the ppc visit must include individuals , families , and communities . the hispanic community , where familial support is of high value , had the highest adjusted rates for postpartum care use . a multitiered approach , targeting individuals , families , and communities may be necessary to improve ppc utilization rates . women who do not have an adequate support system in place may find it exceedingly difficult to set aside time to care for their personal healthcare needs . for the purpose of this analysis , marital status was viewed as a proxy for support . in addition to a supportive spouse , extended family support can also facilitate a woman 's ability to seek ppc . encouragement from one 's spouse and/or family member(s ) can provide the necessary impetus for scheduling a postpartum visit . even gentle nudging from friends , neighbors , and respected individuals in the community can elicit the motivation necessary to seek care . medi - cal is the medicaid program available to residents in the state of california . this public health insurance program provides health care services to low - income individuals and families . in california , certain eligibility requirements apply to receive 60 days of ppc through medi - cal services . if the eligibility requirements are met , covered services include hospital and scheduled office visits during puerperium , assessment of uterine involution , and contraceptive counseling . comprehensive postpartum care including nutrition , psychosocial , or health education services is billed fee for service . furthermore , the postpartum care eligibility period does not cover conditions unrelated to the pregnancy , such as urinary tract infection , respiratory infection , hepatitis , preexisting hypertension , cholecystitis , appendicitis , abnormal pap smear , and cancer . although some postpartum services are available , eligibility restrictions likely limit the accessibility of postpartum care for select subgroups . our findings indicate that women enrolled in medi - cal are twice as likely to lack a ppc visit compared to those with private insurance . these findings further support the need for national and state level policies addressing barriers to postpartum care . the affordable care act addresses this need through the inclusion of provisions to support pregnant and postpartum women . under the affordable care act , furthermore , the affordable care act helps make preventive care affordable and accessible by requiring new health plans to cover and eliminate cost sharing for preventive services . the lamb study was a cross - sectional study based on a sampling of live birth certificates . in addition , the mother 's current relationship with the baby 's father may differ from their relationship status during pregnancy and as a result has a bearing on the survey responses . as this study took place in la county however , this study contributes to the growing body of the literature emphasizing the importance of the postpartum visit and identifying barriers to ppc . in our study population , despite the many barriers to obtaining postpartum care , hispanic women were more likely to receive postpartum services compared with non - hispanic white mothers . an interesting finding of our study was that perceptions about the barriers to postpartum care were the result of a perceived low value of the postpartum visit . this indicates a need for health care facilities and providers to make concerted efforts to increase knowledge about the importance of the postpartum visit , enhance the use or design of medical encounters , identify community resources , and develop targeted interventions . the prenatal care visit is one early opportunity to educate women about the importance of ppc . providers can influence perceptions about ppc , increase its acceptability , and contribute to significant improvements in women 's health . | this study aimed to identify actual and perceived barriers to postpartum care among a probability sample of women who gave birth in los angeles county , california in 2007 .
survey data from the 2007 los angeles mommy and baby ( lamb ) study ( n = 4,075 ) were used to identify predictors and barriers to postpartum care use .
the lamb study was a cross - sectional , population - based study that examined maternal and child health outcomes during the preconception , prenatal , and postpartum periods .
multivariable analyses identified low income , being separated / divorced and never married , trying hard to get pregnant or trying to prevent pregnancy , medi - cal insurance holders , and lack of prenatal care to be risk factors of postpartum care nonuse , while hispanic ethnicity was protective .
the most commonly reported barriers to postpartum care use were feeling fine , being too busy with the baby , having other things going on , and a lack of need .
findings from this study can inform the development of interventions targeting subgroups at risk for not obtaining postpartum care .
community education and improved access to care can further increase the acceptability of postpartum visits and contribute to improvements in women 's health .
postpartum care can serve as a gateway to engage underserved populations in the continuum of women 's health care . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
in the early days of biological research , mutations that caused discernable phenotypes were the primary tool for understanding how a biological system worked in the absence of a mutation a gene was invisible . today , biologists are armed with a whole arsenal of tools to regulate gene , mrna , and protein abundance and activity , thereby promoting the discovery of mechanisms and how a system gone awry can lead to disease ( 1 ) . among these are tools for suppressing the activity of a gene or gene product ( e.g. site - directed mutagenesis , rna interference , small molecule inhibitors ) or enhancing activity ( e.g. activating mutations or receptor agonist ) . for instance , interfering with protein activity using small - molecule inhibitors should have a phenotype similar to reducing the abundance of the corresponding mrna with anti - sense oligonucleotides ( 2 ) . likewise , similar responses are expected whether increases in intracellular protein concentration are achieved via an inducible promoter or by addition of recombinant protein ( 3 ) . as such , perturbations form the core of understanding how biological systems work , how diseases arise , and how they can be treated . any serious attempt to analyze a biological process starts by identification and characterization of perturbations that have been used in prior work . this task requires a framework that can be systematically applied and that is amenable to both manual and automatic means . currently , there is no established categorization that sufficiently represents the range of described experimental manipulations beyond high - level semantic and grammatical classifications ( 4,5 ) or description of techniques ( 6 ) . for example , the closest concept we have found is altered expression, defined as altered expression level of a gene / protein ( 7 ) . we believe that this concept is overly specific and fails to cover important phenomena , among others , changes in protein activity or gene mutations . we propose , instead , taking the existing concept of perturbation and broadening it to comprise the range of terms used in text to indicate changes in the abundance or activity of dna , rna and proteins . perturbations , in this new formulation , would refer to a collection of phenomena in a manner analogous to the way protein protein interactions refer to biological phenomena of different type ( e.g. bind , activate , inhibit ) . since this proposition , like any other , needs to be tested for validity and utility , we have applied it to a case study involving gene phenotype associations in disease and have developed a mining algorithm that detects the diverse forms in which perturbations appear in text . therefore , we are introducing in this work both a new way to understand a crucial part of biology and a new text - mining method tailored to its extraction . design , test and analysis. as initial step , we created the design corpus to develop an analytical framework for annotation . the purpose of this corpus was to identify challenges in the annotation process and to refine guidelines that would help the annotators in choosing their evaluations . annotating perturbations requires at times thorough knowledge of experimental biology , which can only be captured and organized within a solid framework . therefore we sought to perform a preliminary analysis on a test corpus to improve on subsequent annotations . this corpus was limited to sentences that included disease - related gene phenotype relationships . using the semantic relationship nomenclature of tsai et al . ( 8) , we selected reports in which the agent that deliberately performs an action is represented by a gene or protein , and the patient that is the recipient of the action corresponds to disease phenotypes . the information we sought stands in contrast to associative relationships , such as elevated protein levels correlating with disease activity . to create the design corpus , our initial query matched medline sentences containing ordered triplets of a gene or protein name , a causative verb and a phenotype related to cancer or diabetes . each member of the triplet was separated within the sentence by a maximum of three words . this software package has the ability to retrieve sentences from text that include word patterns established by user queries . semantically , term classes can be defined combining external ontologie and adding term morphological variations and regular expression patterns . syntactically , it recognizes part - of - speech and shallow parsing constructs such as noun phrases , verbs and prepositions . in terms of scope , queries can be confined to different document or text sections , including abstracts , titles or sentences . for example , a sentence - level query may comprise a term class protein , a list of verbs ( i2e can automatically generate morphological variants ) and a list of phenotype objects . the gene / protein thesaurus was internally developed and based on biothesaurus ( 9 ) . forty verbs that signal causality ( e.g. inhibition , stimulation ) were compiled manually , as was a set of disease - related phenotypes relevant to cancer ( e.g. tumorigenesis , vascularization ) and diabetes ( e.g. serum glucose levels , weight gain ) . a set of 100 retrieved sentences and relationships were annotated by three phd - level evaluators for relevance and direction of perturbation ( see table 1 for examples ) . relevance annotation was used to mark relationships that should be eliminated from the corpus for being irrelevant to the intent of the retrieval query , e.g. if the gene was not acting as an agent . direction indicated the type of perturbation ( increased , decreased or unknown ) relative to the starting state . for example , if a gene is added back to cells that carry a deletion in that gene to restore the wild - type state , it is noted as an increase , because the abundance of the gene was increased relative to the starting state . unknown if there was no stated perturbation , or direction could not be inferred at the sentence level . it is worth noting that although an unknown direction could be frequently resolved by reviewing the abstract or the full text of the article , we strictly limited our scope to evidence found at the sentence level . table 1.examples of relevance and direction annotationrelevancedirectionsentenceexplanationpmidirrelevantdifferent members of bcl-2 family may promote or inhibit apoptosis by synthesizing anti- and proapoptotic proteinsmembers of a gene family , not a gene , are causing apoptosis phenotype17404013irrelevantthe atf6 and ire1 pathways cooperatively caused apoptosis via induction of chop , activation of xbp1 and phosphorylation of jnk , and the perk - eif2 pathway counteracted the proapoptotic processespathways , not genes , are causing apoptosis phenotype17464326irrelevantthis result suggests that toxic products such as reactive oxygen species and aldehydes liberated by the action of polyamine oxidase on the acetylated polyamines formed by ssat may enhance tumor developmentmetabolites , not proteins that generate them , are causing tumor development phenotype17675337relevantunknownthese results indicate that survivin is important for optimal development of bovine blastocysts and confirm that survivin expression suppresses apoptosis of pre - implantation embryossurvivin expression does not indicate if there is a state change in amount17075833relevantincreasefinally , we showed that after 79 days of incubation , mch also inhibits proliferation of non - stimulated pbmcincubation indicates mch was added exogenously17537530relevantincreasetaken together , ldm induces apoptosis in a p53-dependent manner when given at low doses , but in a p53-independent manner when given at high dosesgiven at low doses indicates ldm was added exogenously17534142relevantdecreasesoluble flt-1 abolished hypoxia / vegf - induced hyperpermeabilitysoluble forms of receptors create inactive complexes17311300 examples of relevance and direction annotation while most sentences contained straightforward descriptions of perturbations ( e.g. mutations in gene a or protein b inhibition ) , the broad range of perturbations in the literature , combined with the complex grammatical structures found in biomedical text , made some sentence relationship pairs difficult to annotate consistently , mainly due to differences in knowledge of experimental descriptions and settings by the evaluators . when inter - annotator agreement proves to be challenging , other groups have adopted strategies to further improve the final gold standard annotation set ( 10 ) . for this work , sentences deemed difficult to evaluate were set aside for later annotation by discussion and group consensus . therefore , the gold standard was comprised of annotations with three - way agreement from the individual assessment , plus the consensus annotations . when each evaluator 's set of straightforward independent annotations ( n = 237 out of 300 annotations , 79% ) was compared to the gold standard , agreement averaged 92.9% . sentence relationships for which there was no agreement or only two - way agreement were discussed and annotated by consensus ( 14% , n = 14 ) . while overall inter - annotator agreement can not be measured with the annotation procedure devised , the agreement metrics described are helpful as proxies to the level of ambiguity of the task . pairs that are only evaluated by consensus differ from the rest largely in the knowledge required from the evaluator to elucidate the annotation , rather than in the factual ambiguity of the sentence . hence , discussion and consensus assessment of pairs yields a better annotation set than individual annotation . only 7% ( n = 7 ) of sentence relationship pairs were marked irrelevant . following the same guidelines , we created the test corpus . each of the three evaluators assessed different sets of 500 sentence sentences deemed not straightforward were left without annotation ( n = 126 , 8.4% ) and later annotated by consensus . n = 95 ) were considered irrelevant , demonstrating the high precision of the retrieval query . overall , the procedure used to construct the corpus assured high quality to the 1405 sentence relationship annotation . we performed detection of increased , decreased or unknown perturbations using machine learning techniques . for that purpose , we constructed a vector of features for each relationship sentence pair using the test corpus above . one of them represented token presence as a binary vector of token weights wi , d = { w1, ,w|t| } , where t is the set of sentence tokens : a weight has value 1 if the token is present in the sentence and value 0 otherwise , an approach called set of words ( sow ) ( 11 ) . hyphenated names were considered both as single and separate tokens in the sow in order to capture affixes like since proximity to the gene name can be important in determining a token 's role in describing a perturbation , the sentence was further divided in several sections relative to the gene name 's position . the sections considered were : n tokens before the gene name , where n = { 5 , 10 , all } , and n tokens after the gene name , where n = { 10 , all}. we noted that many perturbation descriptions were adjacent or very close to the gene name ( e.g. overexpression of p53 ) . hence , we included a feature with the distance between the gene name and the beginning of the sentence ( e.g. distance of 0 or 1 may indicate that the perturbation is unknown , e.g. we also created a set of features using a small perturbation ontology developed independently over a disease - agnostic set of retrieved sentences . if a member of the ontology was present in the sentence a feature was added with value 1 , otherwise with value 0 . all the feature sets described were integrated in a single feature vector for each sentence . an algorithm based on the principles of maximum entropy ( 12 , http://homepages.inf.ed.ac.uk/s0450736/maxent_toolkit.html ) was trained using 80% of the test corpus , randomly sampled from the cancer and diabetes sets , and tested over the remaining 20% ( results were averaged over 10 runs ) . the training allowed the algorithm to predict the presence of a perturbation and its direction . performance measures were evaluated and compared favorably to an svm algorithm ( 13 ) . to create the analysis corpus , we extended the scope of our methodology used to retrieve the test corpus by eliminating the disease - specific phenotype constraints in the retrieval query ( hence , phenotypes were not included in the query ) . we applied the query to medline diabetes abstracts after 1996 and retrieved 359 385 relationships related to different conditions and phenotypes . this output was run through the machine - learning algorithm to create a wide - scope , disease - agnostic set of 191 240 perturbation predictions . to create a diabetes - specific subset , only sentences from medline abstracts containing the word diabetes in their mesh descriptors were included . there were significant differences both in technique and direction between the cancer and diabetes perturbations in the test corpus , with decreased perturbations more prevalent in cancer . detection of increased and decreased perturbations had a lower f - measure , 72.9% and 71.2% , respectively . when we excluded the perturbation ontology in feature generation , results were only slightly lower , whereas when we exclusively used the perturbation ontology , results were much lower , notably due to reduced recall . straightforward relationships ( 91.6% of the total ) were those that evaluators annotated without consultation with other annotators . these relationships were less challenging for humans , and the algorithm had better performance over this subset than overall . due to absence of previous work in perturbation detection , these results can not be compared , but they fall in ranges typical of other successful biomedical text mining tools . table 2.perturbation extraction performancecountprecision ( % ) recall ( % ) f - measure ( % ) perturbation69079.279.879.4 no ontology76.479.677.6 only ontology77.259.567.0 straightforward83.378.981.0increased43675.371.172.9 no ontology76.669.972.9 only ontology65.750.956.8 straightforward77.768.872.7decreased25479.065.171.2 no ontology78.563.670.0 only ontology79.858.466.4 straightforward80.467.272.9precision , recall and macro - averaged f - measure were calculated using four different combinations of feature sets : full , no ontology , only ontology and straightforward. baseline frequencies were 32.8% for increased perturbations and 18.1% for decreased perturbations . perturbation extraction performance precision , recall and macro - averaged f - measure were calculated using four different combinations of feature sets : full , no ontology , only ontology and straightforward. baseline frequencies were 32.8% for increased perturbations and 18.1% for decreased perturbations . to determine whether the disease impacted the frequency of perturbation types , we compared cancer and diabetes literature . the diabetes literature was significantly enriched in increased perturbations , whereas the cancer literature showed an even distribution between increased and decreased perturbations . in cancer , more perturbations were performed with antisense oligonucleotide ( n = 9 ) , antibody ( n = 16 ) and rna interference ( n = 26 ) than in diabetes ( antisense , n = 3 ; antibody , n = 2 ; rnai , n = 0 ) . perturbations in diabetes were more frequently described as injections ( n = 132 ) and/or administered by dose ( n = 57 ) than in cancer ( dose , n = 3 ; injection , n = 6 ) . perturbations in cancer were also more frequently described as being in vivo ( n = 12 ) or in vitro ( n = 14 ) than in diabetes ( in vivo , n = 5 ; in vitro , n = 0 ) . we examined the sentences for frequently occurring terms , grouped by their level of regulation ( i.e. protein , mrna or dna ) , if known . many of the terms related to dosing and routes of administration ( e.g. administration , intracerebroventricular , injection ) show a strong dominance in the diabetes literature compared to the cancer literature . although these usually indicate an increasing perturbation , there can be exceptions , such as systemic delivery of an inhibitor . table 3.ontology occurrence count in sentences from the test corpus , separated by diabetes and cancer phenotypesgene and protein perturbationscancerdiabetescancerdiabetestotal increasing modifications266586total decreasing modifications267110general increasing modificationsgeneral decreasing modifications activation6612 down - regulated , -ion120 administered , -ion7120 inactivation50 dose , -age , -dependent1186 inhibition307 ectopic70 repression10 enhanced expression100 suppression103 exogenous623dna decreasing modifications i.c.v.011 deficiency , -ent1512 i.p.07 deletion32 increased2618 dominant - negative30 induction82 knockout12 infused , -ion035 loss1213 injected , -ion766 mutant00 intracerebroventricular051 mutated , -ion03 intraperitoneal116mrna decreasing modifications mg / kg,/kg08 anti(-)sense93 oral64 interference51 overexpressed , -ration380 interfering rna20 peripherally07 knockdown120 recombinant710 rna interference40 restoration20 rnai20 subcutaneous02 short hairpin rna10 systemic19 silenced10 treatment2422 sirna150 up - regulation80protein decreasing modificationsdna increasing modifications antagonist728 adenoviral21 anti-254 adenovirus00 antibody , -ies113 gain - of - function20 blockade , -ing128 gene delivery13 deactivation10 transgenic32 decoy20mrna increasing modifications fc-00 inducible20 inhibitor5315protein increasing modifications inverse agonist01 activator100 mabs10 agonist954 neutralization10 analog / analogue27 reduced soluble40 targeting60note that total increased and decreased perturbations exceeds values in table 2 since multiple perturbation terms may appear in a single sentence . ontology occurrence count in sentences from the test corpus , separated by diabetes and cancer phenotypes note that total increased and decreased perturbations exceeds values in table 2 since multiple perturbation terms may appear in a single sentence . the phenotypes in this study were selected based on prevalence in the literature without regard to their use in vivo or in vitro . for instance , in diabetes a change in blood glucose levels signals a change in disease state clearly a phenotype monitored following perturbation in vivo . likewise , in cancer a change in the number of metastases is exclusively described in in vivo systems . in contrast , insulin sensitivity can be used to describe both in vivo and in vitro systems . for cancer , cell proliferation and apoptosis rates if our hypothesis that perturbed genes and proteins form the underpinnings of disease mechanisms , these entities should be well represented in pathway diagrams and among drug candidates . we applied our trained algorithm to a set of 14 345 sentence relationship facts for genes from our analysis corpus belonging to a diabetes pathway ( figure 1 ) . predictably , our algorithm found a strong correlation between the number of medline abstracts in which a gene is mentioned and the number of times it is described as perturbed ( r = 0.70 ) . the results in figure 1 show the difference in intensity and modality in which each gene is described or pursued experimentally . some genes were typically more increased or decreased , often reflecting their roles in therapeutics . examples of significantly ( p < 10 ) increased were insulin , interleukin 2 or parathyroid hormone . among the decreased were such genes as epidermal growth factor receptor ; caspase 8 and plasminogen activator , urokinase receptor . figure 1.perturbations extracted from genes involved in the diabetes mellitus type ii pathway in the kyoto encyclopedia of genes and genomes ( 21 ) . differences in perturbation direction are large for some members of the pathway , note that the scale bar is logarithmic . perturbations extracted from genes involved in the diabetes mellitus type ii pathway in the kyoto encyclopedia of genes and genomes ( 21 ) . differences in perturbation direction are large for some members of the pathway , note that the scale bar is logarithmic . disease associations included in the omim morbid map ( omim online reference , http://www.ncbi.nlm.nih.gov/omim/ ) . the average abstract mention and gene perturbation counts were higher for genes with morbidmap associations ( t - test , p < 10 ) . this was consistent with our expectation that genes associated to disease would be the subject of deeper study . however , genes that had been perturbed numerous times were not necessarily linked to disease . for example , out of the 100 most perturbed genes , 54 were not linked to disease in omim morbidmap , including at the top such genes as jun oncogene , interleukin 4 , fibroblast growth factor 2 ( basic ) , colony stimulating factor 2 ( granulocyte macrophage ) , colony stimulating factor 3 ( granulocyte ) and mitogen - activated protein kinase 3 . perturbations are relevant for areas like the study of gene disease associations , protein protein interactions ( ppi ) or gene regulatory networks . gene disease association extraction studies have largely focused on simply detecting associations rather than characterizing them ( 7,14 ) . ppi and gene regulation extraction studies have side - stepped perturbation types , without considering anything further than the experimental technique ( 15,16 ) . we note that ppi relationships are frequently devoid of perturbations and the focus is on how a ppi was detected , which does not necessarily involve a causative relationship . the proteomics standards initiative molecular interactions ( psi - mi ) ontology ( 6 ) is a comprehensive effort to describe molecular interactions . this ontology , while including a detailed experimental preparation section , lacks expressivity in describing perturbations generically . similarly to bundschus et al . ( 7 ) , it includes a section on expression level with entries under expressed , over expressed and physiological. the lack of a general framework for recognizing , characterizing and classifying perturbations is surprising when one considers their importance in phenomena encountered experimentally . researchers with interest in characterizing previous and current perturbation work on a biological system face the challenge of a naturalist trying to deal with animal species without a linnaean taxonomy . this is reflected in the methodological landscape that was set in the early literature in the fields of biomedical ontologies and text mining . for example , the comprehensive text mining tool medlee ( 17 ) only considered the state of a gene or protein , where the state has an adjectival role such as mutated in the phrase mutated x. perturbation descriptions , however , should be considered carefully . observe the differences between the sentences ( i ) x activates y. and ( ii ) inhibition of x activates y. from the point of view of classic ppi extraction both relationships are equivalent and can be represented with the triplet x activate y. the perturbation in sentence ( ii ) , however , signals that it is likely that protein x is inhibiting protein y instead . we have called this phenomenon reversal. given the results of the present assessment , a review of the relationship data available should be considered under this model . phenotype associations in disease but the principles shown are applicable to other well - known areas , such as ppi , as well as less explored ones such as identification of biomarkers or cellular processes . a perturbation taxonomy , like the one described in table 4 , could capture the different dimensions that may be of interest to the inquiring scientist . unknown direction annotation is intended for perturbations whose direction can not be inferred at the sentence level . molecule annotation characterizes the type of molecule being primarily affected by the perturbation : gene , rna or protein . expression annotation is used for a change of expression level without clarifying whether the change is in rna or protein . the following examples illustrate these annotations : a gene mutation is a decrease in gene activity , where the function / activity of a gene is specifically understood as making a wild - type transcript . exogenous addition of a gene via viral transfection , plasmid transformation , etc . , is an increase in gene abundance . a gene duplication is an increase in gene abundance while a knockout is a decrease in gene abundance . dominant negative is a mutation in a gene , which indicates that , compared to wild - type , it has defective function . silencing , knock - down and antisense all apply to a decrease in rna abundance . an antibody blocking a protein decreases the protein activity or function . interfering with a protein binding treatment , incubation , recombination , or synthetic refer to exogenous addition of protein . table 4.proposed annotation guidelinesrelevancedirectionmoleculeeffectrelevantincreasegeneactivityirrelevantdecreasernaabundanceunknownprotein expression proposed annotation guidelines perturbations evolve , notably as new techniques are developed and targets are identified . we expect perturbations to be subject to trends and popularity variations similar to those in other aspects of biomedicine ( 1820 ) . | molecular perturbations provide a powerful toolset for biomedical researchers to scrutinize the contributions of individual molecules in biological systems .
perturbations qualify the context of experimental results and , despite their diversity , share properties in different dimensions in ways that can be formalized .
we propose a formal framework to describe and classify perturbations that allows accumulation of knowledge in order to inform the process of biomedical scientific experimentation and target analysis .
we apply this framework to develop a novel algorithm for automatic detection and characterization of perturbations in text and show its relevance in the study of gene phenotype associations and protein protein interactions in diabetes and cancer .
analyzing perturbations introduces a novel view of the multivariate landscape of biological systems . |
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the online version of this article ( doi:10.1007/s00702 - 014 - 1353 - 5 ) contains supplementary material , which is available to authorized users . neurological and psychiatric diseases put a high toll on public and personal health and cause a heavy economic burden ( wittchen et al . 2011 ; smith 2011 ) . the pathological processes involved in neurological diseases such as alzheimer s and parkinson s disease are now becoming better understood . however , effective treatments and prevention measures are yet to be identified ( montine et al . 2012 ) . in psychiatric diseases , the nature of molecular and cellular changes of the brain is still obscure ( deep - soboslay et al . 2011 ) . notwithstanding recent impressive progress in mri , genetic and biomarker studies as well as experimental animal research , validation by histological , cellular and molecular research of the human brain is still required ( lin et al . 2011 ) . state of the art imaging techniques , genomics and proteomics make human brain tissue research more compelling than ever . however , cns tissue samples can generally only be obtained after death and to foster research in human brain diseases , brain banks have been established worldwide ( kretzschmar 2009 , samarasekera et al . post - mortem removal and storage of human organs , research with human brain tissue and genetic research have often been the centre of media attention ( burton et al . 2003 ) and posed a great deal of questions in the fields of law and ethics ( andrews 2006 ; case law 1988 ; greenfield 2006 ) . due to the relative novelty of the brain bank as an entity frequently , brain banks do not fit into well - defined and harmonized legal regimes while existing local legal regimes applicable to donation and use of human organs and tissues for research purposes differ significantly between countries . therefore , many uncertainties arise pertaining to initiation and governance of the brain banks , informed consent , donor confidentiality or tissue commercialization , which hampers the establishment of the valuable resources ( bell et al . the partners of the brain net europe ( bne ) consortium , a consortium of 19 european brain banks , belong to a variety of academic and/or health institutions , established in eleven different european countries . all of the bne brain banks are either affiliated with or are part of ( neuro)pathology departments or neuroscience institutes . the lack of harmonization in european legislation with regard to post - mortem medical procedures and donations of tissue to a biobank for research purposes contributes to the complexity of the field . although a set of uniform exhaustive operating requirements was deemed unfeasible to formulate and perhaps in many cases undesirable to impose , we reasoned that a certain set of minimum ethical standards had to be maintained at all times , regardless of the differences among the brain banks . to formulate these standards , we systematically surveyed various authoritative sources in the disciplines of law and ethics . these include declarations , conventions , recommendations , guidelines and directives issued by the international governmental and non - governmental organizations , such as the council of europe , european commission , world medical association and world health organization ( documents used in the preparation of the code of conduct see : http://www.brainnet-europe.org/index.php?option=com_content&view=article&id=104&itemid=104%29 , http://www.brainbank.nl ) . specifically , the recommendation on research on biological materials of human origin adopted by the committee of ministers of the council of europe was important in the design of the code of conduct and functioned as a template for its first draft . to establish a common ground as well as fundamental differences in the practices and the legal frameworks under which brain banks operate , and to involve more bne members in the development of the first drafts of the code of conduct , a meeting in amsterdam was organized in november 2006 . during this meeting , attendees from different bne brain banks have deliberated on principal points to be included in the code of conduct , thereby setting a certain course . the first draft of the code of conduct was presented during a bne meeting in stockholm , in june 2007 . although it had been established that informed consent of the donor or the next of kin is not always prescribed by law for conducting an autopsy and retaining whole organs or tissue samples for examination or research purposes , the bne members agreed that a brain bank should strive to implement the information and consent procedure in their daily practice ( lunetta et al . declaration of intention in the code of conduct , an aim for a future standard had been set . following this meeting , the final draft of the code of conduct has been circulated among all bne members with a possibility to comment on its contents and suggest textual amendments . during the next meeting in barcelona in june 2008 , the final version of the code of conduct was ratified by all bne brain banks . subsequently , at an international conference on brain banking , organized by the bne network , in munich in december 2008 ( http://www.brainnet-europe.org/index.php?option=com_content&view=article&id=125&itemid=141 ) , a workshop on legal and ethical issues in brain banking was held where the applicability of the code of conduct was discussed . this workshop was attended by the directors of brain banks , scientists as well as representatives of patient organizations from many different countries . practical examples of code of conduct application in daily practice were discussed . the code of conduct consists of four chapters ( for the text of bne 's code of conduct for brain banking see supplementary material ) . the first chapter sets out the objectives , general principles and the scope of the code of conduct , as well as defining key terminology ( indicated in the text by capital letters ) . the second chapter deals with different aspects of material procurement ( e.g. informed consent , authorization , incompetent persons , autopsy ) the third chapter states the principles that should govern the processing of the material ( e.g. financial aspects , confidentiality , data protection measures ) . the fourth chapter is concerned with distribution and the use of the material for research . it also contains articles on research results and guidelines on how to deal with information on hereditary diseases . the code of conduct for brain banking states the principles that should govern brain banking in general . detailed regulations concerning the local governance of a brain bank are to be laid down in a different document such as a brain bank regulations , which should be in concordance with the principles described in the code of conduct . furthermore to support the daily practice of the brain bank documents such as a material transfer agreements , confidentiality agreements , tissue application forms and personal data the scope of the code of conduct could be widened to cover all post - mortem tissue donations for research purposes , whether of cns origin or not ( for instance peripheral lymph nodes or dorsal root ganglia ) , since the same principles apply . bne s code of conduct is being adopted by the bne network prior to being enshrined in official legislation . bne has no legal power to enforce it . however , this code of conduct is a first common european attempt to define ethical standards in brain banking in one document . in case of bne , the code of conduct has stimulated the bne brain banks to reflect on their daily practice and on the governance of their brain banks . we hope that brain banking will receive more attention in future legislation , locally and internationally , and that this code of conduct may lay in part a foundation for more official legislation for brain banking in europe and beyond . | research utilizing human tissue and its removal at post - mortem has given rise to many controversies in the media and posed many dilemmas in the fields of law and ethics .
the law often lacks clear instructions and unambiguous guidelines .
the absence of a harmonized international legislation with regard to post - mortem medical procedures and donation of tissue and organs contributes to the complexity of the issue .
therefore , within the brainnet europe ( bne ) consortium , a consortium of 19 european brain banks , we drafted an ethical code of conduct for brain banking that covers basic legal rules and bioethical principles involved in brain banking .
sources include laws , regulations and guidelines ( declarations , conventions , recommendations , guidelines and directives ) issued by international key organizations , such as the council of europe , european commission , world medical association and world health organization .
the code of conduct addresses fundamental topics as the rights of the persons donating their tissue , the obligations of the brain bank with regard to respect and observance of such rights , informed consent , confidentiality , protection of personal data , collections of human biological material and their management , and transparency and accountability within the organization of a brain bank .
the code of conduct for brain banking is being adopted by the bne network prior to being enshrined in official legislation for brain banking in europe and beyond.electronic supplementary materialthe online version of this article ( doi:10.1007/s00702 - 014 - 1353 - 5 ) contains supplementary material , which is available to authorized users . |
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growing interest in association between periodontal disease and systemic inflammation has led to convergence in oral and medical care . it has been widely accepted that periodontal infections are leading culprits for cardiovascular disease ( cvd ) . this association could partly be explained by c - reactive protein ( crp ) , which is a systemic marker of inflammation . it is receiving the most attention as it best describes the inflammatory status of an individual due of its kinetics . crp is an acute - phase reactant produced by the liver , which is nonspecific and produced in response to various stimuli . crp possesses the ability to reveal inflammation at an early stage as it rises in serum within 48 h. its long plasma half - life of 12 - 18 h is constant under most of the conditions and hence that the sole determinant of circulating crp is the synthesis rate , which directly reflects the intensity of the pathological process stimulating crp production . this property is useful for early detection of patients who are at risk for inflammatory disease . moreover , it can upregulate proinflammatory mediator production . according to the centers for disease control and prevention / american heart association , crp levels > 3 mg / l indicates a higher risk for cvds , whereas crp levels of 1 - 3 mg / l suggests medium risk , and crp levels < 1 mg / l suggests lower risk . a number of studies have demonstrated an association between periodontal disease and the risk of cvd . but , it is not clear whether this association is casual or definite as both the diseases share so many risk factors . there is strong evidence that crp is increased in otherwise healthy adults with poor periodontal status . however , the disparity among them occurs in terms of methods used , criteria of patient selection , confounding of risk factors and moreover some authors denied this association . most studies to date have included patients with chronic periodontitis without correlating serum crp levels with severity of inflammation , and also limited information is available whether systemic inflammation differs in different types of periodontitis as there is scarcity of literature regarding crp levels in subjects with aggressive periodontitis . since both forms of periodontitis show disparity in the rate of progression , the effect of chronic and aggressive periodontitis on crp levels seems to be an appealing area of research . thus , the present study was undertaken to determine the relative levels of serum crp and compare them in aggressive and chronic periodontitis patients and correlating the serum crp levels with severity of disease . following complete medical and dental examination , 75 individuals were selected for the study from department of periodontics , dr . ziauddin ahmad dental college , aligarh . a total of seventy - five systemically healthy subjects were divided into three groups : group i , nonperiodontitis subjects ; group ii , chronic generalized periodontitis ( cgp ) patients and group iii , generalized aggressive periodontitis ( gap ) patients . subjects were placed into three groups according to the following definitions :
group i : nonperiodontitis ( np ) group - clinically healthy periodontal status with probing depth ( pd ) 2 mm along with no evidence of attachment lossgroup ii : cgp patients - probing depth ( pd ) of 5 mm and/or clinical attachment loss ( cal ) > 30% sites with varying degree of disease severity . local factors concomitant with the amount of destruction and with moderate rate of progressiongroup iii : gap - patients under age of 30 years having probing depth ( pd ) of 5 mm and/or cal on 8 or more teeth , at least three of which were not first molars and incisors with varying degree of disease severity . group i : nonperiodontitis ( np ) group - clinically healthy periodontal status with probing depth ( pd ) 2 mm along with no evidence of attachment loss group ii : cgp patients - probing depth ( pd ) of 5 mm and/or clinical attachment loss ( cal ) > 30% sites with varying degree of disease severity . local factors concomitant with the amount of destruction and with moderate rate of progression group iii : gap - patients under age of 30 years having probing depth ( pd ) of 5 mm and/or cal on 8 or more teeth , at least three of which were not first molars and incisors with varying degree of disease severity . current smokers ; pregnant and lactating women ; individuals with acute or chronic medical disorders ; patient under any medication for the past 3 months ; patient undergone any dental treatment for the past 6 months were excluded from the study . periodontal disease status was evaluated at 4 sites per tooth ( mesiobuccal , buccal , distobuccal , lingual / palatal ) by measuring the probing depth ( pd ) , cal , gingival index by loe and silness ) , plaque index ( silness and loe ) using the same periodontal probe ( unc-15 probes hu - friedy 's , usa ) and by same examiner to avoid bias . the probing depth was measured as the distance from the gingival margin to the base of the pocket in millimeters . the clinical attachment levels were calculated from the recession and probing depth measures and represented as the distance in millimeters from the cementoenamel junction to the base of the pocket . they were made to tighten a fist so that vein was more palpable , and antecubital vein was selected for venipuncture . a tourniquet was applied about 1 - 2 inches above the antecubital fossa . after cleansing the puncture site with 10% isopropanol solution tourniquet was released as the blood flow began . after drawing 3 ml of blood , the subjects were instructed to apply mild finger pressure on the site for few minutes to avoid oozing out of blood . samples were centrifuged in the centrifuge machine at 3000 rpm for 10 min to separate the serum from blood . separated serum was collected in eppendrof and stored in the deep freeze at - 20. quantitative determination of crp in patient 's serum was done by double antibody sandwich enzyme - linked immunosorbent assay ( elisa ) method ( icla , usa ) . quantitative determination of crp in patient 's serum was done by double antibody sandwich elisa method . in this assay , the crp present in sample reacts with anti - crp antibodies , which had been adsorbed to the surface of polystyrene microtiter wells . after the removal of unbound sample proteins by washing , anti - crp antibodies conjugated with horseradish peroxidise were added . these enzyme- labeled antibodies form complexes with the previously bound sample crp . following another washing step , the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate , 3 , 3 , 5 , 5-tetramethylbenzidine . the quantity of bound enzyme varies directly with a concentration of crp in the test sample . the quantity of crp in test sample can be interpolated from the standard curve constructed from standard and corrected for serum dilution . data were presented as mean , standard deviation and 95% confidence interval of the mean difference . comparison of three groups ( cgp , gap , and np ) with respect to crp values was done using one - way analysis of variance ( anova ) . subjects were placed into three groups according to the following definitions :
group i : nonperiodontitis ( np ) group - clinically healthy periodontal status with probing depth ( pd ) 2 mm along with no evidence of attachment lossgroup ii : cgp patients - probing depth ( pd ) of 5 mm and/or clinical attachment loss ( cal ) > 30% sites with varying degree of disease severity . local factors concomitant with the amount of destruction and with moderate rate of progressiongroup iii : gap - patients under age of 30 years having probing depth ( pd ) of 5 mm and/or cal on 8 or more teeth , at least three of which were not first molars and incisors with varying degree of disease severity . group i : nonperiodontitis ( np ) group - clinically healthy periodontal status with probing depth ( pd ) 2 mm along with no evidence of attachment loss group ii : cgp patients - probing depth ( pd ) of 5 mm and/or clinical attachment loss ( cal ) > 30% sites with varying degree of disease severity . local factors concomitant with the amount of destruction and with moderate rate of progression group iii : gap - patients under age of 30 years having probing depth ( pd ) of 5 mm and/or cal on 8 or more teeth , at least three of which were not first molars and incisors with varying degree of disease severity . current smokers ; pregnant and lactating women ; individuals with acute or chronic medical disorders ; patient under any medication for the past 3 months ; patient undergone any dental treatment for the past 6 months were excluded from the study . periodontal disease status was evaluated at 4 sites per tooth ( mesiobuccal , buccal , distobuccal , lingual / palatal ) by measuring the probing depth ( pd ) , cal , gingival index by loe and silness ) , plaque index ( silness and loe ) using the same periodontal probe ( unc-15 probes hu - friedy 's , usa ) and by same examiner to avoid bias . the probing depth was measured as the distance from the gingival margin to the base of the pocket in millimeters . the clinical attachment levels were calculated from the recession and probing depth measures and represented as the distance in millimeters from the cementoenamel junction to the base of the pocket . they were made to tighten a fist so that vein was more palpable , and antecubital vein was selected for venipuncture . a tourniquet was applied about 1 - 2 inches above the antecubital fossa . after cleansing the puncture site with 10% isopropanol solution tourniquet was released as the blood flow began . after drawing 3 ml of blood , the subjects were instructed to apply mild finger pressure on the site for few minutes to avoid oozing out of blood . samples were centrifuged in the centrifuge machine at 3000 rpm for 10 min to separate the serum from blood . separated serum was collected in eppendrof and stored in the deep freeze at - 20. quantitative determination of crp in patient 's serum was done by double antibody sandwich enzyme - linked immunosorbent assay ( elisa ) method ( icla , usa ) . quantitative determination of crp in patient 's serum was done by double antibody sandwich elisa method . in this assay , the crp present in sample reacts with anti - crp antibodies , which had been adsorbed to the surface of polystyrene microtiter wells . after the removal of unbound sample proteins by washing , anti - crp antibodies conjugated with horseradish peroxidise were added . these enzyme- labeled antibodies form complexes with the previously bound sample crp . following another washing step , the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate , 3 , 3 , 5 , 5-tetramethylbenzidine . the quantity of bound enzyme varies directly with a concentration of crp in the test sample . the quantity of crp in test sample can be interpolated from the standard curve constructed from standard and corrected for serum dilution . data were presented as mean , standard deviation and 95% confidence interval of the mean difference . comparison of three groups ( cgp , gap , and np ) with respect to crp values was done using one - way analysis of variance ( anova ) . clinical parameters and mean crp levels were summarized in table 2 . the mean crp ( mg / l ) levels for the gap , cgp and np groups were 4.61 0.58 , 2.31 0.56 and 0.96 0.13 , respectively . crp values of the three patient groups were significantly different from each other , with crp levels in the group iii greater than those in the group ii , which were in turn greater than those in the group i subjects . furthermore , comparison of three groups ( cgp , gap , and np ) with respect to crp values by one - way anova showed statistical significance ( p < 0.0001 ) , as shown in table 3a . furthermore , multiple comparisons of all groups with respect to crp values by anova [ table 3b ] illustrated statistical significance . correlation coefficient between crp and pd was 0.939 , 0.957 , 0.902 and with cal 0.742 , 0.945 and 0.920 in group i , ii and iii respectively , thus indicating a positive correlation [ table 4 ] . again , they were significantly correlated , and all the parameters increased with increasing crp levels , thus indicating a positive correlation . subject characteristics clinical parameters and serum crp levels in subjects one - way anova showing comparison between and within groups correlation of crp with pd and cal main finding of the study was that crp levels were higher in the periodontitis group as compared with np group and highest in subjects with aggressive periodontitis demonstrating that periodontitis can have impact on systemic markers of inflammation in a relatively younger subject group . crp levels correlated positively with the severity of inflammation as it is biologically plausible that inflammatory cytokines interleukin-6 ( il-6 ) , il-1 , and tumor necrosis factor - alpha in response to periodontal infection present the capacity to stimulate hepatocyte to produce crp . circulating crp levels are markers of systemic inflammation and are associated with periodontal disease . also of significance are the results of prospective longitudinal trials which indicate that this marker appears to be a useful predictor for future cardiovascular events in a variety of population . all the subjects included in the study were systemically healthy , and all the other risk factors for periodontitis were excluded . the aggressive periodontitis subject group displays younger age range than that seen in studies of chronic periodontitis . strength of the study was that the quantitative determination of crp was done by double antibody sandwich elisa method which is very sensitive method for detecting crp as compared to other methods with a detection limit of 0.0005 - 0.005 another strength of the study was that the results of the present study indicate a significant correlation between attachment loss , probing depth and crp , which is consistent with findings of noack et al . as some studies did not find a correlation with severity of disease . in a study reported by salzberg et al . patients with gap had increased levels of crp ( 3.72 mg / l ) compared to control ( 1.54 mg / l ) . sun et al . reported 1.87 and 0.52 mg / l in patients with gap and control subjects , respectively . the mean crp ( mg / l ) level for chronic periodontitis patients was 2.3 1 0.56 mg comparison of three groups ( cgp , gap , and np ) with respect to crp values by one - way anova showed statistical significance ( p < 0.0001 ) as observed in table 3a , which confirms the results of the previous studies but the controversy regarding the mechanism of linkage between periodontitis and elevated crp remains . our findings were in contrast with findings of kanaparthy et al . where mean levels of crp were greater in chronic periodontitis subjects when compared to aggressive periodontitis , which could be attributed to long standing nature of the disease and its influence on systemic health moreover , there lies genetic susceptibility towards inflammatory hyperactivity , which is beyond the scope of this study . the correlation coefficient between crp levels and pd as well as cal in cgp was found to be positive and significant . with increasing mean pd and cal these findings are at par with those of a study done by beck and offenbacher . thus , as the severity and extent of periodontal disease increase , the systemic component of inflammation also increases . this may be seen as increased production of crp . one more strength of the study was complete periodontal examination had been performed to classify the patients into two groups of periodontitis which ensures that there were less chances of diagnosing false positive cases of periodontitis . none of the subjects in this study had crp levels > 15 mg / l , indicating that it is relatively unlikely that the subjects were experiencing acute or chronic systemic diseases characterized by large increases in serum crp . similar results were found in another study , in which lim et al . concluded that crp was a significant predictor of sites with pd 5 mm and could be a potential risk marker in periodontal destruction . crp tends to increase with age but the elevation of crp in relatively younger subjects of the present study may correlate with periodontal destruction rather than with age . thus , serum crp levels can be a possible marker to depict active periodontal destruction and can differentiate progressive disease from a quiescent one . recently , crp has been proved to be the strongest and most significant predictor of future cardiovascular events . the results of the present study suggest that elevated crp levels in periodontitis patients can contribute at least in part to the increased risk for cvd . this may be of particular concern in younger individuals , as represented by gap patients where elevated levels of crp due to periodontitis may contribute to early or rapider cvd in susceptible individuals . this study shows an association between elevated serum crp level , pd and cal , but due to its cross - sectional nature , effect of each variable upon other ca nt be determined . for better understanding limited sample size , the role of genetics , oral health behaviors , nutrition , stress levels , which have been shown to affect the prevalence of periodontitis , were not considered in our study . from our study , we can conclude that periodontitis is related with the severity of inflammation , so it is empirical to treat the periodontal inflammation but recommending periodontal therapy solely for the purpose of preventing cvd is not supported by current evidence of literature . recommendations by american heart association states that patients should be educated about oral health as no one is systemically healthy without possessing good oral health . thus , there is a complementary need to recognize that apparently healthy individuals may be having extensive periodontal disease , which might be adding to systemic inflammation . hence , periodontal disease assessment and its treatment should be instituted in current health programs to improve the overall health status of an individual . it should be noted , however that an additional study in larger samples of patients might further investigate and confirm this idea . | objective : c - reactive protein ( crp ) is an acute - phase reactant and has been proved to be a significant predictor of future cardiovascular events .
recent studies have demonstrated a correlation between periodontitis and elevated crp levels . however ,
most of the studies have focused on chronic periodontitis and very few studies are done in patients with aggressive periodontitis .
the aim of this study was to determine and compare the relative levels of serum crp in aggressive and chronic periodontitis patients.materials and methods : a total of 75 systemically healthy subjects were divided into three groups : group i , nonperiodontitis subjects ; group ii , chronic generalized periodontitis patients and group iii , generalized aggressive periodontitis patients .
all participants were subjected to quantitative crp analysis using enzyme - linked immunosorbent assay.results:mean crp levels were significantly greater in both group ii and iii as compared to group i and group iii having greater level than group ii .
furthermore , crp levels positively correlated with the amount of periodontal destruction as measured by probing depth and clinical attachment loss.conclusion:the present study indicates a positive correlation between crp and periodontal disease severity with particular concern in younger individuals that could be a possible underlying pathway in the association between periodontal disease and the observed higher risk for cardiovascular disease in periodontitis patients . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
nowadays , the loss of the last remaining teeth is delayed , due to oral prevention ; consequently , the prevalence of edentulousness increases with age . in europe , the prevalence of edentulism of persons between 65 and 74 years old ranges from 12.8% , in italy , to 69.6% , in iceland.1,2 thus , rehabilitation of edentulism occurs later in life , and the integration of complete dentures is increasingly difficult with age.3 indeed , aging reduces adaptive capacity and degrades clinical oral status.4 in particular , bone resorption significantly reduces available bone volume . the buccal mucosa becomes thinner and weakens , creating an additional difficulty for prosthetic tolerance.5 under these conditions , the design of a conventional functional , removable complete denture becomes difficult or impossible . as a result , the edentulous elderly avoid many types of foods , particularly raw vegetables , because chewing is difficult with conventional complete dentures.68 dental status , among other factors , has an impact on the nutritional status of the elderly.9,10 it has been shown that subjects wearing conventional complete dentures have a lower nutritional intake than do subjects having more than 20 teeth.11 worldwide , the prevalence of malnutrition is reported to be high in the elderly , varying between 2% and 10% for autonomous senior subjects.12 multiple factors other than the dental status may have an impact on nutrition , among which is muscular force , which decreases as a function of age , leading to extended mastication time9,13 therefore , the capacity to form a bolus that allows swallowing ( plastic , cohesive , and slippery ) is jeopardized.14 from a technical viewpoint , successful prosthetic integration depends on the stability and maintenance of the prosthesis . in the past , the only strategy for preventing problems involved the use of prosthetic adhesive or retention by attachments.15 when using an attachment system , residual tooth roots can stabilize a denture and allow conservation of the alveolar bone structure . in the absence of roots , a complete denture retained by two implants is considered as the minimal treatment for the rehabilitation of an edentulous mandible.16,17 indeed , the symphyseal region generally allows implant treatments even when the mandibles are strongly resorbed . the dental implant may be compared with an artificial dental root onto which a prosthetic device can be screwed , sealed , or retained ( single crown , bridge , or denture ) . thus , with additional retention , these implants could provide comfort and provide a function similar to those of toothed subjects.18,19 however , for very old or vulnerable patients , a complex surgical procedure and its consequences could compromise standard implant rehabilitation . in addition , given their socioeconomic status , the high cost of implant use can be an additional barrier . in this context , the stabilization of complete mandibular denture by geriatric slim implants these implants have proven useful for orthodontic or temporary treatment.2022 gsi can be inserted via flapless surgical techniques , with no pain and edema , even in the presence of atrophic mandibles.2327 furthermore , the monolithic structure of gsi decreases the risk of contamination of the abutment - implant interface.28 dental implants can be loaded immediately or after a delay . the immediate loading of gsi is a well - documented surgical prosthetic procedure29,30 that instantly restores aesthetics , comfort , and chewing , with significant nutritional consequences.31 its success rate of over 95% is comparable to that of delayed loading.32 indeed , immediate loading enables the transmission of the masticatory forces on the bone , and this endosseous stimulation , when moderate , may favor osteogenesis . the formation of new osseous blood capillaries at the surgical site is stimulated , and the density of the peri - implant bone is then improved compared with that observed in delayed loading . the elimination of the delay between implant setting and prosthetic restoration avoids the wearing of a transitional prosthesis , thereby improving the patient s comfort and providing better chewing and therefore , better treatment acceptability.31 repeated studies have shown that these findings also apply to conventional implants used to retain overdentures.33,34 successful immediate loading is facilitated by the use of a rigid suprastructure , which enables the bone integration of the implant by applying a force along the entire implant - prosthetic structure.35,36 however , none of the studies cited above address the use of gsi for the stabilization of a complete mandibular denture . for this type of rehabilitation , a simple procedure of immediate loading of four gsis , using a flapless procedure , this is a case report of the use of a type of gsi ( obi mini ball implant 2.7 ; euroteknika group , sallanches , france ) in a procedure commonly used at the dental hospital of clermont - ferrand ( france ) . this method of oral rehabilitation is recommended in presence of unstable complete mandibular denture.16,37,38 patients with incompatible oral or general diseases should not be scheduled for this surgical procedure . gsi are not indicated for patients with unstable systemic disorders or rheumatoid polyarthritis treated with corticoid therapy . regarding patients with stable systemic disorders , the use of gsi treatment is accepted after a case - by - case decision , based on the patient s general health status . three steps preceding implantation can be described : ( 1 ) radiopaque plots are inserted in the prosthesis , to the left and right of the canines ; ( 2 ) the prosthesis is placed in the mouth , and an x - ray is taken to measure the space between the radiopaque plot of the prosthesis and the interforamen space ( figure 1 ) ; and ( 3 ) the interforamen space is marked accordingly with a mucosal pencil , indicating to the surgeons the situation of the foramens and delimiting the placement of the implants . in the described case , the implants used were 2.7 mm in diameter and from 9 to 15 mm in length . after drying the mucous membranes , the marks delimitating the emergence of the mental foramen were plotted on the crest , to delimit the area of parasymphyseal implant placement . transmucosal drilling was performed with a single 2 mm diameter drill ( figure 2 ) . the implants had to be placed as parallel an arrangement to each other as possible ( with a tolerance of 10 ) and perpendicular to the occlusal plane ( figure 3 ) . after surgery , the denture was recessed in the parasymphyseal area to avoid interference with the heads of the implants . then , under occlusal pressure , the prosthesis was secured against the female parts of the o - ring prosthesis attachment using a self - polymerizable methacrylic resin ( paladurr , heraeus materials technology gmbh & co. kg , hanau , germany ) ( figure 4 ) . afterwards , the denture was secured by clipping onto the set implants ( figure 5 ) . a panoramic , control radiograph the benefits of the gsi for the elderly and for frail persons are numerous , especially because of the specific characteristics of the surgical procedure . indeed , the flapless technique reduces perioperative stress and surgery duration and therefore , postoperative complications , with no increased surgical risk.39 traditional protocols for implant surgery with flap require direct access to the bone during drilling , and this approach requires subperiosteal elevation of a full - thickness flap . therefore , resorption has been observed during the healing phase , due to the compromised bone vascularization alone . in addition , removing the flap from the bone makes the latter more prone to microbial exposure , and infectious complications can be expected despite the use of aseptic procedures . furthermore , the survival rate of flapless implants is identical to that observed in implants with flaps , and using flapless implants constitutes an advantage for the elderly.40 the use of a single drill reinforces this benefit indeed , heating of the bones related to drilling is a major factor in implant failure . for appropriate bone remodeling , the critical temperature of 47c for 1 minute must not be exceeded.41 the use of a single , small - diameter drill limits the drilling duration and thus , reduces the risk of overheating . due to these features , gsi can be used in almost any clinical situation , even in the presence of a narrow ridge , where they can be implemented without prior adjustment of the ridge . indeed , the stabilization of a denture by gsi can be compared with an immediate loading , which is very beneficial for the patient . the success rate of this technique is probably closely linked to the implant - prosthesis connection mode and to the spatial distribution of the implants . an o - ring system ensures connection , comprising a male part ( a metallic ball ) and a female part ( the o - ring inserted into a metallic ring ) . the attachment of the prosthesis is performed under occlusal pressure to ensure that the denture impression surface is in full contact with the ridge . consequently , during chewing , the elasticity of the o - ring will ensure that a significant fraction of the masticatory forces exerted will be directed to the mucosa and not to the implants some implant complications are related to occlusal overload . therefore , unloading the intraprosthetic cup can disperse the forces on the mucosa and ensure implant health . thus the o - ring system is particularly suitable for immediate loading because of its shock absorption capacity . the distribution , number , and position of the implants are also contributing factors for osseointegration . indeed , a minimum of three implants is necessary to optimize biomechanical behavior . raising the number of implants to four increases the bone - implant surface interface . thus the forces transferred to the bone for the same surface area are decreased , and osseointegration is encouraged . in addition , the four implants form a trapezoid , with the angles varying according to the mandibular anatomy of the patient . in comparison with a two - implant configuration , better prosthesis stability is achieved , due to the decrease in axis rotation . during implantation , it is better to position the side implants at the most posterior site possible ( within the limit of the mental foramen ) to increase the distance between the implants and decrease distal extension . the lever arm of the extension is decreased , and consequently , the nonvertical forces on the implants are reduced , leading to better denture balance . regarding the financial aspect , gsi are less expensive than conventional implants , regardless of the manufacturer , and may be offered for a reasonable cost . this is particularly important when we address the elderly , whose income is often modest . indeed , it has been shown that the chewing parameters of mastication were improved in patients whose prosthesis was stabilized with implants ; when measured , the kinematic parameters and particle sizes were similar to those reported in normally toothed patients.42 the elderly may vary and thus increase the range of foods habitually consumed . additional studies are required to confirm this benefit , as several studies did not report a link between malnutrition and chewing function.43,44 in many cases , the use of gsi is advantageous ; however , a number of shortcomings can be listed . although the implant insertion appears easy , the use of a single drill implies that all implant axes must be correct from the outset , without any error in the estimation of parallelism . indeed , no second drill with larger diameter can be used to correct any axis error . the flapless procedure requires a practitioner with solid experience in conventional implant setting.45 in addition , to maintain sustainability of the implant , a width of 2 mm of keratinized gingiva is necessary.46 in geriatrics , this could particularly restrict the use of the flapless mini - implant . since the gsi is a one - piece implant ( with the abutment integrated in the implant ) , the male section of the attachment can not be changed in the case of wear . retention could then be improved by increasing the hardness of the o - ring rubber , but only to a certain extent . therefore , this technique should not be recommended in patients less than 70 years old . the practitioner performing the denture installation must rely on clinical experience in the use of this technique . therefore it may be necessary to use another implant - prosthesis connection mode that is easier to manage in terms of retention force and monitoring , such as the locator root attachment system ( zest anchors , escondido , ca , usa ) . furthermore , as with any other type of implant , independent acute complications could appear , in particular , cases of primary implant loss or severe inflammatory reaction . some chronic disorders , such as peri - implantitis , can be observed but rarely due to the characteristic of the one - part implant , which decrease this specific risk . a synthesis of the advantages and disadvantages of the gsi procedure is proposed in table 1 . this method of oral rehabilitation is recommended in presence of unstable complete mandibular denture.16,37,38 patients with incompatible oral or general diseases should not be scheduled for this surgical procedure . gsi are not indicated for patients with unstable systemic disorders or rheumatoid polyarthritis treated with corticoid therapy . regarding patients with stable systemic disorders , the use of gsi treatment is accepted after a case - by - case decision , based on the patient s general health status . three steps preceding implantation can be described : ( 1 ) radiopaque plots are inserted in the prosthesis , to the left and right of the canines ; ( 2 ) the prosthesis is placed in the mouth , and an x - ray is taken to measure the space between the radiopaque plot of the prosthesis and the interforamen space ( figure 1 ) ; and ( 3 ) the interforamen space is marked accordingly with a mucosal pencil , indicating to the surgeons the situation of the foramens and delimiting the placement of the implants . in the described case , the implants used were 2.7 mm in diameter and from 9 to 15 mm in length . after drying the mucous membranes , the marks delimitating the emergence of the mental foramen were plotted on the crest , to delimit the area of parasymphyseal implant placement . transmucosal drilling was performed with a single 2 mm diameter drill ( figure 2 ) . the implants had to be placed as parallel an arrangement to each other as possible ( with a tolerance of 10 ) and perpendicular to the occlusal plane ( figure 3 ) . after surgery , the denture was recessed in the parasymphyseal area to avoid interference with the heads of the implants . then , under occlusal pressure , the prosthesis was secured against the female parts of the o - ring prosthesis attachment using a self - polymerizable methacrylic resin ( paladurr , heraeus materials technology gmbh & co. kg , hanau , germany ) ( figure 4 ) . afterwards , the denture was secured by clipping onto the set implants ( figure 5 ) . a panoramic , control radiograph the benefits of the gsi for the elderly and for frail persons are numerous , especially because of the specific characteristics of the surgical procedure . indeed , the flapless technique reduces perioperative stress and surgery duration and therefore , postoperative complications , with no increased surgical risk.39 traditional protocols for implant surgery with flap require direct access to the bone during drilling , and this approach requires subperiosteal elevation of a full - thickness flap . therefore , resorption has been observed during the healing phase , due to the compromised bone vascularization alone . in addition , removing the flap from the bone makes the latter more prone to microbial exposure , and infectious complications can be expected despite the use of aseptic procedures . furthermore , the survival rate of flapless implants is identical to that observed in implants with flaps , and using flapless implants constitutes an advantage for the elderly.40 the use of a single drill reinforces this benefit indeed , heating of the bones related to drilling is a major factor in implant failure . for appropriate bone remodeling , the critical temperature of 47c for 1 minute must not be exceeded.41 the use of a single , small - diameter drill limits the drilling duration and thus , reduces the risk of overheating . due to these features , gsi can be used in almost any clinical situation , even in the presence of a narrow ridge , where they can be implemented without prior adjustment of the ridge . indeed , the stabilization of a denture by gsi can be compared with an immediate loading , which is very beneficial for the patient . both function and comfort the success rate of this technique is probably closely linked to the implant - prosthesis connection mode and to the spatial distribution of the implants . an o - ring system ensures connection , comprising a male part ( a metallic ball ) and a female part ( the o - ring inserted into a metallic ring ) . the attachment of the prosthesis is performed under occlusal pressure to ensure that the denture impression surface is in full contact with the ridge . consequently , during chewing , the elasticity of the o - ring will ensure that a significant fraction of the masticatory forces exerted will be directed to the mucosa and not to the implants some implant complications are related to occlusal overload . therefore , unloading the intraprosthetic cup can disperse the forces on the mucosa and ensure implant health . thus the o - ring system is particularly suitable for immediate loading because of its shock absorption capacity . the distribution , number , and position of the implants are also contributing factors for osseointegration . indeed , a minimum of three implants is necessary to optimize biomechanical behavior . raising the number of implants to four increases the bone - implant surface interface . thus the forces transferred to the bone for the same surface area are decreased , and osseointegration is encouraged . in addition , the four implants form a trapezoid , with the angles varying according to the mandibular anatomy of the patient . in comparison with a two - implant configuration , better prosthesis stability is achieved , due to the decrease in axis rotation . during implantation , it is better to position the side implants at the most posterior site possible ( within the limit of the mental foramen ) to increase the distance between the implants and decrease distal extension . the lever arm of the extension is decreased , and consequently , the nonvertical forces on the implants are reduced , leading to better denture balance . regarding the financial aspect , gsi are less expensive than conventional implants , regardless of the manufacturer , and may be offered for a reasonable cost . this is particularly important when we address the elderly , whose income is often modest . indeed , it has been shown that the chewing parameters of mastication were improved in patients whose prosthesis was stabilized with implants ; when measured , the kinematic parameters and particle sizes were similar to those reported in normally toothed patients.42 the elderly may vary and thus increase the range of foods habitually consumed . additional studies are required to confirm this benefit , as several studies did not report a link between malnutrition and chewing function.43,44 in many cases , the use of gsi is advantageous ; however , a number of shortcomings can be listed . although the implant insertion appears easy , the use of a single drill implies that all implant axes must be correct from the outset , without any error in the estimation of parallelism . indeed , no second drill with larger diameter can be used to correct any axis error . the flapless procedure requires a practitioner with solid experience in conventional implant setting.45 in addition , to maintain sustainability of the implant , a width of 2 mm of keratinized gingiva is necessary.46 in geriatrics , this could particularly restrict the use of the flapless mini - implant . since the gsi is a one - piece implant ( with the abutment integrated in the implant ) , the male section of the attachment can not be changed in the case of wear . retention could then be improved by increasing the hardness of the o - ring rubber , but only to a certain extent . therefore , this technique should not be recommended in patients less than 70 years old . the practitioner performing the denture installation must rely on clinical experience in the use of this technique . therefore it may be necessary to use another implant - prosthesis connection mode that is easier to manage in terms of retention force and monitoring , such as the locator root attachment system ( zest anchors , escondido , ca , usa ) . furthermore , as with any other type of implant , independent acute complications could appear , in particular , cases of primary implant loss or severe inflammatory reaction . some chronic disorders , such as peri - implantitis , can be observed but rarely due to the characteristic of the one - part implant , which decrease this specific risk . a synthesis of the advantages and disadvantages of the gsi procedure is proposed in table 1 . the protocol presented here allows the oral rehabilitation of vulnerable subjects , whose general health excludes overly invasive surgery . in addition , the cost of conventional implant treatment is often out of reach for elderly patients who have been edentulous for long periods . thus , although the mcgill consensus16,38 on the two implants setting remains the reference in terms of mandibular complete rehabilitation , the protocol developed with four gsis could be an interesting alternative for the care of very old and completely edentulous patients . however , the reliability of this procedure needs to be validated in a follow - up study . in parallel , assessment of the impact of such rehabilitation on masticatory and dietary behaviors and of its nutritional consequences | backgroundadvances made in prevention have helped postpone complete edentulism in older patients . however , in the elderly , the physiological state reduces patients ability to adapt to oral rehabilitation and degrades the patient s oral condition .
consequently , elderly edentulous subjects avoid many types of foods , which can lead to substantial nutritional consequences .
complete dentures retained by implants are , currently , the treatment of reference in prosthodontic mandibular rehabilitation .
indeed , the mandibular symphysis generally tolerates implantation , even when the mandible is strongly resorbed .
however , in the elderly , implant rehabilitation is compromised by the complexity of the surgical protocol and possible postoperative complications . in this context , the use of geriatric slim implants
( gsi ) offers an interesting alternative.methodsin the present study , the surgical and prosthetic procedures for the use of gsi in a french dental hospital are presented .
the objective was the stabilization of a complete mandibular denture in an elderly person , with the immediate implantation of four gsi.resultsthe operating procedure was found to be less invasive , less expensive , simpler , and more efficient than the conventional procedure.conclusionthe result strongly suggests that this protocol could be used systematically to treat complete edentulism in very elderly patients .
long - term monitoring and the evaluation of the reliability of this type of rehabilitation should be undertaken . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
non - alcoholic fatty liver disease ( nafld ) is a benign disease often associated with central obesity and insulin resistance and , in general , with factors of metabolic syndrome . nafld contains a spectrum of pathologic conditions , ranging from simple steatosis to nonalcoholic steatohepatitis and cirrhosis . the effect of nafld on the autonomic nervous system ( ans ) is still unknown . however , previous studies showed that both sympathetic and parasympathetic pathways are impaired , either singly or together . the autonomic nervous system plays a central role in regulating cardiovascular function , and cardiovascular autonomic nervous system dysfunction is related to significantly increased cardiovascular mortality . heart rate recovery ( hrr ) is an important measure of autonomic nervous system dysfunction and is directly correlated with parasympathetic activity . the value for the hrr index is defined as the reduction in the heart rate from the rate at peak exercise to the rate at the 1 , 2 , 3 , and 5 minutes after the discontinuation of an exercise stress test . the impairment of hrr provides useful clinical information about evaluating patients with nafld for higher risk of serious arrhythmias . previous studies investigated the effect of nafld on autonomic dysfunction but none have evaluated the heart rate recovery index . the aim of our study was to evaluate the heart rate recovery index in patients with nafld . the study population included 59 patients with nafld ( mean age=42.39.3 years ) and 22 healthy subjects as controls ( mean age=40.76.5 years ) . all patients were admitted to the gastroenterology clinic with nafld and the cardiology department for general health examination and were evaluated with detailed history and physical examination . we recorded age , sex , and body mass index , as well as biochemical measurements ( fasting blood glucose , total cholesterol , low - density lipoprotein cholesterol , high - density lipoprotein cholesterol , and triglyceride levels ) . demographic characteristics and clinical features of the patients and the controls are given in table 1 . in all subjects , we performed 12-lead electrocardiography ( ecg ) at 25 mm / sec ( paper speed ) , and we performed transthoracic echocardiography by means of a ge - vingmed vivid 7 system ( ge - vingmed ultrasound as , horten , norway ) using a 2.5-mhz transducer . the control subjects had no cardiovascular or any other organ system disease and presented with normal physical examination , chest roentgenogram , electrocardiogram , and 2-dimensional and doppler echocardiogram . patients were excluded from the study if they had < 10g / dl hemoglobin ; had diseases interfering with the autonomic nervous system , including diabetes mellitus , renal diseases , parkinson s disease , porphyria , and amyloidosis ; had cardiovascular diseases , including hypertension , ischemic heart disease , left ventricle ( lv ) ejection fraction lower than 50% , severe valvular regurgitation , moderate or severe valvular stenosis , cardiomyopathy , and cardiac arrhythmia ; or had neurological diseases and chronic obstructive pulmonary disease . this study complied with the declaration of helsinki and was approved by the local ethics committee . the predicted peak heart rate was calculated as ( 220 age ) , and the aim was to reach at least 85% of the age - predicted heart rates . the end of exercise was flagged , and at least 5 min of postexercise heart rate recorded with the subject at rest . qualified exercise physiologists and/or cardiology fellows prospectively collected physiologic and hemodynamic data during testing , including symptoms , heart rate , heart rhythm , blood pressure , and estimated functional capacity in metabolic equivalents ( mets ; where 1 met=3.5 ml / kg per min of oxygen consumption ) . heart rate recovery indices were defined as the reduction in heart rate from the rate at peak exercise to the rate at the 1 , 2 , 3 , and 5 minutes after the cessation of the exercise stress test , and these results were indicated as hrr1 , hrr2 , hrr3 and hrr5 , respectively . continuous variables are given as mean sd and categorical variables are given as a percentage . an independent - samples t test was used to compare the study variables between nafld patients and control subjects . a probability value of p<0.05 was considered significant , and 2-tailed p values were used for all statistics . all statistical analyses were carried out using statistical software ( spss , version 15.0 for windows ; spss , chicago , il ) . the study population included 59 patients with nafld ( mean age=42.39.3 years ) and 22 healthy subjects as controls ( mean age=40.76.5 years ) . all patients were admitted to the gastroenterology clinic with nafld and the cardiology department for general health examination and were evaluated with detailed history and physical examination . we recorded age , sex , and body mass index , as well as biochemical measurements ( fasting blood glucose , total cholesterol , low - density lipoprotein cholesterol , high - density lipoprotein cholesterol , and triglyceride levels ) . demographic characteristics and clinical features of the patients and the controls are given in table 1 . in all subjects , we performed 12-lead electrocardiography ( ecg ) at 25 mm / sec ( paper speed ) , and we performed transthoracic echocardiography by means of a ge - vingmed vivid 7 system ( ge - vingmed ultrasound as , horten , norway ) using a 2.5-mhz transducer . the control subjects had no cardiovascular or any other organ system disease and presented with normal physical examination , chest roentgenogram , electrocardiogram , and 2-dimensional and doppler echocardiogram . patients were excluded from the study if they had < 10g / dl hemoglobin ; had diseases interfering with the autonomic nervous system , including diabetes mellitus , renal diseases , parkinson s disease , porphyria , and amyloidosis ; had cardiovascular diseases , including hypertension , ischemic heart disease , left ventricle ( lv ) ejection fraction lower than 50% , severe valvular regurgitation , moderate or severe valvular stenosis , cardiomyopathy , and cardiac arrhythmia ; or had neurological diseases and chronic obstructive pulmonary disease . this study complied with the declaration of helsinki and was approved by the local ethics committee . the predicted peak heart rate was calculated as ( 220 age ) , and the aim was to reach at least 85% of the age - predicted heart rates . the end of exercise was flagged , and at least 5 min of postexercise heart rate recorded with the subject at rest . qualified exercise physiologists and/or cardiology fellows prospectively collected physiologic and hemodynamic data during testing , including symptoms , heart rate , heart rhythm , blood pressure , and estimated functional capacity in metabolic equivalents ( mets ; where 1 met=3.5 ml / kg per min of oxygen consumption ) . heart rate recovery indices were defined as the reduction in heart rate from the rate at peak exercise to the rate at the 1 , 2 , 3 , and 5 minutes after the cessation of the exercise stress test , and these results were indicated as hrr1 , hrr2 , hrr3 and hrr5 , respectively . continuous variables are given as mean sd and categorical variables are given as a percentage . an independent - samples t test was used to compare the study variables between nafld patients and control subjects . a probability value of p<0.05 was considered significant , and 2-tailed p values were used for all statistics . all statistical analyses were carried out using statistical software ( spss , version 15.0 for windows ; spss , chicago , il ) . the baseline characteristics of the study groups are shown in table 1 . according to the basic clinical and demographic characteristics , both groups were similar with respect to age , sex , and fasting glucose levels . all subjects were normotensives , and no significant differences were observed in systolic or diastolic blood pressures and resting heart rates between the 2 groups ( sbp ; 128.715 , 122.112.8 , p : 0.06 , dbp ; 75.68.6 , 73.75 , p : 0.11 ; hr ; 79.610.4 , 77.18.2 , p : 0.07 , respectively ) . all patients and healthy participants had sinus rhythm and normal 12-lead ecg results at rest . all completed the exercise stress test without rhythm abnormalities , ischemic changes , or other complications . comparisons of the baseline echocardiographic values among nafld patients and healthy controls are summarized in table 2 . comparisons of the maximal heart rate , maximal systolic and diastolic blood pressure , exercise duration , and metabolic equivalents attained during the exercise stress test values among nafld patients and healthy subjects are also summarized in table 2 . according to these results , the 1- and 2-minute hrr indices of patients with nafld were significantly lower than those of the healthy control group ( 19.98.2 vs. 34.19.6 ; p<0.001 and 24.35.4 vs. 40.59.1 ; p=0.006 , respectively ) . similarly , hrr indices after the 3 and 5 minutes of the recovery period were significantly lower in patients with nafld compared with those indices in the control group ( 32.38.5 vs. 58.46.5 ; p=0.001 and 5818.2 vs. 75.115.8 ; p<0.001 ) ( table 2 ) . however , we found that metabolic equivalents attained during the exercise stress test of patients with nafld were significantly lower than those of the healthy control group ( 111.9 vs. 12.51.5 mets ; p=0.001 ) ( table 2 ) . in this study we have demonstrated that heart rate recovery indices impaired in the 1 , 2 , 3 , and 5 minutes of the recovery period after maximal exercise testing in patients with non - alcoholic fatty liver disease compare to healthy control subjects . nafld might be considered as a manifestation of metabolic syndrome , and insulin resistance plays a role in both conditions . the components of metabolic syndrome perform sympathetic activation , and several studies have demonstrated the association between metabolic syndrome and decreased heart rate variability . the autonomic nervous system plays a central role in regulating cardiovascular function in both disease and health . the rise in heart rate during exercise is considered to be due to activation of the sympathetic nervous system and the simultaneous suppression of the parasympathetic nervous system . on the other hand , the fall in heart rate immediately after exercise is considered to be the parasympathetic reactivation together with sympathetic withdrawal . previous studies showed that heart rate recovery is a marker of autonomic system function and is directly associated with parasympathetic nervous system activity . another study found that an increment in vagal activation as well as a decrement in sympathetic activation was detected during the first minutes of the recovery soon after exercise cessation . showed that short- and mid - term hrr indices ( 30 s to 2 min ) are mediated primarily by vagal reactivation . demonstrated that parasympathetic effect on heart rate was defined by the difference in heart rate with and without atropine . these data showed that parasympathetic system effects continued during high - intensity exercise , and a large parasympathetic system effect on heart rate was noted at 1 min , increased at 4 min , and then stayed stable until 10 min in the recovery period . heart rate recovery has been studied in various diseases such as heart failure , coronary artery disease , diabetes mellitus , obstructive sleep apnea , systemic lupus erythematosus , and psoriasis . in the present study , we excluded subjects with coronary artery disease , neurologic diseases , prior cardiovascular event presence , and current smoking to avoid the effects of these variables on heart rate recovery index . several studies have reported that impaired heart rate recovery index after exercise is a powerful and independent predictor of cardiovascular and all - cause mortality . additionally , previous studies reported that a delayed decrease in heart rate during the 1 minute after exercise is a potent predictor of overall mortality , of the presence or absence of myocardial perfusion defects , and of changes in heart rate during exercise . jouven et al . reported that an hrr of less than 25 beats / min after the 1 min of the recovery period increased the relative risk for sudden cardiac death by 2.2-fold compared with the hrr group rate of more than 40 beats / min . various laboratory methods to assess autonomic nervous system function may help . however , they often require special equipment and training , and are not easily used in routine evaluation . our results show that nafld patients have decreased heart rate recovery index compared with healthy controls . hence , autonomic dysfunction might be a risk factor for cardiac death in patients with nafld , and heart rate recovery index analysis might be used for risk stratification . the heart rate recovery index is impaired in patients with non - alcoholic fatty liver disease . when the prognostic significance of the heart rate recovery index is considered , these results may explain the increased occurrence of cardiac death in patients with nafld . this study calls attention to the importance of the heart rate recovery index , which is simple to use and may be clinically useful in the identification of high - risk patients . the major limitations of the present study are that it is based on a single - center experience and may be limited by the small number of patients . however , our population contained a homogeneous mix of unselected patients with nafld , therefore mirroring the real - world situation . the major limitations of the present study are that it is based on a single - center experience and may be limited by the small number of patients . however , our population contained a homogeneous mix of unselected patients with nafld , therefore mirroring the real - world situation . | backgroundnon - alcoholic fatty liver disease ( nafld ) has been considered as a benign disease often associated with central obesity and insulin resistance and , in general , with factors of the metabolic syndrome .
heart rate recovery after exercise is a function of vagal reactivation , and its impairment is an independent prognostic indicator for cardiovascular and all - cause mortality .
the aim of our study was to evaluate the heart rate recovery index in patients with nafld.material/methodsthe study population included 59 patients with nafld ( mean age=42.39.3 years ) and 22 healthy subjects as controls ( mean age=40.76.5 years ) .
basal electrocardiography , echocardiography , and treadmill exercise testing were performed on all patients and controls .
the heart rate recovery index was defined as the reduction in the heart rate from the rate at peak exercise to the rate at the 1st minute ( hrr1 ) , 2nd minute ( hrr2 ) , 3rd minute ( hrr3 ) , and 5th minute ( hrr5 ) after stopping exercise stress testing.resultsthere were significant differences in hrr1 and hrr2 indices between patients with ed and the control group ( 19.98.2 vs. 34.19.6 ; p<0.001 and 24.35.4 vs. 40.59.1 ; p=0.006 , respectively ) .
similarly , hrr indices after the 3rd and 5th minutes of the recovery period were significantly lower in patients with nafld compared with those indices in the control group ( 32.38.5 vs. 58.46.5 ; p=0.001 and 5818.2 vs. 75.115.8 ; p<0.001 ) .
effort capacity was markedly lower ( 111.9 vs. 12.51.5 mets ; p=0.001 ) among the patients with nafld.conclusionsthe heart rate recovery index is deteriorated in patients with nafld .
when the prognostic significance of the heart rate recovery index is considered , these results may help explain the increased occurrence of cardiac death .
it points to the importance of the heart rate recovery index in the identification of high - risk patients . |
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activation of the immune system , through either infection or administration of cytokines , causes significant changes in eating , drinking , social , and sleeping behaviors in both rodents ( hart , 1987 ; oreilly et al . , 1988 ; crestani et al . , 1991 ; yirmiya , 1996 ) and humans ( capuron et al . , 2002 ; vollmer - conna et al . , 2004 ) . for example , depressed individuals have disturbed sleep patterns ( coble et al . , 1979 ; pigeon et al . , 2004 ; armitage , 2007 ) , greater fatigue ( demyttenaere , 2005 ) , fewer social interactions ( george et al . , 1989 ) , and anhedonia ( pizzagalli et al . , 2007 ; sherdell et al . , 2012 ) . indeed many of these behavioral alterations are diagnostic criteria for major depression as outlined by the dsm . initially depression was thought to be associated with a suppression in immunity ( schleifer et al . , 1984 ) . investigators came to this conclusion after finding that blood lymphocytes of depressed individuals had an attenuated proliferative response when stimulated with mitogens ( schleifer et al . , 1984 ; kronfol et al . , 1986 ; kronfol and house , 1989 ) . this was associated with reduced natural killer cell activity ( nerozzi et al . , 1989 ) . in addition , depressed patients were known to have elevated glucocorticoids , specifically cortisol ( carroll et al . , 1976 ) , and a dysfunctional stress feedback system ( carroll et al . , 1968 ) . since glucocorticoids were well known to dampen immune responses ( crabtree et al . , 1979 ) , the immunosuppression found in depressed patients seemed logical and corresponded nicely with the endocrine abnormalities . contrary to the suggestion that depression was immunosuppressive , smith ( 1991 ) proposed the macrophage theory of depression that drew on research demonstrating interleukin ( il)-1 can lead to endocrine abnormalities and significantly alter behavior . in addition , inflammation was seen as a common link between depression and other diseases that were often comorbid with depression . in short , smith s theory proposed that in depressed patients activated macrophages produced cytokines , which lead to depression ( smith , 1991 ) . soon , evidence began accumulating that depressed patients were actually showing patterns of an activated inflammatory response . depressed patients were reported to have an increase in leukocytes , monocytes , and other inflammatory factors , including prostaglandins ( ohishi et al . , 1988 ; maes et al . , 1992 ) and increased nk cells ( seidel et al . , 1996 ) . maes ( 1995 ) followed up with his own studies on inflammation and depression and described several ways that inflammation could influence depression , including decreased bioavailability of tryptophan for the synthesis of serotonin . during the same time , animal studies were documenting the relationship between inflammation and sickness behavior ( bluthe et al . , 1991 , 1992a , 1992b , 1994 , 1995 ; kent et al . , 1992 ; nadjar et al . , in addition to reducing motor activity and food intake and increasing slow wave sleep , the cytokine inducers lipopolysaccharide ( lps ) ( bluthe et al . , 1992a ) , il-1 ( kent et al . , 1992 ) , and tumor necrosis factor ( tnf ) ( bluthe et al . , 1991 , 1994 ) were found to decrease social interaction in rodents . importantly , these effects were obtained whether lps or cytokines were administered at the periphery or in the brain , indicating a possible central site of action for peripheral cytokines . yirmiya ( 1996 ) first reported that endotoxin caused depressive - like behaviors in rodents that were sensitive to the effects of antidepressant drugs . since the macrophage theory was proposed , the last 2 decades have seen an abundant amount of investigation into the relationship between inflammation and depression at both the clinical and preclinical levels . patients with depression are now reported to have elevated levels of inflammatory markers , including proinflammatory cytokines ( kim et al . , 2008 ; shelton and claiborne , 2010 ) , c - reactive protein ( danese et al . , 2008 ; vogelzangs et al . , 2012 ; morris et al . , 2014 ) , and myeloperoxidase ( vaccarino et al . , 2008 although some studies have reported negative results ( carpenter et al . , 2004 ; basterzi et al . , 2005 ) , several meta analyses support the association between depression and proinflammatory cytokines ( howren et al . furthermore , the literature indicates that only specific subtypes of depression are associated with inflammation . for example , patients with atypical depression have an increase in plasma c - reactive protein ( hickman et al . , 2014 ) , proinflammatory cytokines ( lamers et al . , 2013 ) , and leukocyte numbers ( rothermundt et al . , 2001 ) compared with healthy controls or patients with melancholic depression , although one report supports both melancholic and atypical patients having elevated biomarkers of inflammation ( karlovic et al . , 2012 ) . interestingly , atypical depression is also characterized by symptoms of fatigue , hypersominia , and lethargy ( gold and chorousos , 2002 ) , which match the known behavioral effects of cytokines . although not all traits of atypical depression align well with sickness behaviors , including weight gain and hypoactivity of the hypothalamic pituitary adrenal system , it is possible that these are due to mechanisms other than inflammation . rodent models of depression are also associated with elevated levels of inflammation in the periphery and brain ( grippo et al . , 2005 ; goshen et al . , 2008 ; you et al . , 2011 ) . ( 2008 ) have demonstrated that cytokine signaling is essential for the development of depressive - like behaviors in stress - based animal models of depression . further animal research has demonstrated that antidepressants may have antiinflammatory effects ( tynan et al . , 2012 ) , and antiinflammatory drugs can prevent depressive - like behaviors ( kreisel et al . , 2014 ) . while a complete review of this literature is too large to be completed here , comprehensive reviews of the research on inflammation and depression can be found elsewhere ( felger and lotrich , 2013 ; furtado and katzman , 2015 ; lotrich , 2015 ; yirmiya et al . , 2015 ) . importantly , a direct effect of immune stimulation on mood has been demonstrated in a clinical population . capuron and ravaud ( 1999 ) report that cancer patients treated with interferon - alpha and/or il-2 developed depressive - like symptoms , and this has been replicated by others ( bonaccorso et al . , 2002 ; kraus et al . in addition , it has been highlighted that patients afflicted with disorders associated with inflammation , including diabetes , multiple sclerosis , and cardiac disease , show higher rates of depression . physically ill patients with chronic inflammation have an improvement in mood when given treatments that target inflammatory cytokines , such as the tnf - antagonist infliximab ( tyring et al . , 2006 ; feldman et al . , 2008 ) . for example , rodents administered proinflammatory cytokines show depressive - like behaviors , such as increased immobility in the forced swim and tail suspension tests and reduced sucrose preference ( brebner et al . , 1999 ; makino et al . , 2000 ; dunn and swiergiel , 2005 ; wu and lin , 2008 ) . our research group has focused on understanding inflammation - induced depression and the downstream mechanisms . we have taken great care in developing an animal model of depression that is based on the clinical literature demonstrating that depression develops on a background of sickness behavior ( dantzer et al . , 2008 ) . this review will discuss the current state of research on inflammation - induced depression , what is known about the mechanisms , and possible novel targets for the treatment of inflammation - induced depression . inflammation - induced depression can be studied using a variety of inflammatory agents , including lps , the viral mimetic poly i : c , and bacillus calmette - guerin ( bcg ) . treated animals lose weight , eat and drink less , and decrease their motor activity for several hours to days , depending on the nature of the inflammatory agent and dose . these sickness behaviors correspond with elevations in proinflammatory cytokines at the periphery and in the brain ( laye et al . , 2000 ; in the lps - induced model of depression , sickness behaviors will typically resolve within 24 hours , whereas bcg inoculation will result in sickness behavior lasting several days ( oconnor et al . interestingly , when sickness behaviors have resolved , the rodents display depressive - like behaviors ( figure 1 for schematic ) . for example , 24 hours after injection of lps , when motor activity is back to normal and appetite present , treated animals show increased immobility in the forced swim test and tail suspension test as well as decreased sucrose preference ( frenois et al . , 2007 ; oconnor et al . , 2009c , sulakhiya et al . , 2016 ; ge et al . , 2015 ) sickness and depressive behaviors depend on the initial inflammation , as antiinflammatory agents can attenuate both ( bluthe et al . interferon - gamma is a crucial component in this model , as transgenic animals with deletion of its receptor do not show depressive - like behaviors ( oconnor et al . , 2009b ) . likewise , many laboratories have continued to demonstrate that a variety of antiinflammatory compounds abrogate or attenuate depressive - like behaviors using this model ( ferreira mello et al . , 2013 ; ji et al . , 2014 ; an et al . , 2015 ; ge et al . , 2015 ; li et al in addition , this model has been modified in a variety of ways , including altering inflammatory agents ( fischer et al . , 2015 ) , initiating chronic inflammation ( kubera et al . , 2013 ; lps administration increases peripheral cytokines and leads to sickness behaviors , including lack of movement and decrease in food and water intake . around 24 hours postinjection , rodents begin eating and moving similarly to control animals as their sickness behaviors have resolved . proinflammatory cytokines will cause an elevation of indoleamine 2,3 dioxygenase ( ido ) 24 hours after lps injection . during the same time that ido is elevated , animals will be anhedonic and spend more time immobile in the forced swim test . while the primary focus of this review is depressive behaviors , there is a growing literature demonstrating inflammation - induced increases in anxiety - like behaviors in rodents ( salazar et al . , 2012 ; bassi et al . , 2012 ; gibney et al . , 2013 ; baganz et al . , 2015 ; sulakhaiya ( 2016 ) found that lps - treated animals have an increase in anxiety - like behavior , as measured by the elevated plus maze . here , the lps - treated animals spent more time in the closed arms and a reduction in time spent in the open arms . however , the animals were tested only a few hours after lps administration , probably still at the peak of the sickness response . testing behavioral measures of anxiety shortly after lps injection may confound the results , as the rodents will most likely be moving less due to sickness . however , some papers have demonstrated anxiety behaviors after sickness behaviors have resolved ( salazar et al . , 2012 ; gibney et al . , 2013 ) . anxiety and depression often appear together in the clinical population ( brown et al . , 2001 ) , and modeling both of these symptoms together can both be beneficial and problematic . on one hand , the presence of both anxiety and depression could reflect a more relevant model of depression , but it is difficult to distinguish the contribution of anxiety to depressive behaviors and vice versa . healthy human subjects given an inflammatory agent , such as endotoxin or typhoid vaccination , show a significant reduction in mood and increased anxiety ( reichenberg et al . , 2001 ; wright et al . , 2005 ; eisenberger et al . , 2009 , 2010 ) the reduction in mood is correlated with plasma il-6 levels ( brydon et al . , 2008 ; eisenberger et al . , interestingly , imaging studies found that typhoid vaccination alters neural activity of the cingulate cortex and its connections with other brain areas associated with mood ( harrison et al . , 2009 ) . ( 2010 ) found that endotoxin treatment decreases ventral striatum responding to reward , further supporting that inflammation can alter critical neural circuits involved in depression . insights about mechanisms downstream of inflammation have come from clinical studies that examined the effects of interferon - alpha on mood of cancer and hepatitis c virus - infected patients . in both groups , the development of depressive symptoms was associated with decreased circulating levels of tryptophan ( bonaccorso et al . , 2002 ; capuron et al . , furthermore , a significant correlation between variations in tryptophan levels and changes in depression scores was reported . specifically , the greater the fall in tryptophan levels , the more severe the symptoms of depression as measured by the montgomery and asberg depression rating scale ( capuron et al . , 2002 ) . the relationship between tryptophan , serotonin , and depression has a long history in psychiatry . tryptophan is the precursor of serotonin , and because tryptophan hydroxylase is not saturated by its substrate , the bioavailability of tryptophan regulates the amount of serotonin formed in the brain . rodents fed a low tryptophan - containing diet show a decrease in serotonin in their brain ( biggio et al . , 1974 ) . similarly , human subjects on a tryptophan - restricted diet have decreased levels of tryptophan and serotonin metabolites in their cerebrospinal fluid ( perez - cruet et al . , 1974 ) . depressed patients have high levels of tryptophan metabolites in their urine ( curzon and bridges , 1970 ) , and a decrease in tryptophan in the blood and cerebrospinal fluid ( maes et al . , 1987 , 1993 ) . the depletion of tryptophan alone is also associated with a depressed mood ( young et al . , 1985 ) . however , this is only apparent in subjects who are at risk of being depressed either because of a family history of depression or because they are in a remission phase ( riedel et al . , 2002 ) . lastly , tryptophan administration in conjunction with an mao - inhibitor potentiates the antidepressant drug effect ( coppen et al . , 1963 ) , which suggests that tryptophan depletion may be a critical biological component of depression . tryptophan is mainly metabolized via the kynurenine pathway ( figure 2 ) , beginning with conversion of tryptophan to kynurenine by 1 of 2 enzymes , primarily tryptophan 2,3 , dioxygenase or indoleamine 2,3 , dioxygenase ( ido ) . these enzymes are regionally divided in the periphery but are both found in the brain ( saito et al . , 1991 ; haber et al . , 1993 ; alberati - giani et al . , tryptophan 2,3 , dioxygenase predominately metabolizes tryptophan in the liver ( nakamura et al . , 1980 ) and responds to hormonal regulations , such as cortisol and glucagon ( nagao et al . , 1986 ; nakamuras et al . , ido activity is induced by the cytokines interferon - gamma and tnf - alpha ( yoshida et al . , 1981 ; yoshida and hayaishi , 1987 ) . lps administration , hiv infection , or interferon treatment increases metabolites of kynurenine in the periphery , brain ( heyes , 1988 ; saito et al . , 1991 ) , and csf ( heyes et al . , 1989 ) . the first demonstration of a role for ido activation was in the field of reproductive immunology . pregnancy is associated with a significant decrease in tryptophan in the plasma ( schrcksnadel et al . , 1996 ) , which is secondary to activation of ido at the level of the trophoblast . cytotoxic cells that are involved in the recognition of the trophoblast as antigenic become anergic because of the local depletion of tryptophan ( munn et al . , 1998 ) . therefore , it is possible that an increase in ido following inflammation could deplete tryptophan , resulting in a decreased serotonin concentration in the brain , and cause depressive - like behaviors . tryptophan is most commonly metabolized into kynurenine via one of two enzymes : indoleamine 2,3 dioxygenase ( ido ) or tryptophan 2,3 , dioxygenase . kynurenine can be further broken down into a variety of metabolites that are neuroactive , including 3-hydroxykynurenine ( 3hk ) , quinolinic acid , and kynurenic acid . the conversion from 3-hydroxyanthranillic acid to quinolinic acid , represented by a dashed line , requires 2 reactions . , we used the lps and bcg model of depression to show that activation of ido is a late event , which is in agreement with the late development of depressive - like behavior . more specifically , we observed that lps increased ido in the brain 24 hours after injection ( lestage et al . , 2002 ) at which time depressive - like behavior this increase in enzymatic activity of ido was associated with an increase in the ratio of kynurenine to tryptophan in the periphery and brain 24 hours post lps ( oconnor et al . other inflammatory agents , including poly i : c and interferon alpha , cause similar behavioral and biochemical results ( gibney et al . , 2013 ; fischer et al . , 2015 ) . rats injected with poly i : c have elevated ido and ratio of kynurenine to tryptophan in the brain ( gibney et al . , 2013 ) . similarly , rats administered recombinant human interferon alpha displayed increased immobility in the forced swim test ( fischer et al . , 2015 ) . however , their brain ratio of kynurenine to tryptophan was increased but not significantly ( p<.10 ) . imipramine blocked the behavioral effects of interferon but had no effect on the kynurenine to tryptophan ratio . these results are difficult to interpret for several reasons , including the species - specific activity of human interferon alpha . earlier studies on the behavioral effects of human interferon alpha showed that these are due to activation of brain opioids ( e.g. , dafny et al . , 1988 ) . secondly , rats are not a good species for studying ido activation , because the large quantities of nitric oxide they produce inhibit ido ( thomas et al . , 1994 ) . to determine if ido was important , we used pharmacological and genetic tools to demonstrate that activation of this enzyme was necessary for the development of depressive - like behavior , but not sickness behavior . in particular , we found that the ido inhibitor , 1-methyl tryptophan , blocked the depressive - like behavior but did not significantly alter inflammation or sickness behaviors ( oconnor et al . , 2009c ) . in a similar manner , deletion of the gene coding for ido , ido1 , abrogated the development of bcg - induced depression - like behavior ( oconnor et al . , 2009a ) . after demonstrating that ido is necessary for the development of depression in our model , the next logical question was whether serotonin depletion was ultimately responsible for the alteration in behavior . however , there was no evidence of a decrease in serotonin levels or serotonin turnover in the brain in response to inflammatory mediators . lps resulted in an increase , not a decrease , in serotonin turnover ( oconnor et al . , 2009c ) , and similar results have been obtained in models using poly i : c in rats ( gibney et al . , 2013 ) . the effect of lps on serotonin levels has not been universal , with some papers reporting a decrease in serotonin following lps at similar time points ( ji et al . , 2014 ; yeh et al . , ( 2009c ) examined whole brain , whereas others report differences in specific regions , including the nucleus accumbens ( yeh et al . it is also possible that different strains of mice may produce different results , as strains vary in serotonin levels at baseline and in response to stress ( shanks et al . , 1991 ) . the majority of kynurenine found in the brain during inflammation comes from the periphery ( gl and sherman , 1980 ; kita et al . , 2002 ) . it is transported into the brain via the l - type amino acid transporter , also known as solute carrier family 7 . once within the brain , kynurenine produces a variety of neuroactive metabolites , including quinolinic acid and kynurenic acid . both quinolinic acid and kynurenic acid act at the level of the nmda receptors , kynurenic acid is an antagonist of the nmda receptors ( but evidence also suggest it binds to acetylcholine nicotinic receptors ) and decreases glutamate release in the brain ( birch et al . , 1988 ) . quinolinic acid is an agonist of the nmda receptor ( stone and perkins , 1981 ) , which can cause excitotoxicity and significant damage to neurons ( foster et al . , 1983 ; schwarcz et al . , 1983 ; these 2 metabolites are produced in different cell types in the brain , with quinolinic acid primarily produced in microglia in a kynurenine monooxygenase ( kmo)-dependent pathway ( espey et al . , 1997 ) and kynurenic acid produced in astrocytes in a kynurenine aminostransferase - dependent pathway ( guidetti et al . , 2007 ) . with our previous data demonstrating that serotonin was not depleted in our model , we hypothesized that the elevations in kynurenine metabolites could contribute to depressive - like behaviors . indeed , lps administration caused an elevation in quinolinic acid but not kynurenic acid in the brain as well as other kmo - dependent kynurenine metabolites ( walker et al . blockade of quinolinic acid access to nmda receptors by ketamine was effective at eliminating depressive - like behaviors in lps - induced depression , but did not alter sickness behaviors or inflammation due to lps ( walker et al . , 2013 ) . ketamine did not alter ido activity itself either , which suggests depressive behaviors are due to the overactivation of the nmda receptor , which could be modulated by quinolinic acid . walker et al . ( 2013 ) also reported elevations in another potentially harmful metabolite , 3-hydroxykynurenine ( 3hk ) . 3hk is known to be neurotoxic ( okuda et al . , 1996 ) , causes mitochondrial dysfunction ( reyes - ocampo et al . , 2015 ) , and elevates reactive oxygen species ( shoki et al . while 3hk itself may alter behavior through neurotoxic effects , it can cause substantially more damage in the presence of quinolinic acid ( guidetti and schwarcz , 1999 ) . so it is possible that both of these metabolites work in concert to alter neuronal functioning and change behavior . for example , the ratio of kynurenic acid to quinolinic acid was significantly reduced in depressed patients , and this ratio was significantly correlated with both anhedonia ( savitz et al . , 2015b ) and volume reductions in mood - related brain regions ( savitz et al . , 2015a ) . in addition , patients who attempted suicide have elevated quinolinic acid levels in the csf compared with healthy controls ( bay - richter et al . , 2014 ) . in humans with traumatic brain injury , there is an increase in ido and quinolinic acid in the csf and brains of patients who had a worse recovery ( yan et al . , 2015 ) . adding further evidence to a role for ido and quinolinic acid in depression , the metabolites of kynurenine are elevated in depressed patients in the csf without any corresponding change in csf tryptophan ( raison et al . , 2010 ) . other papers have failed to show alterations in kynurenine or its metabolites in the plasma of depressed patients ( dahl et al . , 2015 ; meier et al . , 2015 ) , and one reported a decrease in plasma kynurenine levels ( hennings et al . , 2013 ) . while meier et al . ( 2015 ) did not see changes in kynurenine , they did report that depressed patients have altered kynurenic acid / quinolinic acid and kynurenic acid/3hk ratios . upon examination of their data , they report that differences in males were driving the significant effects , suggesting that sex may be a contributing factor . another possible explanation for the negative findings is that only a subpopulation of patients may show elevated kynurenine levels . this corresponds well with the data reporting only a portion of depressed individuals have elevated proinflammatory cytokines ( as discussed above ) . likewise , plasma kynurenine levels were increased in individuals who attempted suicide compared with healthy controls and other patients with depression ( who had not attempted suicide ) , so severity of depression may be relevant ( sublette et al . , 2011 ) . it is important to note that kynurenine data need to be interpreted with caution , since they do not necessarily reflect ongoing low - grade inflammation . cortisol - driven activation of the liver enzyme tryptophan 2,3 dioxygenase can also lead to marked elevations in circulating kynurenine . the best way to distinguish inflammation - driven elevations of kynurenine from other causes would be to measure the levels of neopterin , or other markers of immune activation , which is never done ( wildner et al . , 2002 ) . it is also important to mention that mechanisms other than the kynurenine metabolites are being investigated in the inflammation - induced depression model , including brain derived neurotrophic factor ( zhang et al . , 2014 ) , leptin ( kurosawa et al . , 2015 ) , glucocorticoids ( adzic et al . , 2015 ) , and alterations in neurotransmitters , such as adrenaline / noradrenaline ( sekio and seki et al . , 2014 ; zhu et al . , inflammation is at the origin of the biological cascade that causes depressive behaviors , but it may not always be possible or reasonable to treat inflammation . by exploring downstream mechanisms , it is possible to uncover novel targets for antidepressant therapies ( figure 3 ) . so far , a number of possible targets have been uncovered , including kynurenine metabolites and enzymes , blood - brain barrier transport mechanisms , and glutamatergic neurotransmission . note all of these targets are working under the assumption that inflammation - induced depression is due to kynurenine metabolites , specifically quinolinic acid , that act on glutamatergic neurons in the brain . this figure demonstrates several possible therapeutic targets ( green circles with red font ) in our lps - induced model of depression . one potential antidepressant route would be to block the production of kynurenine by administering inhibitors of indoleamine 2,3 dioxygenase ( ido ) , such as 1-methyl tryptophan . downstream kynurenine metabolites are damaging to neurons , and therapies that inhibit their production would also be effective . for example , kynurenine 3-monoxygenase ( kmo ) inhibitors may be used to decrease 3-hydroxykynurenine ( 3-hk ) or quinolinic acid production . as the majority of kynurenine in the brain is transported from the periphery , a reasonable intervention would be to prevent the transport of kynurenine . tryptophan and kynurenine are both transported into the brain by l - type amino acid transporter . l - type amino acid transporter has a high affinity for leucine , and leucine administration could result in a decrease in kynurenine transport and thereby reduce downstream kynurenine metabolites in the brain . lastly , upon activation microglia can release glutamate and inhibit the uptake of glutamate by astrocytes that can cause excessive glutamatergic activity . administration of inhibitors to prevent the release of glutamate from microglia would reduce glutamate receptor activation and possibly prevent depressive - like behaviors . likewise , lps and quinolinic acid can interfere with glutamate uptake in astrocytes . the use of riluzole has been demonstrated to increase astrocyte uptake of glutamate , and block glutamate release . glt-1 , glutamate transporter ; tlr , toll - like receptor . directly targeting kynurenine production and therefore decreasing the most direct method would be to prevent kynurenine accumulation by blocking ido activity with an inhibitor of the enzyme . currently , 3 ido inhibitors , d1-mt , incb024360 , and gdc-0919 ( formerly nlg-919 ) , are being tested in patients with solid tumors to suppress immunotolerance of the tumor and enhance response to cancer therapy ( table 1 ) . the specificity of action on ido1 varies between the drugs ( li et al . , incb024360 has high specificity for ido1 ( li et al . , 2010 ) , while d-1mt has broader effects ( for review , see lob et al . , 2009 and moon et al . , 2015 . these clinical trials will give insight into the feasibility of administering ido inhibitors in the long term , which , if successful , could be considered as possible therapeutics in specific populations of depressed patients . kmo catalyzes kynurenine into 3-hk and can be used to create quinolinic acid in microglia cells . recently , kmo inhibitors have been proposed as potential therapeutic targets for huntington s disease ( toledo - sherman et al . , 2015 ) , and it may be worthwhile to explore these molecules in inflammation - induced depression . kynurenine can alternatively produce kynurenic acid if it is metabolized by kats instead of kmo , and because kynurenic acid has opposing roles of quinolinic acid , it could counteract its effects . indeed , studies have demonstrated that administering nicotinylanaline will increase kynurenic acid and have a protective effect on neurons ( russi et al . , there is already evidence of antidepressant effect of exercise in rodents ( duman et al . , 2008 ; marais et al . overexpression of peroxisome proliferator - activated receptor- coactivator 1 , a protein elevated in skeletal muscle after exercise , made mice resistant to stress - induced depression by enhancing the enzymatic activity of kynurenine amino transferases and increasing production of the neuroprotective kynurenine metabolite kynurenic acid ( agudelo et al . clinical trials with drugs targeting the kynurenine pathway abbreviations : ido , indoleamine 2,3 dioxygenase ; 1-mt , 1-methyl tryptophan . this transport is mediated through l - type amino acid transporter ( fukui et al . , 1991 ) . tryptophan , kynurenine , and other large neutral amino acids , such as leucine , compete at the transporter for entry into the brain . recent data from our laboratory indicate that it is possible to block depressive - like behaviors following lps injection with the amino acid l - leucine . it is hypothesized that leucine would outcompete kynurenine for transport into the brain and thus decrease central kynurenine levels . this does seem to be the case , as our preliminary findings demonstrate that there is a significant reduction in the ratio of kynurenine to tryptophan of the brain in leucine - treated rodents who were administered lps ( walker et al . , 2015 ) . these data indicate that blood - brain transport mechanisms are a viable target for antidepressant treatment . excessive levels of glutamate are excitotoxic to neurons ( lucas and newhouse , 1957 ) , and glutamate dysfunction is a possible cause of depression ( for review , see paul and skolnick , 2003 ; sanacora et al . , 2012 ) . in both the lps - induced and stress - based models of depression , ketamine , an nmda antagonist , can block depressive - like behaviors ( li et al . targeting glutamate activity can be done by enhancing glutamate reuptake or decreasing glutamate release by facilitating gaba or other molecules that can decrease glutamatergic activity . astrocytes play an important role in the uptake of glutamate , which helps limit and prevent damage due to excitotoxicity . astrocytes uptake glutamate through transporters and convert glutamate into glutamine , which can then be released back into the extracellular space and picked up by neurons . microglia can also take up glutamate from the environment , but , importantly , upon immune activation microglia will release glutamate , which may contribute to excitotoxicity ( takaki et al . , 2012 ; thomas et al . , 2014 ) a variety of factors , including lps or tnf , can increase glutamate release from microglia ( thomas et al . , 2014 ) , and glutamate exits through two systems in microglia . the first is through the cell adhesion hemichannel ( takeuchi et al . , 2006 ) , and the other is system xc , the glutamate / cysteine antiporter ( piani and fontana , 1994 ; kigerl et al . , 2012 ) . in addition , microglial release of glutamate decreases astrocytes ability to uptake glutamate , which results in greater neuronal damage ( takaki et al . , 2012 ) . in addition , lps alone can cause glutamate efflux from astrocytes through the release of atp ( pascual et al . , , quinolinic acid can prevent the uptake of glutamate by astrocytes through a decrease in the glutamate transporter ( tavares et al . , 2002 ) . there is an indication that both the release of glutamate from microglia and the modulation of astrocytes due to lps can be reversed when treated with antagonists of 1 of the 2 transporters that microglia use to export glutamate ( domercq et al . , 2007 2012 ) , and decreases glutamate release from neurons ( wang et al . , 2004 ) . riluzole has antidepressant effects as determined by the forced swim test ( gourley et al . , 2012 ) and can reverse depression due to chronic stress in rodents ( banasr et al . , 2010 ) . indeed , riluzole has been tested humans with depression with promising results ( zarate et al . , 2005 ; , 2012 ; brennan et al . , 2010 ) . this drug could be worth investigating in inflammation - induced depression . overall , targeting the glutamate transport systems within microglia and astrocytes could be another viable method to eliminate depressive - like behaviors . depression is a debilitating and recurring disorder that is estimated to affect nearly 20% of the population . the most common antidepressant therapies are oral medications that show variable response rates and take weeks to improve the moods of patients . while antiinflammatory agents have been tested as antidepressants , with some success in special populations of patients , targeting a more specific mechanism could have a greater response rate and be applicable to a wider range of patients . the inflammation - induced model of depression has uncovered the tryptophan - kynurenine pathway as critical for depression , and it has provided a variety of new targets for antidepressant therapies . with further analysis of these downstream pathways , it is possible to also discover mechanisms that are more broadly applicable to other models of depression . | inflammation and depression are closely inter - related ; inflammation induces symptoms of depression and , conversely , depressed mood and stress favor an inflammatory phenotype .
the mechanisms that mediate the ability of inflammation to induce symptoms of depression are intensively studied at the preclinical level .
this review discusses how it has been possible to build animal models of inflammation - induced depression based on clinical data and to explore critical mechanisms downstream of inflammation .
namely , we focus on the ability of inflammation to increase the activity of the tryptophan - degrading enzyme , indoleamine 2,3 dioxygenase , which leads to the production of kynurenine and downstream neuroactive metabolites . by acting on glutamatergic neurotransmission ,
these neuroactive metabolites play a key role in the development of depression - like behaviors .
an important outcome of the preclinical research on inflammation - induced depression is the identification of potential novel targets for antidepressant treatments , which include targeting the kynurenine system and production of downstream metabolites , altering transport of kynurenine into the brain , and modulating glutamatergic transmission . |
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the protective and tooth resisting modalities against dental decay are incorporating fluoride ions in developing hydroxyappatite lattice and incipient caries . it has been known as a major reason of remarkable decline in dental caries incidence and prevalence in developed countries.1 in addition , the inherent properties of fluoride toothpaste , biological and behavioral factors , are the reasons of anticaries efficacy of fluoride products . in a systematic review of seventy trials , the average of reduction in dental caries attributed to fluoride dentifrices was twenty percent1 . the daily tooth brushing frequencies , length of brushing time and availability of fluoride ions content are effective dependent factors . degree of enamel fluorosis during permanent teeth development and application of dentifrices with high concentration of fluoride are important factors.24 systematic reduction of fluoride content in children 's dentifrices and supervised application of pea sized toothpastes are the practical and reasonable strategies in decreasing dental fluorosis.4 recently fluoridated dentifrices and mouth rinse products are sold over - the - counter without formal prescription . it is ordinarily recommended not to use more than 10 mg / kg fluoride to avoid undesirable degrees of dental fluorosis.56 however , it has been proven in vitro that at least 500 ppm fluoride content is essentially needed to protect the enamel of permanent teeth against demineralization ( artificial lesion).78 the exact mechanisms of preventive effects of locally used fluoride products are debated but , arresting the progress of dental caries , diminishing the organic acid attacks , decreasing free charge enamel surfaces , changing the bacterial flora of biofilm , modifying surface hardness , and accentuation of remineralization dynamic are hypothesized and challenged.9 it is confirmed that fluoride ions are essentially contributed to de / remineralization processes in white spot lesions and artificial caries . conclusive evidence in literature has shown the efficacy of fluoride dentifrices in primary dentition . it seems that fluoride ions do not pass through the placenta , however , the efficacy of fluoride protection during pregnancy is dismissible . deciduous teeth exhibit a different behavior in de - remineralization processes in comparison with permanent teeth . surface microhardness of enamel is a physical property that permits the researcher to assess the effects of chemical and physical agents on enamel , dentin , and cementum.911 ph - cycling systems evaluate caries lesion and mineral changes in dental hard tissues . according to ten cate and duijsters8 , the best way to simulate the environment in vitro is to apply ph - cycling models that is equivalent to dynamics dental decay . the purpose of surface microhardness evaluation was supported by the finding of a strong correlation between remineralization measured by this technique and radiographic techniques8 . featherstone5 reported a good correlation between enamel microhardness and mineral loss in caries lesions . over the counter children 's toothpastes are not so common . generally , all toothpastes have monofluoride phosphate ( mfp ) , sodium fluoride ( naf ) as anticaries and silica as abrasive agents.12 the aim of this study was the evaluation of changes of surface microhardness following ph - cycling of crest ( adult type ) ( naf , 1100 ppm f ) ( standard ) , crest ( children type ) ( naf , 500 ppm f ) ( positive control ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride ( negative control ) . fifty six blocks of coronal portion of extracted primary incisors without dental caries , hypoplasia , crack , and malformation were collected . the crowns were mounted in cylindrical plastic tubes with epoxy resin stick exposing 2 4 mm of enamel surface . preparation of enamel surface was done by boehler abrasive instrument ( buehler , chicago , usa ) , with 600 - 1200 grits al2o3 paper and water . the smooth surfaces were examined under light microscope ( siemens , germany ) using x 100 objective lens in order to evaluate glassy and transparent appearance of enamel surfaces . the surface microhardness was measured by buehler microhardness machine ( buehler , chicago , usa ) . the force action of silver diamond was projected to enamel surface , 100 g/20 in three points distances of 500 , 1000 , 1500 micrometers . the amount of surface microhardness was measured with vickers index hardness unit.1315 through this equation , vickers hardness number ( vhn ) = f1.85/d where f= kg / m and d = mean diameters . in this step , 56 blocks with surface microhardness of 311 - 330 vhn were selected . the remieralizing and demieralizing solutions and four dentifrices suspensions were freshly provided in biochemical laboratory of school of pharmacy and pharmaceutical sciences , isfahan university of medical sciences , isfahan , iran . the demineralizing solution contained 2.2 mm nahpo4 , 1.5 m cacl2 , 0.5 m kcl with ph = 71621 and reminerlizing solution contained 0.9 mm nahpo4 , 1.5 cacl2 , 0.m kcl . the suspensions of crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride were prepared by diluting them with distilled water to 1/3 . ph - cycling and remineralization process were done in 8 days with cyclic pattern . in each ph - cycle , blocks were immersed and shook in 20 ml of each solution as follow:16
1)at the beginning of each cycle , in each group 20ml of one of dentifrices , after homogenizing , affected on samples for one minute.2)each sample remained in 20 ml of deminerlising solution for 2 hours.3)finally , after applying 20ml of each dentifrice for minutes , samples remained in 20 ml of reminerlising solution for 22hours .
at the beginning of each cycle , in each group 20ml of one of dentifrices , after homogenizing , affected on samples for one minute . finally , after applying 20ml of each dentifrice for minutes , samples remained in 20 ml of reminerlising solution for 22hours . after changing each solution , the 56 blocks were randomly and blindly divided equally into four groups as crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride groups . each group was immersed in remineralizing solution and suspension of related dentifrice in ph - cycling process . we obtained the dentifrices crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) from drug stores , but pooneh without fluoride was provided by goltash company in the city of isfahan . finally , surface microhardness of enamel blocks were measured , at the end of process , again . the following equation was used for calculating the percentage of surface microhardness recovery : ( vhn1 is initial vhn , vhnd is deminerization vhn , and vhnf is final vhn).22 spss soft ware , version 11.5 was used to analyse the data . the mean , standard deviation , maximum , and minimum measures in three steps for four groups were described . we examined the findings of this study by , anova , tukey 's tests and paired t - test where < 0.05 was considered significant . fifty six blocks of coronal portion of extracted primary incisors without dental caries , hypoplasia , crack , and malformation were collected . the crowns were mounted in cylindrical plastic tubes with epoxy resin stick exposing 2 4 mm of enamel surface . preparation of enamel surface was done by boehler abrasive instrument ( buehler , chicago , usa ) , with 600 - 1200 grits al2o3 paper and water . the smooth surfaces were examined under light microscope ( siemens , germany ) using x 100 objective lens in order to evaluate glassy and transparent appearance of enamel surfaces . the surface microhardness was measured by buehler microhardness machine ( buehler , chicago , usa ) . the force action of silver diamond was projected to enamel surface , 100 g/20 in three points distances of 500 , 1000 , 1500 micrometers . the amount of surface microhardness was measured with vickers index hardness unit.1315 through this equation , vickers hardness number ( vhn ) = f1.85/d where f= kg / m and d = mean diameters . in this step , the remieralizing and demieralizing solutions and four dentifrices suspensions were freshly provided in biochemical laboratory of school of pharmacy and pharmaceutical sciences , isfahan university of medical sciences , isfahan , iran . the demineralizing solution contained 2.2 mm nahpo4 , 1.5 m cacl2 , 0.5 m kcl with ph = 71621 and reminerlizing solution contained 0.9 mm nahpo4 , 1.5 cacl2 , 0.m kcl . the suspensions of crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride were prepared by diluting them with distilled water to 1/3 . ph - cycling and remineralization process were done in 8 days with cyclic pattern . in each ph - cycle , blocks were immersed and shook in 20 ml of each solution as follow:16
1)at the beginning of each cycle , in each group 20ml of one of dentifrices , after homogenizing , affected on samples for one minute.2)each sample remained in 20 ml of deminerlising solution for 2 hours.3)finally , after applying 20ml of each dentifrice for minutes , samples remained in 20 ml of reminerlising solution for 22hours .
at the beginning of each cycle , in each group 20ml of one of dentifrices , after homogenizing , affected on samples for one minute . finally , after applying 20ml of each dentifrice for minutes , samples remained in 20 ml of reminerlising solution for 22hours . after changing each solution , the 56 blocks were randomly and blindly divided equally into four groups as crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride groups . each group was immersed in remineralizing solution and suspension of related dentifrice in ph - cycling process . we obtained the dentifrices crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) from drug stores , but pooneh without fluoride was provided by goltash company in the city of isfahan . finally , surface microhardness of enamel blocks were measured , at the end of process , again . the following equation was used for calculating the percentage of surface microhardness recovery : ( vhn1 is initial vhn , vhnd is deminerization vhn , and vhnf is final vhn).22 spss soft ware , version 11.5 was used to analyse the data . the mean , standard deviation , maximum , and minimum measures in three steps for four groups were described . we examined the findings of this study by , anova , tukey 's tests and paired t - test where < 0.05 was considered significant . fifty six blocks of coronal portion of extracted primary incisors without dental caries , hypoplasia , crack , and malformation were collected . the crowns were mounted in cylindrical plastic tubes with epoxy resin stick exposing 2 4 mm of enamel surface . preparation of enamel surface was done by boehler abrasive instrument ( buehler , chicago , usa ) , with 600 - 1200 grits al2o3 paper and water . the smooth surfaces were examined under light microscope ( siemens , germany ) using x 100 objective lens in order to evaluate glassy and transparent appearance of enamel surfaces . the surface microhardness was measured by buehler microhardness machine ( buehler , chicago , usa ) . the force action of silver diamond was projected to enamel surface , 100 g/20 in three points distances of 500 , 1000 , 1500 micrometers . the amount of surface microhardness was measured with vickers index hardness unit.1315 through this equation , vickers hardness number ( vhn ) = f1.85/d where f= kg / m and d = mean diameters . in this step , the remieralizing and demieralizing solutions and four dentifrices suspensions were freshly provided in biochemical laboratory of school of pharmacy and pharmaceutical sciences , isfahan university of medical sciences , isfahan , iran . the demineralizing solution contained 2.2 mm nahpo4 , 1.5 m cacl2 , 0.5 m kcl with ph = 71621 and reminerlizing solution contained 0.9 mm nahpo4 , 1.5 cacl2 , 0.m kcl . the suspensions of crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride were prepared by diluting them with distilled water to 1/3 . ph - cycling and remineralization process were done in 8 days with cyclic pattern . in each ph - cycle , blocks were immersed and shook in 20 ml of each solution as follow:16
1)at the beginning of each cycle , in each group 20ml of one of dentifrices , after homogenizing , affected on samples for one minute.2)each sample remained in 20 ml of deminerlising solution for 2 hours.3)finally , after applying 20ml of each dentifrice for minutes , samples remained in 20 ml of reminerlising solution for 22hours .
at the beginning of each cycle , in each group 20ml of one of dentifrices , after homogenizing , affected on samples for one minute . finally , after applying 20ml of each dentifrice for minutes , samples remained in 20 ml of reminerlising solution for 22hours . after changing each solution , the 56 blocks were randomly and blindly divided equally into four groups as crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride groups . each group was immersed in remineralizing solution and suspension of related dentifrice in ph - cycling process . we obtained the dentifrices crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) from drug stores , but pooneh without fluoride was provided by goltash company in the city of isfahan . finally , surface microhardness of enamel blocks were measured , at the end of process , again . the following equation was used for calculating the percentage of surface microhardness recovery : ( vhn1 is initial vhn , vhnd is deminerization vhn , and vhnf is final vhn).22 spss soft ware , version 11.5 was used to analyse the data . the mean , standard deviation , maximum , and minimum measures in three steps for four groups were described . we examined the findings of this study by , anova , tukey 's tests and paired t - test where < 0.05 was considered significant . four groups were exposed to demineralization , re - mineralization , and toothpaste suspensions in ph - cycling model . mean , standard deviation , maximum and minimum surface microhardness before and after demineralization , and after remineralization are shown in table 1 . kolmogorov - smirnoph and levene 's test were examined the normality of data distribution with homoscedasticity . we applied anova to evaluate the proportional changes of surface microhardness in four groups , and also we used the tukey 's test for paired group comparison ( table 2 ) . distribution of surface microhardness of enamel blocks according to toothpaste comparison of changes of surface microhardness in four groups ( p value ) the purpose of the present study was the comparative evaluation of changes in surface microhardness following application of different fluoride concentration dentifrices . data were provided in initial stage , after demineralization and after remineralization through ph - cycling process . however , it has been reported that their unsupervised use in young children may result in an undesirable high fluoride uptake . the clinical efficacy of these toothpastes in deciduous teeth is matter of dispute and particularly whether a lower caries preventive effect can be expected for the fluoride concentration below 500 ppm . . ammari et al.17 compared low fluoride toothpaste containing 600 ppm or less with toothpastes containing 1000 ppm or more in children and adults and reported that 250 ppm fluoride dentifrices are not as effective as dentifrices containing 1000 ppm in caries prevention . bjarnason et al.18 in a randomized clinical study found that the progression of already existing carious lesions was similar after using 250 ppm or 1000 ppm containing fluoride dentifrice . our findings showed that the crest ( naf , 1100 ppm f ) , crest(naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride increased surface microhardness after remineralization through suspension in toothpaste solutions . in contrast to our study , dunipace and wefel et al.1920 found that mineral acquisition by initial white spot lesions in permanent teeth were increased with increasing fluoride levels in situ . recent studies showed that 500 ppm fluoride containing dentifrices led to a similar caries reduction compared to standard and adult toothpastes in permanent teeth,21 contradictive to our study , crest ( naf , 1100 ppm f ) , ( as the standard toothpaste ) increased surface microhardness more than other tooth pastes in primary tooth enamel samples . the abrasive contents of our applied toothpastes were silica . in the study of maia et al.21 , duraphat ( 1.26% ) cury et al.22 reported that the amount of increased surface microhardness was attributed to the abrasive systems in dentifrices . we could not approve or reject the role of abrasive systems on increasing remineralization in all our dentifrices groups . in the study of queroz et al.23 , there were no significant differences between surface microhardness produced by apf and mfp contained dentifrices , that is not agree with our results . bothenberg et al.24 reported that the depth of indentation in the surface of enamel after application of fluoride dentifrices was more than that of dentifrices without fluoride . tukey 's test showed that the crest ( naf , 1100 ppm f ) increased the surface microhardness significantly more than crest ( naf , 500 ppm f ) and pooneh ( naf , 500 ppm f ) ; however , the increased surface microhardness of using pooneh 500 ppm was significant . in the present study , surface micro hardness recovery after application of crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride was 45.4% , 28.6% , 35.4% and 23.7% respectively . in the present study , the effect of crest ( naf , 500 ppm f ) was similar to the effect of pooneh without fluoride . the possible reasons could be the interaction of ingredients , low fluoride concentrations in the packs , inconsistent distribution and solubility of fluoride , existence of an incompatible abrasive agent with fluoride . from the findings of this study , it can therefore be concluded that crest ( naf , 1100 ppm f ) , crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) , and pooneh without fluoride can increase the surface microhardness in primary teeth enamel . the average of changes of surface microhardness of crest ( naf , 1100 ppm f ) , was higher than that of crest ( naf , 500 ppm f ) , pooneh ( naf , 500 ppm f ) and pooneh without fluoride . | background : surface microhardness is a physical property which access the effect of chemical and physical agents on hard tissues of teeth , and a useful way to examine the resistance of fluoride treated enamel against caries . the purpose of this study was to evaluate microhardness of enamel following ph - cycling through demineralization and remineralization using suspensions of dentifrices with different fluoride contents.methods:in this in vitro study 56 enamel blocks of primary incisors were soaked in demineralizing solution and four dentifrices suspensions including : crest 1100 ppm f ( naf ) , crest 500 ppm f ( naf ) , pooneh 500 ppm f ( naf , ) and pooneh without fluoride . the means and percentage changes of surface microhardness in pre - demineralization , after demineralization and remineralization stages in four groups were measured .
the findings of four groups in three stages were compared by , anova , tukey and paired t - tests .
( =0.05)results : average surface microhardness changes of crest 1100 ppm f , was higher than crest 500 ppm f , pooneh 500 ppm f , and pooneh without fluoride . the percentages of surface microhardness recovery for crest 1100 ppm f , crest 500 ppm f , pooneh 500 ppm f , and pooneh without fluoride were 45.4 , 35.4 , 28.6 , and 23.7 respectively .
demineralization treatment decreased the surface microhardness of enamel ( p<0.05 ) and the surface microhardness recovery in all groups were significant ( p<0.0001).conclusion : surface microhardness of enamel after remineralization by crest 1100 ppm f was higher than crest 500 ppm f , pooneh 500 ppm f , and pooneh without fluoride . |
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altered membrane permeability is a hallmark of inflammation and incipient ischemia , as demonstrated by experimental , radiologic , and pathologic studies . often , this is a systemic condition , involving retinal , cerebral , cardiac , pulmonary , and renal vessels . the presence of microalbuminuria is a risk factor both in the general population or in elderly people.13 altered kidney function parameters further worsen the clinical course and prognosis of cardiovascular , cerebrovascular , and metabolic diseases.211 they are associated with white matter lesions,12,13 silent brain infarction,14 a high prevalence of stroke,15,16 worse prognosis , in terms of both short- and long - term morbidity and mortality,9,10 and recurrence of vascular events ( stroke , myocardial infarction , heart failure).10 pathologic renal findings are also described in young patients affected with stroke , mainly related to hypertensive cardiopathy , nephroangiosclerosis , and chronic pyelonephritis.17 lastly , renal dysfunction is related to cognitive impairment and dementia.1821 urinary polyclonal light chains are low to middle molecular weight proteins , filtered at the glomerular level and reabsorbed in the tubules by megalin - cubulin receptors.2225 in diabetes , the presence of polyclonal light chains is predictive of nephropathy in the absence of albuminuria.26 they herald systemic inflammation and progression of renal injury to chronic renal failure.27 the aim of the present study was to evaluate proteinuria and urinary polyclonal light chains in acute and chronic cerebrovascular disease . five - hundred and fifty patients came under our observation from august 2008 to february 2010 , 36/553 ( 0.06% ) of whom bounced back . they were recruited at the department of emergency and assistance or through the ambulatory service . all the patients underwent routine blood and urine examinations , and had an electrocardiogram and computed tomography within 2448 hours . the diagnoses were acute stroke in 139 ( 58 males , mean age 75.01 , standard deviation [ sd ] 10.85 , 81 females , mean age 80.27 9.04 ) , glasgow coma scale ( gcs ) 11.67 2.94 ; chronic cerebral vascular disease in 361 ( 162 males , mean age 73.78 10.33 , 199 females , mean age 77.61 8.67 ) , gcs 13.8 2.03 ; and other neurologic diseases in 50 patients ( 23 males , mean age 51.79 4.24 , 27 females , mean age 51.72 20.43 ) , gcs 14.56 1.61 . serum parameters , ie , natremia , kalemia , glycemia , urea nitrogen , and creatinemia ( cre ) were detected by cobas integra 800 analyzer . urinary samples were treated with 0.1 m hepes 0.1 m and 0.2 g / l sodium azide , then frozen and stored at 20c . proteinuria and urinary light chains were analyzed by high - resolution agarose gel electrophoresis on sebia hydragel 2 if and hydragel 2 bence jones immunofixation with anti - light chain antibodies and detection by a semiautomatic hydrasis system . some samples were analyzed by nephelometric immunoassay with specific anti - free light chain antibodies . urinary tract infection and bence jones - positive samples were excluded from the statistical analysis . this was performed using the unpaired t - test , for standard description of baseline characteristics and differences among the study groups , and by pearson correlation test and logarithmic regression analysis for identification of association between the study parameters . serum parameters , ie , natremia , kalemia , glycemia , urea nitrogen , and creatinemia ( cre ) were detected by cobas integra 800 analyzer . urinary samples were treated with 0.1 m hepes 0.1 m and 0.2 g / l sodium azide , then frozen and stored at 20c . proteinuria and urinary light chains were analyzed by high - resolution agarose gel electrophoresis on sebia hydragel 2 if and hydragel 2 bence jones immunofixation with anti - light chain antibodies and detection by a semiautomatic hydrasis system . some samples were analyzed by nephelometric immunoassay with specific anti - free light chain antibodies . urinary tract infection and bence jones - positive samples were excluded from the statistical analysis . this was performed using the unpaired t - test , for standard description of baseline characteristics and differences among the study groups , and by pearson correlation test and logarithmic regression analysis for identification of association between the study parameters . our data showed proteinuria in 116/149 ( 78% ) acute stroke patients ( 26.85 42.39 mg / dl ) , 265/384 ( 69% ) chronic cerebrovascular disease patients ( 21.49 40.74 mg / dl ) , and 35/53 ( 66% ) patients with other neurologic diseases ( 17.81 37.17 mg / dl , see table 1 ) . no statistically significant difference was found between proteinuria in acute stroke , chronic cerebrovascular disease , and other neurologic diseases . urinary polyclonal light chains were mainly detected in cerebrovascular patients , with the highest significant levels of chains in acute stroke ( 24.16 26 l ; acute stroke versus other neurologic diseases , p 0.001 , and chronic cerebrovascular disease versus other neurologic diseases , p 0.01 . mg / l in acute stroke , 11.97 51.70 mg / l in chronic cerebrovascular disease , and 4.98 4.27 mg / l in other neurologic diseases ( acute stroke versus other neurologic diseases , p 0.004 and chronic cerebrovascular disease versus other neurologic diseases , p 0.02 , figure 1 ) . a trend towards a difference in the urinary polyclonal light chain ratio was found between acute stroke and other neurologic diseases ( acute stroke 2.62 1.0 , other neurologic diseases 2.15 0.74 , p 0.05 ) . the polyclonal light chain / cre ratio was increased in acute stroke ( acute stroke /cre 0.42 0.47 ; acute stroke /cre 0.12 0.10 ) , but was relatively decreased in chronic cerebrovascular disease ( chronic cerebrovascular disease /cre 0.09 0.02 ; chronic cerebrovascular disease /cre 0.05 0.02 ) compared with acute stroke and other neurologic diseases ( other neurologic diseases /cre 0.17 0.11 ; other neurologic diseases /cre 0.06 0.01 ; and acute stroke /cre versus other neurologic diseases /cre , p 0.001 ; acute stroke /cre versus chronic cerebrovascular disease /cre , not statistically significant ; chronic cerebrovascular disease /cre ratio versus other neurologic diseases /cre , p 0.02 ; acute stroke /cre versus other neurologic diseases /cre , p 0.01 ; acute stroke /cre versus chronic cerebrovascular disease /cre , not statistically significant ; chronic cerebrovascular disease /cre versus other neurologic diseases /cre , p 0.04 , figure 2 ) . at case history , hypertension was referred by 127/149 ( 85.23% ) of acute stroke patients , 293/384 ( 76.3% ) of patients with chronic cerebrovascular disease , 18/53 ( 34% ) patients with other neurologic diseases , and diabetes was present in 29/149 ( 19.46% ) patients with acute stroke , 65/384 ( 16.92% ) with chronic cerebrovascular disease , and 1/53 ( 0.01% ) with other neurologic diseases . no significant correlation was found for proteinuria , urinary polyclonal light chains , systolic , diastolic , and mean arterial pressure values , and natremia , kalemia , glycemia , urea nitrogen , and creatinemia . our finding of a significant increase in urinary polyclonal light chains in acute and chronic vascular patients compared with those having other neurologic diseases , in the absence of a significant correlation with other systemic and renal parameters , suggests that urinary polyclonal light chains represent an early systemic marker of altered membrane permeability . the higher and more significant level of compared with chains in acute stroke can be explained by their higher physiologic production , up to twice the rate of .22 their further increase in pathologic conditions is counterbalanced by augmented elimination and reduced reabsorption at the tubular level . we observed a relatively insignificant increase in urinary chains in chronic cerebrovascular disease compared with acute stroke . in tubular proteinuria , an increase of urinary low molecular weight proteins , as dimers , was detected . this is attributed to an increased excretion , a failure of proximal tubule reabsorption , and reduced catabolism.23 unfolding intermediates are found after exposure to denaturing conditions , such as urea , heat , and ph variation.25 higher seric concentrations of polyclonal light chains and a higher urinary polyclonal light chain / cre ratio , together with increased glomerular filtration and reduced reabsorption at the tubular level have been reported.24,27 serum and urinary concentrations of polyclonal light chains are related to the severity and progression of renal dysfunction.27 taken together , our data suggest that the increase in urinary polyclonal light chains may be a negative prognostic sign and represent the boundary between a reversible stage of renal dysfunction , namely nephrotic syndrome , in which there is still an opportunity for therapeutic intervention , and a definite progression to chronic kidney disease , indicated by a decreased polyclonal light chain / cre ratio . this precedes an irreversible cascade of events characterized by reduction of glomerular filtration,27 systemic interstitial and parenchymal edema , infection , tubular necrosis , progression to uremia and irreversible renal failure , and need for emergency treatment , such as ventilatory assistance and hemodialysis . therefore , it is important to prevent systemic complications that threaten the lives of patients , prolong hospitalization , cause frequent bounce - backs , diminish quality of life , and add further costs for sanitary assistance . the sensibility and specificity of such an assay for urinary polyclonal free light chains , their predictive role concerning short- and long - term prognosis , and the association with cardiac and renal echographic and magnetic resonance imaging parameters are under evaluation . | altered membrane permeability is a hallmark of inflammation and ischemia with systemic spreading .
renal dysfunction is a risk factor for cardiovascular , cerebrovascular , and metabolic diseases .
the aim of the present study was to assess proteinuria and urinary polyclonal light chains in acute stroke and chronic cerebrovascular disease compared with other neurologic diseases .
our results showed significantly increased levels of urinary polyclonal light chains in cerebrovascular disease compared with other neurologic diseases .
the highest values of urinary polyclonal chains were found in acute stroke compared with chronic cerebrovascular disease and other neurologic diseases , while the level of chains was mainly increased in chronic cerebrovascular diseases .
the shift to chronic renal failure seems to be signaled by a decreased polyclonal light chain / creatinemia ratio .
the absence of a significant correlation with blood pressure and other seric parameters suggests that polyclonal light chains are an early marker of reversible vascular impairment with renal dysfunction before progression to irreversible renal failure and need for dialysis and/or intensive care . |
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much of the literature discussed within the present review uses tca - insoluble fractions as an experimental result . in order to understand this , we need to appreciate what the tca precipitation is measuring . the tca precipitation method involves the addition of equal volumes of tca solution ( 510 % ) to the sample , the precipitate is then removed by filtration or centrifugation and the liquid fraction can be measured by the same methodology as the unprecipitated sample . the tca - insoluble fraction can either be assessed by difference or by digestion of the precipitate / filtrate . as early as 1975 , dick et al . showed that the in vitro addition of increasing amounts of di- ( mos_{2 } o _ { 2}^{2 - } ) , tri- ( mos3o ) , or tetra - thiomolybdate ( mo s _ { 4}^{2 - } ) , to plasma ( which was derived as the filtrate fraction from previous tca precipitation ) resulted in decreased cu within the tca - soluble filtrate until at approximately 3 g / ml plasma all of the cu was found in the tca precipitate . in vitro addition of molybdate with or without sulfate had no effect on the cu in the tca precipitate . in the absence of thiomolybdates , the cu was in the tca - soluble fraction with cu in the tca this suggested that tca - insoluble cu can be used as an indirect measure of thiomolybdate , differentiating thiomolybdate effects from mo and s in non - thiomolybdate form . . showed that duodenal administration of thiomolybdates increased the cu content in the tca precipitate of blood plasma , whereas a duodenal infusion of molybdate ( with or without s ) had no effect whatsoever on the blood plasma tca - insoluble cu . this study indicates but does not prove that the thiomolybdate must be absorbed to affect the tca - insoluble cu in plasma and also that thiomolybdates are not formed in the duodenum . a suspension of rumen micro - organisms was incubated in a solution containing molybdate and sulfate ; this resulted in the production of a mixture of di- , tri- and tetra - thiomolybdates which were identified by their absorption spectra . this led to dick et al . suggesting the following mechanism : sulfates are first reduced to sulfides by ruminal micro - organisms , the sulfide then reacts with the molybdate to form the thiomolybdates , the thiomolybdates then react with cu to form insoluble cu thiomolybdates , thus limiting the absorption of dietary cu . in the field , the reduction of sulfate to sulfide in the proposed mechanism explains the increased potency found with increased dietary or water sulfide intakes . bray et al . found tri- and tetra - thiomolybdates to be the predominant species found in cultured rumen fluid . using non - fermentation ruminal conditions , clarke & laurie showed the formation of the relative thiomolybdates to be ph- and s ( given as sulfide):mo molar ratio - dependent . clarke & laurie showed at ph 70 that molar s : mo ratios of < 3 only produced mono - thiomolybdates , 510:1 resulted in di - thiomolybdates , above 10 quickly transformed to tri - thiomolybdates , but found total conversion to tetra - thiomolybdate occurring at about 300:1 . an s : mo molar ratio of 62:1 at ph 80 resulted in the di - thiomolybdate being the predominant form ( with no significant tetra - thiomolybdate ) , whereas at ph 65 the tri form dominated , and as ph decreased the tetra - thiomolybdate form increased . the modern dairy cow has a much lower ruminal ph than the older - type hay - fed dairy cow . they also reported s : mo ratios found in the field and in supplementation trials to range from 6:1 up to 8000:1 , suggesting that many field conditions would produce mainly tetra - thiomolybdates . showed that the actual thiomolybdate production in rumen fermentation conditions , especially the production of tetra - thiomolybdate , was much greater than the clarke & laurie work predicted . a third of molybdate was found to be converted to tetra - thiomolybdate over a wide range ( 412 g mo per ml ) of mo inputs and at s : mo molar ratios of < 10 . the molar ratios of s : mo required for production of the tetra - thiomolybdate in this fermentative culture system were much lower than clarke & laurie found for their non - fermentation culture . the system used by clarke & laurie was also a closed system with no removal or binding of the product produced and was therefore allowed to come to equilibrium . incubations of sieved ( through a 09 09 mm muslin mesh ) rumen fluid ( from cattle fed no mo ) with molybdate ( 01 mm ) and sulfate ( 24 mm ) by elgallad et al . resulted in the production of an absorption spectra indicative of tetra - thiomolybdate ( 239 , 317 and 467 nm ) with no peaks indicative of di- or tri - thiomolybdates ( 392 and 394 nm ) , indicating that only tetra - thiomolybdate was produced in significant amounts . however , when cu was added to the culture before the incubation there was no evidence of tetra - thiomolybdates in the rumen fluid culture . transferring their work into the whole animal , elgallad et al . showed that in cattle fed 100 mg mo per kg dm , 3 g s per kg dm and 200 mg cu per kg dm , there was no tca - insoluble cu and little albumin - bound cu . however , when cu was reduced to 1 mg / kg dm the tca - insoluble cu content increased and greater than 80 % of this was associated with albumin , mo and cu ; the caeruloplasmin concentrations were also reduced . when a cu injection was given to these low - cu ( 1 mg / kg dm)-fed cattle , the plasma cu increased , as did the plasma mo , and both the total amount and proportion of cu in the tca - insoluble fraction were increased . the mechanism by which the thiomolybdates are formed is a stepwise dehydrolysis of molybdate with an o being replaced by an s from a sulfide donor at each stage , the o being released as a water molecule . the direction is driven by the availability of molybdate and sulfide and the removal of the formed thiomolybdates by absorption and binding to the solid phase of forming the copper thiomolybdate complex within the rumen . the direction is driven by the availability of molybdate and sulfide and the removal of the formed thiomolybdates by absorption and binding to the solid phase of forming the copper thiomolybdate complex within the rumen . the study by price et al . in which a large dose of mo - labelled tetra - thiomolybdate was administered intra - ruminally and mo - labelled tri- and di - thiomolybdates were recovered from the rumen digesta indicated the reversibility of this reaction in the rumen . whether the thiomolybdates are associated with the liquid or the solid phase of the rumen digesta is also of importance , as association with the solid phase imparts some stability on the thiomolybdates formed in the rumen , whereas the thiomolybdate in the liquid phase when remaining unbound , or unabsorbed is readily hydrolysed , reversing the reaction outlined above . available cu within the digesta is also largely associated with the solid phase , therefore potentially facilitating the intraruminal formation of cu thiomolybdate complexes . it is doubtful that the equilibria are reached under rumen conditions due to the availability of the substrates , molybdate and s ( as sulfide , or after rumen bug conversion to sulfide ) , along with the constant removal of the tetra - thiomolybdate . the tetra - thiomolybdate would seem to have the following potential fates : ( 1 ) absorbed through the rumen wall ; ( 2 ) passed with digesta through to the abomasum ; ( 3 ) intra - ruminally bound to cu , forming insoluble copper thiomolybdates which are egested ; ( 4 ) bound to the solid phase which seems to incur some chemical stability ; ( 5 ) hydrolysed in the liquid phase ( i.e. involved in the equilibria ) . kelleher et al . showed that mo - radiolabelled tetra- and tri - thiomolybdates administered directly into the rumen of sheep by stomach tube appear within the plasma within 5 min , mainly in a tca - insoluble form . however , when mo - radiolabelled molybdate was administered by the same route , only small traces were found in the plasma over the first 60 min , with these predominantly in the tca - soluble plasma fraction ; after 2 h the amounts of mo in the plasma increased rapidly and from 4 h the radiolabel was predominantly in the tca - insoluble fraction , suggesting that appearance of the label into the tca - soluble or -insoluble fraction is rate dependent rapid uptake from a large ruminal thiomolybdate dose gives a large insoluble tca fraction , whereas a slower digestive trickle feed gives more in the tca - soluble fraction . further , this indicates that more of the thiomolybdate from the large dose appears in plasma in a non - protein - bound form , whereas the trickle dose results in a greater proportion of the thiomolybdate being protein bound . it must be remembered that the above effects are ruminal , as these sheep had duodenal re - entrant cannulae fitted , which allowed the digestion and absorption from the rumen to be totally separate from the intestinal digestion and absorption , as digesta was exchanged between the rumen output of one sheep and the duodenal input of the partner sheep and vice versa . initially after starting to receive the exchange digesta , the proportion of tca - soluble mo in plasma for both tetra- and tri - thiomolybdate for the rumen administered / duodenal absorbed sheep was increased in comparison with the rumen administered / rumen absorbed partner ( first 80 h ) , but the tca - insoluble fraction was similar after 30 h. for the molybdate - administered sheep the digesta - exchanged sheep had virtually entirely tca - soluble mo for the first 4 h , but between 4 and 6 h the tca - insoluble fraction increased rapidly but to only half of the level of the tca - soluble fraction . hynes et al . showed , using mo - labelled molybdate administered to cattle , that there was maximal plasma concentration of labelled mo after 20 h , although similar to kelleher et al . this was initially mainly in the tca - soluble fraction and the tca - insoluble fraction lagged behind but remained more persistent as the tca - soluble labelled mo declined . hynes et al . also showed that protein - bound mo - labelled compounds were present . they eluted this protein with unlabelled tetra - thiomolybdate and displaced significant proportions of di- and tri - thiomolybdate and trace amounts of molybdate . they were unable to elute any tetra - thiomolybdate using this technique ; however , whether this was as none was present or that unlabelled tetra - thiomolybdate was unable to displace the labelled tetra - thiomolybdate ( both would have similar affinities ) remains unclear . . showed similar effects in sheep with no apparent production of tetra - thiomolybdate ; however , they used the same elution technique and no account was made of the cu content of the animal or diet . in steers either given or not given a cu injection immediately before an injection of radiolabelled tri - thiomolybdate , the steers with the cu injection had virtually all of the radiolabel remaining in the plasma , whereas the steers not injected with cu only had 4050 % of the administered radiolabel ( from tri - thiomolybdate ) in the plasma ( mainly in the tca - insoluble fraction ) . the work of others ( discussed above ) concluded that tetra - thiomolybdate was produced in the rumen , but remained absent from the liquid fraction and available for absorption until the solid fraction became saturated . the liquid fraction was the supernatant fraction of strained rumen fluid ( through a single layer of muslin ) after centrifugation at 2200 g , the solid fraction being the fine particulate material . the saturation of the solid fraction is probably related to the ruminally available cu content of the solid fraction and this is at least partially shown by the cu supplementation in the work of hynes et al . this is supported by work by price & chesters that showed available cu in the digesta to be mainly in the solid phase of the rumen fluid . it is further supported by the tetra - thiomolybdate having a greater affinity for cu and therefore the tetra - thiomolybdates will react with available cu before the tri- and subsequently di - thiomolybdates and therefore not be available for absorption whereas the less reactive species are . showed the presence of cu(i)tetra - thiomolybdate ( cus2mos2cu ) in rumen digesta as well as cu(i)tri - thiomolybdate and cu(i)di - thiomolybdate . this cu(i)tetra - thiomolybdate was also found to have completely exchangeable tetra - thiomolybdate given excess copper iodide . suttle & field also concluded that tetra - thiomolybdates caused effects of cu and mo antagonism , whereas the di - thiomolybdate was shown not to play an important role in the cu and mo antagonism . chidambaram et al . showed tetra - thiomolybdate to inhibit in vitro all of the following cu - containing oxidase enzymes ( value in parentheses is the concentration of tetra - thiomolybdate required for 50 % inhibition ) : caeruloplasmin ( p - phenylenediamine 30 m ; o - dianisidine 27 m ) ; cytochrome oxidase ( 20 m ) ; superoxide dismutase ( 50 m ) ; ascorbate oxidase ( 10 m ) ; tyrosinase ( dopachrome formation 30 m , catecholase acivity 45 m ) . phillippo et al . measured plasma mo concentrations ranging from below the detection limit of 0015 g / ml ( 0156 m ) for control and fe - supplemented treatments up to 016 g / ml ( 167 m ) for mo - supplemented groups which would indicate that these levels could potentially be achieved in plasma , with the large caveat that it is not possible to show how much of the plasma mo is in the form of thiomolybdates . . also showed using atomic absorption that caeruloplasmin and tetra - thiomolybdate - treated caeruloplasmin both contained six coppers , i.e. tetra - thiomolybdate does not remove cu from caeruloplasmin to inactivate it . the tetra - thiomolybdate was suggested to bind to the caeruloplasmin through the sulfide ( s ) groups , reducing the cu ( ii ) to cu ( i ) ( fig . chidambaram et al . also showed that , on a mole basis , ten tetra - thiomolybdate molecules bind to each caeruloplasmin , which suggests that some non - specific binding of tetra - thiomolybdate to caeruloplasmin must also occur ; this had been previously shown to occur with non - specific binding of tetra - thiomolybdate to albumin . this reaction was reported to be irreversible . however , both hynes et al . and lannon & mason reported reversibility of tri- and tetra - thiomolybdate effects both in vivo and in vitro . in vitro , lannon & mason showed the reaction to reverse after gel filtration of the plasma , whilst in vivo the reaction began to reverse on cessation of the tetra - thiomolybdate infusion . lannon & mason showed that infusion of tetra- and tri - thiomolybdates both reduced the caeruloplasmin p - phenylenediamine oxidase activity and tca solubility of cu , with the tetra form more potent than the tri form . the reaction was apparently reversible , as full p - phenylenediamine oxidase activity of caeruloplasmin returned in vitro after gel filtration of the plasma , and in vivo as activity began to return upon cessation of thiomolybdate infusion . it was also noted that during infusion the total cu content of the blood increased ( 24 % over 24 h ) ; however , there was no reduction in caeruloplasmin cu associated with the increased cu , which is in agreement with chidambaram et al . the increase in plasma cu is something also shown by kendall et al . , who found the total cu concentration of plasma to be increased by a continuous infusion of 1 mg tetra - thiomolybdate per h in sheep . by 1-week post - cessation of infusion , thiomolybdate and control infused sheep had similar concentrations of plasma cu . the tca - soluble plasma cu concentration remained consistent with the tetra - thiomolybdate infusion having a significant tca - insoluble fraction . the caeruloplasmin p - phenylenediamine oxidase activity was also decreased by the tetra - thiomolybdate infusion , but at the 1-week post - infusion sample full activity was regained . it was only the measure of whole blood superoxide dismutase activity which remained depressed at 1 week after cessation of the 5 d tetra - thiomolybdate infusion . kumaratilake & howell found in cu - loaded sheep ( fed 2 % solution of copper sulfate at 10 ml / kg body weight for 5 d per week until a rise in alkaline phosphatase was observed ) ( range 55149 d ) that the administration of tetra - thiomolybdate ( 50 g intravenously twice per week ) for 11 weeks significantly reduced liver cu concentrations from 81376 ( sd 7477 ) to 30578 ( sd 4825 ) mg / kg wet weight ( ww ) ; cu concentrations also decreased in the non - thiomolybdate - treated controls but only to 59457 ( sd 10429 ) also there were significantly increased liver mo concentrations ( 093 to 8725 ( sd 16 ) mg / kg ww ) in the cu - loaded animals . haywood et al . found a pituitary endocrinopathy in sheep associated with therapeutic administrations of thiomolybdates for the treatment of cu toxicity . phillippo et al . showed fe and s supplementation to reduce the blood plasma cu content of cattle to hypocupraemic status ( 189267 m and 315378 m compared with control group minimums of 1054 and 1573 m for two experiments , respectively ; the internationally recognised threshold for cu deficiency is 9.4 m ) without inducing the symptoms of clinical cu deficiency ( reduced weight gain , decreased food intake , reduced efficiency of food conversion , alteration in hair texture , delayed puberty , reduced conception rate and inhibition of oestrus ) . bremner et al . showed that fe supplementation ( 4.5 , 9 and 135 mmol fe / kg dm ) had no effect on cu status in preruminant calves , but decreased plasma and liver cu when fed post - wearing although this did not induce a the effects of fe and mo were additive at the relatively low levels administered ; previous trials with high supplement rates did not indicate an additive effect . showed fe supplementation ( 30 mg / kg live weight ) to reduce liver cu from 179 mg / kg dm down to 7 mg / kg dm , with plasma cu concentrations starting to reduce after depletion of liver cu to 40 mg / kg dm . the cu - dependent enzymes amine oxidase and caeruloplasmin were measured in this trial only after blood cu had already been significantly reduced and were lower for the fe - dosed group compared with the control group . these animals had a basal diet containing 15 mg mo / kg dm and 107 mg cu / kg dm . no effect of high fe supplementation ( 1 g / kg diet ) was noted on serum , liver or spleen cu concentration by rosa et al . g / kg fe , 028 % sulfate ) to significantly reduce the blood cu concentration when compared with iron citrate ( 16 g / kg fe ) and no supplement . however , blood cu concentrations when fed sodium sulfate ( 028 % sulfate ) were reduced but not significantly , and not to the degree that the iron sulfate reduced the blood cu concentration . in the rumen the fe can react with sulfide and cu either to produce an fe cu s complex which is not absorbed by the animal ; an alternative hypothesis is that the fe and sulfide combine to form an iron sulfide , and the cu then exchanges with the fe to form a copper sulfide ( comment by c. f. mills in suttle & peter ) . both will therefore reduce the amount of cu available within the rumen and will hence reduce the cu available to participate in the mo and s ( thiomolybdate ) and cu interactions . however , there is little direct evidence in the literature to support either hypothesis , although what is clear is that there is an fe cu s interaction and it does decrease cu availability . the complex interaction between these elements is explained above , but it is important to remember how these elements reach the rumen and in what form . the diet includes the elemental composition of the plant material consumed , dietary inclusion and supplements containing minerals ( for example , mineral licks ) and soil consumption , both as feed contamination and direct consumption . soil can account for up to 25 % of dm intake in certain situations ; for example , in sheep grazing pasture with short grass either in very wet conditions ( poaching ) or with very dry and dusty conditions , where the annual soil consumption can exceed the live weight of the animal . other sources confirm this , with over - wintered grazing cattle ingesting between 140 and 1400 g soil per d. drinking water can also be an important elemental entry route , whether it is mains water , spring water , stream water or from a borehole . borehole water , especially , tends to be individual to the farm and can have a high sulfide content which as outlined above makes the s much more potent in the thiomolybdate formation reactions . the fe in borehole water also often tends to be in the highly reactive ferrous form . water - logging of soil in addition to increasing poaching and increasing soil consumption will also increase the uptake of fe to plant material ( four- to five - fold in young green shoots ) , with the fe in the highly bioactive ferrous form . when soil ( contamination ) is ensiled with forages the fermentation process appears to increase the fe bioactivity . the mo uptake by plants , especially legumes , is also increased when grown in wet soils . in general , mo content is higher in vegetable protein supplements ( for example , soya ) and legumes ( for example , clover ) . mo uptake is also increased with increasing ph of the soil ( liming pasture increases mo uptake ) and generally increases throughout the growing season . there are also differences found between continuous and discontinuous feeding on sulfide production ; this will also affect rumen ph . therefore , a continuous feeding system ( for example , total mixed ration ; tmr ) will give a more consistent but moderately higher rumen ph with a stable sulfide content whereas a single or twice - daily feed will give a rapid decrease in rumen ph and peak production of sulfide , both of which will revert over time before the next meal . within the present review we have been able to provide evidence that : all classes of thiomolybdates are formed in the rumen ; in the absence of available cu all thiomolybdates can be absorbed into the animal rapidly though the rumen wall or more sedately via the small intestine ; thiomolybdates can bind to cu in biological compounds and are able to cause problems ; effects of thiomolybdate are reversible in vivo and in vitro on cessation of thiomolybdate challenge ; the tetra - thiomolybdate form is the most potent cu binder with decreased potency with decreased s in the compound . | the rumen is the site of significant interactions between cu , s and mo
. it also shows reactions between cu , s and fe . the interaction between mo and s results in the formation of thiomolybdates , which in the absence of adequate quantities of rumen cu are absorbed into the animal and bind to cu in biological compounds .
this is the cause of thiomolybdate toxicity , often misleadingly called cu deficiency .
the effects of thiomolybdates being absorbed into the animal are considered , especially how thiomolybdates bind to cu - containing compounds such as enzymes , decreasing their activity without removing the active cu component .
the sources of cu , mo , fe and s are examined , including the impacts of water and soil on the animal 's intake . within the present review
we have been able to provide evidence that : all classes of thiomolybdates are formed in the rumen ; in the absence of available cu all thiomolybdates can be absorbed into the animal rapidly though the rumen wall or via the small intestine ; thiomolybdates bind to cu in biological compounds and are able to cause problems ; effects of thiomolybdate are reversible in vivo and in vitro on cessation of thiomolybdate challenge ; the tetra - thiomolybdate form is the most potent cu binder with decreased potency with decreasing s in the compound .
fe will exacerbate a thiomolybdate problem but will not directly cause it . |
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a 29-year - old male , driver by occupation , came with complaints of redness , pain , and diminution of vision in the right eye from 1 month . on general examination , there was no edema , pallor , organomegaly , or lymphadenopathy . the visual acuity in the right eye was 6/9 , and the visual acuity in the left eye was 6/6 . the right eye showed mild ciliary congestion and pigments on the endothelium and anterior lens capsule . the iris had multiple atrophic patches , 2 iris holes were present , and the pupil reacted normally . there was a live , motile worm hooked to the midperipheral iris [ fig . 1 ] . magnetic resonance imaging of the brain and orbit and ultrasonography b - scan of both eyes were normal . depicting live worm hooked to midperipheral iris under peribulbar anesthesia the worm was extracted with plain forceps from the anterior chamber and was sent for microscopy to the department of microbiology and parasitology . the worm was a short and stout reddish brown , cylindrical structure measuring approximately 1 cm in length . on the examination of wet mount , it showed bulbous head with four rows of hooklets ; the anterior part of the worm is covered with rows of cuticular spines . the patient was given oral albendazole 400 mg once daily for 21 days and tapering dose of oral prednisolone ( 50 mg od for a week followed by tapering dose by 10 mg every week , 5 mg od for a week after the 5 week , and then stopped ) . third - stage larva of gnathostoma spinigerum cephalic bulb of gnathostoma larva with rows of hooklets body cavity of gnathostoma spinigerum h and e stained section eye of the patient after removal of the gnathostostoma larva of the 12 known species within the genus gnathostoma , only 4 species have been known to infect humans namely gnathostoma nipponicum , gnathostoma hispidum , gnathostoma doloresi , and g. spinigerum . g. spinigerum is found in wild and domestic cats and dogs in southeast asia , china , japan , and india . humans become accidental host when they consume raw or undercooked meat of the definitive host or the second intermediate hosts such as brackish water fish , chicken , snails , and frogs or paratenic hosts such as birds . cutaneous lesions such as migratory panniculitis or serpiginous eruptions caused by the migration of the third - stage larvae are the most common manifestation of this infection , but their onset may be delayed for months and even years . migration to unexpected sites leads to visceral involvement of the lungs , gastrointestinal tract , genitourinary tract , ear , central nervous system , and eye . among the various forms of visceral gnathostomiasis , central nervous system infestation causes fatal eosinophilic myeloencephalitis whereas ocular involvement is rare . the most common manifestation of intraocular gnathostomiasis is anterior uveitis and intraocular parasite because it mostly localizes itself in the anterior segment of the eye . the other common manifestations are eyelid edema , conjunctival chemosis , hyphema , retinochoroidal , vitreous hemorrhage , and rarely , central retinal artery occlusion leading to blindness . the portal of entry into the eye may be posterior retina because intraocular gnathostomiasis has been associated with macular scarring , rupture of nasal branch of central retinal artery , or retinal tear with choroidal hemorrhage near the optic disc . iris holes , uveitis , and subretinal hemorrhage with subretinal tract can be characteristic features of intraocular gnathostomiasis . clinical symptoms of gnathostomiasis are due to the inflammatory reaction provoked by migrating larvae . clinically , it may present as painful cutaneous larva migrans , gastrointestinal , genitourinary , and central nervous system infections , with or without peripheral blood eosinophilia ( eosinophil count > 0.4 10/l ) , or undiagnosed eosinophilia with nonspecific symptoms . because of the avascularity of the anterior chamber of the eye , eosinophilia which is the hallmark of parasitic infections is evidently absent and usually mild if at all present . once the disease is diagnosed , management of gnathostomiasis is straightforward . hence , eosinophilia can not be considered a screening tool . however , moore et al . have commented that it could be used as a marker of treatment response in those with eosinophilia at baseline . in the present case high - dose albendazole , given for 3 weeks , has been used as specific chemotherapy for treatment of patients with gnathostomiasis . till now , 14 intraocular gnathostomiasis cases have been documented from india 3 from calcutta , 2 from west bengal , 3 from chennai , one each from meghalaya , assam , odisha , kerala , pondicherry , and maharashtra [ table 1 ] . with increasing reports of intraocular gnathostomiasis from india , the clinicians should be familiar with this disease ; therefore , diagnosis is not missed or delayed , avoiding potentially serious complications . | we report the first case of intraocular gnathostomiasis from central india . a 29-year - old male from indore , madhya pradesh , presented with pain and redness of the right eye since 1 month .
slit lamp examination revealed anterior uveitis , multiple iris atrophic patches , and a live worm hooked on iris .
the worm was removed through a small sclerocorneal tunnel .
microscopy confirmed gnathostoma spinigerum .
the patient was treated with oral albendazole and steroids .
the case is reported because of its rarity . |
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a continuous hydrolipidic film , representing the actual interface between the epidermal viable layers and outer environment , covers human skin . skin surface lipids ( ssls ) are a mixture of sebaceous and epidermal lipids , displaying a very peculiar composition as compared to lipid fractions of serum or internal tissues . this peculiarity is originated by the unique contribution of sebum secreted from the sebaceous glands , unevenly distributed in all areas of the body with the exception of the palms and foot soles , and becoming extremely specialized in local districts , like the eye , where the meibomian glands exert highly efficient protective functions . ssl composition in the skin areas with the highest concentration of sebaceous glands ( forehead , upper chest , and dorsum ) mainly reflects sebaceous secretion , flowing from those sites also to areas with lower concentration , where the contribution of cellular lipid components , rich in oleic and linoleic acid , becomes more relevant [ 2 , 3 ] . the keratinocyte membrane lipid contribution and the continuous metabolic action of resident microbial flora hosted at skin surface in healthy conditions are key determinants of the uniqueness of this complex mixture . major lipid components in human sebum include squalene ( sq(2,6,10,15,19,23,-hexamethyl-2,6,10,14,18,22-tetracosahexaene ) , wax esters , and triglycerides . as a whole , the ssl fatty acids fraction is relatively poor in polyunsaturated fatty acids ( pufas ) . typically , sebum is rich in long - chain fatty acids ( up to 26 carbon atoms ) , linear or branched , mainly saturated or monounsaturated [ 4 , 5 ] . these are partly present in the free form , secondary to the microbial and epithelial lipase activity on sebum triglycerides , and are responsible for antimycotic and antibacterial properties of the skin [ 57 ] . for the most part , these specialized fatty acids are esterified with cholesterol , or with fatty alcohols , to form the fraction of wax mono- and diesters , crucial for skin insulation [ 810 ] , available uniquely on the skin and hair shaft . this fraction has been extensively investigated by analytical lipidomic approach in recent years , allowing , for example , the identification of more than 160 different wax esters ranging from 24 to 42 total carbons , and 73 species of ceramides , in the human proximal hair [ 11 , 12 ] . a most peculiar component of ssl is sq , a key biosynthetic precursor of cholesterol . in humans , about 60 percent of dietary sq is absorbed , transported in serum by very low - density lipoproteins , and distributed ubiquitously in human tissues , with the greatest accumulation in the skin through sebocyte concentration . sq levels , being negligible in other organs , normally range about 12% of total ssl in adult life and can reach up to 20% [ 14 , 15 ] . in the liver and in other tissues , this linear 30-carbon triterpenoid compound is entirely metabolised to sq 2,3-epoxide to be subsequently converted to lanosterol . sq overproduction in sebocytes may be due to altered expression and activity of two key oxygen - regulated enzymes involved in sq metabolism , squalene synthase , and squalene oxidocyclase , in response to the anaerobic environment occurring locally inside the sebaceous gland . this biochemical peculiarity bears important biological implications , in that the peroxidable sq molecule has been extensively proven to be a key mediator of skin reactions to environmental stressors . in defence towards oxidative events occurring on the skin , vitamin e of nutritional origin , actively secreted from sebaceous glands and probably cosecreted with sq and coenzyme q10 of endogenous origin and partly co - synthesized with sq by the sebaceous gland , provide necessary antioxidant protection to the skin lipid film . the cellular - derived component of skin surface lipids consists of phospholipids derived from the plasma membrane of corneocytes , also contributing as the unique component of ceramides characterizing cell envelope [ 7 , 20 ] . lipids from keratinizing cells account for the rather limited contribution of polyunsaturated fatty acids , namely linoleic acid , available in the surface hydrophobic film . more deeply into the cornified envelope , ceramides and proteins concur to form the unique intercellular matrix determining most of the functions of the skin barrier . the overall composition of ssl differs therefore sensibly from lipids of the viable skin epithelial layers , and from systemic lipids , both in the relative percentage and type of lipid fractions , and in the relative amount of the pufa oxidable fraction . following the rather extensive studies on the composition and role of ssl started by the pioneer observations of nicolaides , the research on epidermal surface lipids and their prevalent sebaceous component has been thereafter almost entirely dropped , in favour of the studies on lipids of keratinocyte origin , thus unravelling the essential functions of ceramides in cell signalling and skin disease [ 23 , 24 ] . nevertheless , the role of ssl in mediating the biologic effects of uv irradiation and other environmental stimuli / stressors has been at least partly identified , and , most importantly , alterations in sebum secretion have been recognised as a common feature of several inflammatory chronic skin diseases , finding their main localization in cutaneous areas where sebaceous glands are more concentrated . this is the case of acne [ 2628 ] , atopic and seborrheic dermatitis , pityriasis versicolor [ 31 , 32 ] , and androgenic alopecia . sebum must in fact also be regarded as vehicle transporting and transmitting several endogenous and exogenous molecules to the skin , including potential regulatory factors of hair follicles . all the above - mentioned dermatoses are so far lacking satisfactory aetiopathogenic elucidation and consequently established and effective therapies . among the different categories of environmental stressors affecting the skin , uv irradiation is the field of most intense research targeting the increased risk of tumor development connected with overexposure and the effects on cutaneous pigmentation of medical / aesthetic concern . based on the capability of uv rays to penetrate at different depths in the epidermal and dermal layers , most of the studies have been so far focused on the photoreceptors localised in the skin viable compartments beneath corneum , such as dna , urocanic acid , and endogenous or bacterial porphyrins . these molecules are able to selectively absorb in the uv wavelenght range and thus catalyse oxidative cytostatic , cytotoxic , or imunomodulating photoreactions , widely employed for medical applications in skin phototherapy . indeed , the very first target of uv and other environmental radiations during natural , professional , or therapeutic exposure is represented by skin surface lipids . nevertheless , their possible role as mediators of cutaneous and systemic biological effects has been so far not adequately highlighted . the uv - r absorbance spectrum for ssl has been assessed spectrometrically , showing significant absorption , with a maximum at 215 nm . the hydrolipid layer present on forehead skin in healthy conditions is likely to reduce transmission at 300 nm by about 10% . apart from representing a first line of defence by direct uv absorption , ssl constitutes a suitable target and scavenger for all reactive species generated at skin level by different molecular mechanisms , in the course of uv irradiation . this indirect photo - oxidation is definitely the most relevant biological effect of uv on the ssl mantel and is the main mechanism operating in ultraviolet a- ( uva- ) and visible light - induced photodynamic stress induced on skin for therapeutical purposes . due to the scavenging action of ssl under atmospheric oxidative chemical stress , photoirradiation , or microbial oxidative metabolism , the hydrolipid superficial mantel becomes a relevant source of peroxidated intermediates on the skin , certain molecules of the resulting composition being clearly cytotoxic , irritant , or immunogenic . among cutaneous ssl components , it was previously demonstrated that when human sebum is subjected to high - dosage uvb irradiation in vitro , sq is markedly degraded as compared to cholesterol , sebum triglycerides , or mono-/diunsaturated free fatty acids ( ffas ) [ 43 , 44 ] . uvr , alone or in combination with photosensitizers or other physicochemical stimuli including microbial peroxidizing metabolism , induces oxidative degradation of sq with the generation of a wide range of by - products of varying polarity and reactivity , that have been clearly characterised by ion mass spectrometry . these include sq monohydroperoxide , different isomers of squalene epoxide , and shorter chain reactive aldehydes , in particular formaldehyde and malonyl dialdehyde ( mda ) [ 4648 ] ] ( figure 1 ) . more recent studies have demonstrated that sq is oxidized at a much higher rate in physiological conditions by uva either than uvb , and that probably most previous results obtained with experimental uvb irradiation must be ascribed to the effects of minimal doses of uva , always contaminating uvb emission sources . following the initial studies concentrated on sq oxidation dynamic , conclusive works have indicated that the very first molecules undergoing oxidation in vivo under uv irradiation of skin are alpha - tocopherol and ubiquinol-10 , both in sebum and viable keratinocyte membranes [ 17 , 19 , 5052 ] . these redox - cycling phenols have been undoubtedly recognised as the first targets of uv oxidation in both three - dimensional skin experimental models [ 50 , 51 ] or cell - free sets of sebum ex vivo irradiation [ 53 , 54 ] . only once these borderline lipophilic antioxidant defences of ssl are overcome , there occurs accelerated sq oxidation ; this leaves unchanged the remaining lipid fractions and protects other unsaturated lipids such as cholesterol and , most importantly , the minimal amount of pufa contributed by pure sebum , namely sebaleic acid ( 5,8-octadecadienoic acid ) . this special di - unsaturated fatty acid has been recently proven in vitro to be a feasible source of oxidized , biologically active chemoattractant and proinflammatory species , when metabolized by neutrophils and keratinocytes in the skin . in vivo instead , the protective effect of sq on pufa and the major physiological relevance of sq peroxides as compared to sebaleic acid oxometabolites were already indirectly proven by the early observations of morello et al . , documenting the absence of significant sebaleic acid depletion in sebum of patients with severe acne , as compared to healthy controls . based on the elevated proneness of sq to photodecomposition , and the many biological effects of uv - induced sq peroxides it has been recently reproposed that these reactive by - products may be principal physiological molecular mediators of the biological effects of uv irradiation and other pro - oxidants targeting skin . as described above , the protective effects of sq are counterbalanced by the generation , by the same peroxidative mode , of low - molecular weight and relatively hydrosoluble reactive species [ 57 , 58 ] , chemically derived from the antioxidant action of the triterpene . these by - products are able to diffuse from the skin outer corneum mantel into viable layers and hence target keratinocyte plasma membrane pufa , thereby causing arachidonic acid decrease and a sequel of consequent biological effects , including the production of a cascade of lipid oxidation reactive nucleophiles , such as 4-hydroxy-2 nonenal ( 4-hne ) . sq peroxides display the typical bimodal biological behavior of reactive species , exerting in vitro a paradoxical effect on keratinocyte cells . at low concentrations and short times of incubation , they stimulate dna and protein synthesis , whilst inducing cellular damage and inhibiting mitotic activity at higher exposure times and dosages . sq peroxides also stimulate keratinocytes to release increased amounts of pro - inflammatory cytokines in vitro whereas they induce uv - like immunologic effects on the guinea pig ear model in vivo . topical application in concentrations likely produced under physiological uv irradiation of the skin is in fact able to suppress contact hypersensitivity to topically applied haptens like dinitrofluorobenzene ( dnfb ) , as demonstrated by the reduction of lymphocyte infiltrate and the depletion of atpase positive immunocompetent cells . among ssl fractions , sq is the most efficient quencher of singlet oxygen , produced under uv irradiation of cutaneous photo - sensitizers or under conditions of phagocyte - driven oxidative burst at skin inflammation sites . sq peroxide by - products generated by this scavenging action are able to upregulate the release of pge and cytokines from keratinocytes , and through this mechanism they possibly activate melanocyte dendricity and stimulate melanin synthesis in the lower skin layers , as demonstrated on the guinea pig ear model . in this direction , sq peroxide effects on melanogenesis can in part explain also changes in skin pigmentation in vivo . taken together , all these results confirm earlier classification of sq as a sacrificial antioxidant , being extensively degraded under different types of peroxidant stimuli and very prone to photodecomposition . this way , this unique molecule is able to afford protection of human skin surface from further damage to the lipid mantel and to cellular peroxidable targets of the viable layers , induced by the exposure to uv and other sources of ionizing radiation , both natural and iatrogenic . in view of the many ascertained biological effects of uv - induced sq peroxides , it has been recently re - proposed that these reactive by - products may be principal physiological molecular mediators of the biological effects of uv irradiation and other pro - oxidants targeting skin [ 39 , 62 ] . the recognition of sq as a principle target for oxidative stressors on the hydrolipid mantle has been acknowledged in the last decade also for applicative purposes , in that the quantification of sq oxidative rate , or of sq hydroperoxide formation , has become a commonly used method to test the protective / antioxidant efficacy of natural or artificial ingredients of formulations directed towards skin protection or sunscreening [ 48 , 57 , 61 , 63 ] . in conclusion , though , from the biological point of view , the reason why sq molecule occurs in high concentration in human , ssl is still considered an enigma . on the basis of all described biochemical properties , it can be sensibly hypothesized that the natural deficiency of squalene oxido - cyclase activity in human sebaceous glands represents an evolutionary advantage , in that sq is capable of neutralizing reactive oxygen species induced by uv irradiation on the skin , thus behaving as an antioxidant and , indirectly ( sq does not absorb in the uv range ) , as a natural sunscreen . the skin of monkeys , unlike that of humans , is covered by a large quantity of hair , protecting from uv rays . in the far less hairy human skin , the shield function is reasonably carried out by sq , in association with the physical defences of stratum corneum and melanin . in contrast to human sebum in fact , ssl of other hominidae contains higher levels of cholesterol , and surprisingly no sq at all . our group has performed a so far unique study on surface lipid composition in the primate superfamily , proving that sq is unique to human sebum , and completely missing in the main genera of non - human primates , including those closer to man , the hominoidea [ 65 , 66 ] . human sebum also contains higher levels of triglycerides and their hydrolysis products and far lower levels of cholesterol . in addition , sq terpene typical of human sebum is also a principal surface lipid of different aquatic mammals , namely otter , beaver , kinkajou , and at least one species of mole [ 6770 ] . in these species , sq accounts for the essential properties of water repellence and thermal insulation . other nonaquatic mammals or birds have evolved different cutaneous fats , such as wax esters and wax di - esters , ensuring the same vital properties . the relevant distance of human sebum in composition and function from the nearest primates , and the close similarity with semiaquatic mammals , bears interesting evolutionary implications and may offer some support to the discussed hypothesis of the origin of man from some semi - aquatic hominids , feeding on fish . marine food , especially microalgae and seaweeds , as well edible seeds of plants dwelling in mangrovian habitats , like the genus amarantus , display in fact an unusual rich sq content . in the light of the so - called aquatic ape theory , that evidences features of convergence among different semi - aquatic species , such as proboscis monkeys , beavers , sea - otters , hippopotamuses , seals , sea lions , and walruses , these new biochemical data on human sebum squalene offer indeed new space for speculation . as previously mentioned , the epidermal surface hydrolipid layer can also be viewed as the growing medium for residential saprophytic microbial skin flora . the distribution and density of bacterial and yeast population at cutaneous surface is dependent on host age and on environmental factors such as sebum secretion , occlusion , temperature , and humidity [ 73 , 74 ] . sebum produced in the pilo - sebaceous gland is composed purely of squalene , waxes , and triglycerides . once secreted , this rich medium is immediately colonized by various lipotrophic biologic agents , the development of which is controlled by several defensive humoral mechanisms and by the contact with ambient oxygen . bacterial / yeast lipase activity is the main responsible of ffa presence on the skin . oxygen and micro - organisms transform sebaceous moiety and hydrolyse triglycerides to ffa , with consequent relevant alterations of the ssl pattern in cases of pathological microbial colonization of the skin . this is the case of acne , where a role for propionibacterium infection is claimed among the main aetiopathogenic triggers . here , ffas metabolically generated by this lipophilic bacterium account for the chronic inflammatory reaction and the fibrogenetic action on infundibulum epithelium , thus sustaining comedones , pustules , and nodules formation . similar mechanisms have been claimed to explain at least partially the chronic inflammatory process characterizing atopic ( ad ) and seborrheic dermatitis ( sd ) [ 76 , 77 ] . pityrosporum ovale is a lipophilic saprophyte belonging to the normal skin flora , mainly localizing in the horny layers , and in the upper tract of the sebaceous follicle . in the scalp , for instance , p. ovale constitutes up to 46% of the cutaneous flora in the healthy subject , while it may increase up to 82% in sd patients . the evidences of defective cell - mediated response to p. ovale and to candida albicans in sd patients , and the elevated incidence of sd in hiv and aids patients , support the hypothesis that a deficit of cell - mediated immunity may play an aetiological role in the disease . a long - lasting pro - inflammatory action of p. ovale is to be taken into account , the yeast being highly immunogenic , able to activate complement and to produce pro - inflammatory reactive oxidized metabolites of selected polyunsaturated ssl , including sq . alterations of ssl composition , along with oxidative by - products of ssl irradiation during antitumor puva or narrow - band uv - b phototherapy , induce similar immunologic impairment locally on skin . this causes an increased incidence of parasite skin colonization , like , for example , the infection caused by the demodex mite , an intrafollicular parasite feeding on sebum , and also frequently causing blepharitis . antimicrobials may reduce the density of the resident pathogenic or saprophytic skin flora , but they do not completely eliminate it . while antimicrobials may cause irritant and allergic contact dermatitis , no evidence exists that their use may change the ecology of resident bacteria on the skin , thereby leading to the overgrowth of pathogenic bacteria . as a consequence , antibiotic / antimycotic treatment is not the elective approach to most of these skin diseases . the antimicrobial function of ffa generated on sebum is an essential factor for the homeostasis of skin microbial colonies , and changes in sebum fatty acid composition are a main cause of microbial alterations on the pathological skin [ 81 , 82 ] . in this respect , skin lipidomics are definitely expected to offer important diagnostic and therapeutic solutions in the near future . due to its high levels of unsaturation , sq in particular has been proposed as the precursor of highly toxic pro - inflammatory mediators , produced by bacterial or yeast lipoperoxidase activity . a further well - characterized example of an aetiologic involvement of ssl in skin pathology is offered by pityriasis versicolor , a pigmentation disease featured by large achromial spots occurring on skin areas with highest concentration of sebum lipids , where pityrosporum orbiculare finds optimal dwelling conditions . this skin saprophyte in some individuals becomes pathogenic , due to still unknown factors . in vitro cultures of p. orbiculare have documented a markedly increased peroxisomal lipid oxidation activity induced by the same unsaturated fatty acids present on the skin , namely linoleic acid , giving rise to hydrogen peroxide through a fenton mechanism and the subsequent generation of hydroxyl radicals . sq , which is not a substrate for lipoxygenase and is not metabolized by pityrosporum , may reasonably be peroxidized in vivo due to the metabolic activities of yeast peroxisomes . in yeast cultures supplemented with linoleic acid plus sq , a marked increase in lipoperoxide formation was observed , with the generation of the same toxic and unstable oxidation products ( trans - trans farnesal and sq epoxides ) formed under experimental high - dosage uv irradiation of sebum and also identified on skin in vivo . this equivalence substantiates the role of sq peroxides in the development of the clinical features of pityriasis , possibly accounting for melanotoxicity and cutaneous depigmentation . in this connection , the early demonstration that vitamin e supplementation suppresses the induction of peroxisomal beta - oxidation and catalase activity induced by linoleic acid offers promising clues for new treatment approaches all surface lipids examined so far play a role in the complex signaling network originating at the epidermal level , so that skin can no more be viewed as a specialized wrapping material protecting internal organs from environment and guaranteeing the main function of permeability barrier homeostasis , but more extensively as a complex organ actively communicating with the external world . as a consequence , thorough revision has been made of the traditional concept that only mere quantitative alterations of sebum , without any concern for sebum quality and composition , accounted for skin diseases associated with seborrhea or sebostasis . the multifaceted functions of the sebaceous gland are hence gaining momentum [ 25 , 87 ] , as the dysfunction of enzymes synthesizing or metabolizing ssl has been found in different disease states , along with altered sebum antioxidant levels . as widely discussed above , ssls are subjected to hydrolysis and oxidative processes , giving rise to biologically active by - products , which are critically modulated by local lipid soluble antioxidant levels , actively transported to the skin surface at the level of the pilosebaceous unit . indeed , ssl composition has been found significantly altered in amount and quality in different skin diseases as well as in the aged skin . in both atopic and seborrheic dermatitis , we and other authors reported a marked reduction of skin total surface lipids [ 29 , 30 ] . it was shown that ssl of children and adults with atopic dermatitis present depleted levels of the lipid fractions of sebaceous origin , sq , and wax esters and correspondingly increased levels of free and esterified cholesterol . analogous alterations were found in seborrheic dermatitis , in hiv - negative and hiv - positive patients , these latter suffering from sd with increased frequency as compared to healthy population . these alterations of ssl composition , summarized in table 2 , were associated with significant systemic depletion of the main lipophilic antioxidant levels and detoxifying enzymatic activities , mainly vitamin e , ubiquinol-10 , and erythrocyte glutathione peroxidase [ 88 , 89 ] . in the acne pathogenic process , sq peroxides most likely play a role , in that sq monohydroperoxide has been proven to be highly comedogenic when topically applied on skin in the animal model . sebum sq fraction was found increased by 2.2-fold in a group of patients affected with moderate to severe acne , as compared to controls , reaching 20% of the total sebaceous lipids . the ssls undergo important qualitative and quantitative modifications due to photo - aging , where sq peroxides seem again to play a major role , mimicking the effects of chronic uv irradiation when applied experimentally on the skin [ 93 , 94 ] . ssl may hence represent a very efficient marker of the sun - protecting efficacy of chemical sunscreens . experimental uv irradiation of a series of commercial chemical filters in the presence of sq in physiological concentrations led to the conclusion that all most common uv filters protect sq from uvb - driven oxidation at different extents ( benzophenone-3 > octylsalicylate > parsol mcx > parsol5000 > octyldimethyl p - hydroxybenzoic acid ( paba ) > parsol 1789 ) , depending on filter dose and irradiation energy . conversely , upon uva irradiation , filters like parsol 1789 and 5000 and octyl - dimethyl paba exerted a marked pro - oxidant effect , enhancing sq peroxides formation possibly through the action of their self - decomposition by - products induced by uva , deserving careful attention for safety concerns [ 64 , 95 ] . the external lipid film hence represents a reliable in vivo marker of skin disease , easy to monitor through noninvasive analytical techniques [ 30 , 96 ] . being the first target of environmental stressors , ssls also represent a suitable research tool for in vitro screening , to study drug delivery through skin , as well as in vitro assessment of the chemical reactions of jewellery , textiles , cosmetics , drugs , industrial chemicals , and particles in direct and prolonged contact with human skin . to provide meaningful results , the composition of artificial ssl should accurately mimic human sweat and sebum , and the conditions of the in vitro test system should accurately reflect in vivo skin conditions . the wealth of results obtained employing artificial sebum formulations is characterized by the poor physiological value of most models , lacking essential components and presenting unrealistic percent ratios of the single molecules . variables like sweat composition , ph , temperature , and so forth need careful standardization , in order to guarantee reliability of the in vitro test system . the combination of tridimensional skin tissue models with ssl formulations bears promising results for in vitro applications , providing both corneum and sebum components to the skin barrier functional model . in view of the physiological relevance of the surface lipid composition for an optimal performance of the skin organ , the modulation of skin surface lipids composition may represent a powerful approach to enhance skin photo - protection , to prevent skin ageing , and to control microbial symbiotic and pathogenic colonization and consequent skin diseases . this goal is attempted traditionally through local application of lipid and/or lipophilic antioxidant - enriched cosmeceutical formulations , with immediate beneficial effects , albeit in most cases not durable after treatment discontinuation . the possibility to control ssl composition and skin function in general by diet or nutriceutical intervention is far more intriguing and promising a perspective . the administration of skin - tropic lipid - based oral supplements has been claimed to be effective , though available reports are sparse [ 98 , 99 ] . the analysis of the existing literature brings to the conclusion that nutritional factors provide benefits to skin physiology , but information on the effects of low - to - moderate doses of nutrients consumed in the long term by healthy individuals is lacking , as well as are the data on the direct effects on basal skin properties , including hydration , sebum production , and elasticity , up - to - date scant , and often contradictory . the largest part of information refers to pufa administration as a treatment for scaly skin , the observation that the formation of pufa oxidation products on the skin can be suppressed by linoleic acid supplementation through vegetable oil sources demonstrates that skin lipid film can be modulated through the diet more efficiently that through topical application . a low - glycaemic diet regimen also proved effective in the normalization of sebum production and composition in acne patients , confirming the feasibility of the control of disease concurrent factors influencing sebaceous gland physiology through the systemic approach . reported no significant alteration of ssl after a daily oral dosage 200 mg of coq10 for 10 days . oral supplementation of sq to mice resulted in a marked dose - dependent upregulation of cellular and nonspecific immune functions . in healthy volunteers , low- or high - dosage ( 13 or 27 g daily ) sq per os , per 90 days resulted in significant improvement of antiaging effects on photoaged skin , with facial wrinkle decrease , as confirmed by molecular markers of uv - induced skin damage . facial erythema decrease and pigmentation increase were observed though high dose oral administration of fat induced some side effects . data discussed so far allow us to consider skin surface lipids , and in particular its polyunsaturated component squalene , as a main target for pro - oxidant agents on the skin . sq peroxides generated locally upon natural or therapeutic uv irradiation are mediators of the immunological response and the melanogenic process in the skin . the ssls represent suitable markers for the diagnosis of skin disease or aging , and for treatment efficacy monitoring , provided that the results are combined with the analysis of the strictly interconnected systemic biomarkers of oxidative damage and antioxidant defences . | skin surface lipid ( ssl ) film is a mixture of sebum and keratinocyte membrane lipids , protecting skin from environment .
its composition is unique for the high percentage of long chain fatty acids , and of the polyterpenoid squalene , absent in other human tissues , and in non - human primates sebum . here ,
the still incomplete body of information on ssl as mediators of external chemical , physical , and microbial signals and stressors is revised , focusing on the central event of the continuous oxidative modification induced by the metabolic activity of residential and pathological microbial flora , natural or iatrogenic uv irradiation , exposure to chemicals and cosmetics .
once alpha - tocopherol and ubiquinol-10 antioxidant defences of ssl are overcome , oxidation of squalene and cholesterol gives rise to reactive by - products penetrating deeper into skin layers , to mediate local defensive inflammatory , photo - protective , immune reactions or , at higher concentrations , inducing local but also systemic immune depression , ultimately implicating skin cancerogenesis .
qualitative modifications of ssl represent a pathogenetic sign of diagnostic value in dermatological disorders involving altered sebum production , like pytiriasis versicolor , acne , atopic or seborrheic dermatitis , as well as photo - aging .
achievements of nutriceutical interventions aimed at restoring normal ssl composition and homeostasis are discussed , as feasible therapeutic goals and major means of photo - protection . |
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despite recent improvements in adhesive dentistry , materials and esthetic demands of the patients have contributed the frequent use of composite materials in posterior teeth , post - operative sensitivity still remains a problem . this complication usually fades within the first few weeks , but may last for a longer period of time , and occasionally it results in the failure of the restoration . according to brannstrm , post - operative sensitivity is related to the bacteria and the microleakage between the pulp and the oral cavity . beside this theory , polymerization shrinkage of the resin - based adhesive materials result in internal stress and gap formation , especially for the multistep bonging agents , because gaps would be formed in the interface between the bonding resin and dentin , which are more prone to microleakage and post - operative sensitivity between the tooth and the material . when using recently developed dentin adhesives , one of two strategies to interact with the dentin smear layer can be used : the total - etch technique or the self - etch technique . for those using the total - etch technique , the quality of the resin - dentin adhesion can be greatly affected by the duration of the acid - etching process and the amount of surface wetness present during the adhesive application . self - etching primers use non - rinsing acidic monomers that simultaneously dissolve the smear layer and prime the dentin and enamel . the latest generation of all - in - one self - etching systems combines the etchant , primer , and adhesive in two containers or in a single component , which do not require a separate etching step result in a more uniform penetration of resin into the etched dentin maintaining a better seal , excellent clinical effectiveness with a reduced clinical application time and technique sensitivity . within the clinical application technique , sensitivity discrepancies of the adhesive bonding strategies , clinical experience , and skill might also affect the performance of a restoration unless the standard instructions are strictly followed . the aim of this study was to evaluate the effect of different adhesive strategies and operator skill on the development of post - operative sensitivity in class i resin composite restorations . the null hypothesis tested was that the operator variability would not affect the prevalence and severity of post - operative sensitivity when cavities for posterior composites were tested using a three - step total - etch dentin bonding system , a two - step self - etch bonding system , and a single - step self - etch system . this clinical study was conducted at the university of istanbul , faculty of dentistry , department of operative dentistry under the approval of the ethical research committee of the university of istanbul , faculty of medicine , turkey ( ethics i d : 2008/1212 ) . patients having occlusal primary caries or needing occlusal restorations were chosen and provided informed consent to participate in the study . a total of 188 molar teeth in 39 patients ( males and females , ages 18 to 30 years ) required occlusal composite restorations . each patient received minimum three restorations including one of each adhesive system , and maximum 2 of each material were applied in each quadrant by only 1 and 2 molars . compositions and application procedure according to the manufacturer 's instructions of the adhesive systems used in the study inclusion criteria were the presence of molar teeth requiring composite restorations either for replacement of an existing amalgam or composite filling ( secondary caries or aging ) or for treatment of primary carious lesions . occlusal carious lesions and existing restorations were adjudged according to a periapical radiograph to be no more than one - half the distance from the dentino - enamel junction to the pulp . exclusion criteria were negative reactions to the vitality test , having non - carious lesions or loss of dental hard tissues , increased hypersensitivity and a pre - operative sensitivity in the carious tooth or associated restorations , allergies to composite materials , pregnancy , systemic diseases , undesired para - functional habits ( bruxism , clenching , etc . ) , and malocclusion . restorations were randomly applied by either a 12-year specialist post - doctorate dentist ( pdd ) or a final - year dental student ( ds ) who were provided with the guidelines about the procedure and materials prior to the start of the study and were told that the guidelines to be strictly followed . cavities were prepared with a high - speed hand piece with water coolant and finished using a low - speed hand piece without beveling , and all restorations were done using cotton roll isolation with no lining materials under the composite restorations . three different dentin bonding systems- optibond fl ( kerr hawe ) ; n = 62 teeth ( 32 by the pdd , 30 by the ds ) , clearfil protect bond ( kuraray , japan ) ; n = 63 teeth ( 32 by the pdd , 31 by the ds ) , and ibond ( herauskulzer , germany ) ; n = 62 teeth ( 32 by the pdd , 30 by the ds ) were used following the manufacturer 's instructions . restorations were completed using a combination of the same manufacturer 's composite resin materials ( herculite xrv ; micro hybride composite ; kerr dental corporation , orange , ca , usa ; clearfil ap - x ; micro hybride composite ; kuraray medical inc . , okayama , japan ; charisma , universal composite ; heraeus kulzer gmbt , hanau , germany ) . color matching was disregarded , and the same color of composite material ( a2 ) was applied incrementally for each patient . each layer was light - cured for 40 seconds ( vip light curing unit , 500 - 600 mw / cm , bisco , schaumburg , usa ) following the manufacturer 's recommendations . articulation was checked carefully , and occlusal adjustments were performed using water spray with pear- and flame - shaped carbide , diamond finishing burs and further polishing was performed using a sequence of disks ( sof - lex pop - on ; 3 m espe , st . post - operative sensitivity was assessed at 24 hours , 1 month , 3 months , and 6 months using the vas score . post - operative sensitivity of each restored tooth was evaluated with a standardized cold - ice stimulus applied with ice stick and air stimulus by air blown from air syringe with a standardized distance of 5 mm ( 45 psi ) . ( 0 cm ) at one end and intolerable sensitivity ( 10 cm ) at the other end . we asked each patient to place a vertical mark on the vas line to indicate the intensity of sensitivity level after the administration of the stimuli for each tooth . a blinded co - author determined the sensitivity scores and quantified each patient 's response to each restoration by measuring the distance in cm from the anchor word ( 0 cm ) to the mark . during the statistical analysis , we used teeth rather than participants as the statistical units to evaluate the intensity of post - operative sensitivity to the materials tested . post - operative sensitivity was statistically analyzed by comparing dependent variables , such as type of adhesive system , operator skill , kind of stimuli , and time . descriptive statistics are expressed as mean standard deviation ( sd ) based on the 10 cm vas scores . as the mean scores of groups did not show the normal distribution , the differences between the treatment groups were analyzed using the kruskal - wallis one - way analysis of variance . , wilcoxon signed rank test was also used to determine the differences between participants responses to each material . commercially available software ( spss 11.0 for windows , spss , chicago ) was used to perform the statistical analysis , and the significance was set at p = 0.05 . during the statistical analysis , we used teeth rather than participants as the statistical units to evaluate the intensity of post - operative sensitivity to the materials tested . post - operative sensitivity was statistically analyzed by comparing dependent variables , such as type of adhesive system , operator skill , kind of stimuli , and time . descriptive statistics are expressed as mean standard deviation ( sd ) based on the 10 cm vas scores . as the mean scores of groups did not show the normal distribution , the differences between the treatment groups were analyzed using the kruskal - wallis one - way analysis of variance . in addition , wilcoxon signed rank test was also used to determine the differences between participants responses to each material . commercially available software ( spss 11.0 for windows , spss , chicago ) was used to perform the statistical analysis , and the significance was set at p = 0.05 . one hundred and eighty - eight restored teeth in 39 subjects were evaluated at 24-hours , 1 month , 3 months , and 6 months [ figure 1 ] . table 2 shows the mean and standard deviations for the severity of response to air and cold stimuli following restorations with each bonding system performed by either the pdd or a less experienced ds . the mean vas scores and standard deviations of the groups treated with three adhesive systems by the pdd or ds are also shown graphically in figures 25 . during the 6-month evaluation period , no tooth exhibited signs of pulpitis , and none of the patients complained of severe pain or strong and intolerable sensitivity . within the treatment group comparisons , the results of the kruskal - wallis test showed that participants in all groups were significantly more sensitive to cold than to air stimuli at data collection points ( p < 0.05 ) . the mean visual analog scale scores for the three adhesive system applied by both post - doctorate dentist and dental student after recieving air and cold stimuli across six months the mean visual analog scale scores and standard deviations of the groups treated with three adhesive systems by the pdd in subsequent evaluations of post - operative sensitivity evaluations for air stimuli by time groups treated with three adhesive systems by the ds in subsequent evaluations of post - operative sensitivity evaluations response to air stimuli by time groups treated with three adhesive systems by the pdd in subsequent evaluations of post - operative sensitivity evaluations for cold stimuli by time groups treated with three adhesive systems by ds in subsequent evaluations of post - operative sensitivity evaluations ; response to cold stimuli by time the vas scores decreased through the 6-month evaluation period in response to air and cold stimuli for all three materials used by both operators . in response to air stimuli , vas scores of optibond fl , clearfil protect bond , and ibond decreased during the time intervals without any statistical differences when applied by the ds ( p > 0.05 ) ; protect bond showed a significant decrease in the intensity of post - operative sensitivity between the 24-hours and third month ( p < 0.05 ) . when the restorations were performed by the pdd using protect bond and optibond fl , there was no significant difference ( p > 0.05 ) in vas scores in response to air stimuli ; ibond showed an insignificant lower score at 24-hours than the 6-month recall ( p > 0.05 ) . a regular decrease in post - operative sensitivity to cold response for each of the 3 materials applied by either the pdd or ds was demonstrated without any significant differences ( p > 0.05 ) . the mean vas scores for teeth treated by the pdd with protect bond to cold stimuli at the first month were significantly lower than the baseline score ( p < 0.05 ) , whereas the scores for optibond fl applied by the pdd showed a statistically significant decrease in response to cold stimuli between the 24-hours and 6-month measurements ( p < 0.05 ) . the null hypothesis tested that there would not be a difference in the prevalence and severity of post - operative sensitivity when cavities in posterior composites were restored with a three - step total - etch dentin bonding system , a two - step bonding system , and a single - step , self - etch system was rejected . included in the measurements was the presupposition that performance of these restorations by a post - doctorate dentist ( pdd ) and a less experienced dental student ( ds ) would make a difference . in the current study , a total of 188 restorations in 39 subjects were performed by pdd or ds who was working under the close supervision of faculty members , and the patients were assessed for 6 months for post - operative sensitivity . only the teeth ( primary carious lesions or secondary caries ) requiring medium - sized composite restoration without any spontaneous pain or severe pulpal sensitivity we did not group the composite restorations by the cavity depth as deep or shallow preparations and used only the lesions not to be more than half - way between the dentino - enamel junction and the pulp and excluded deep caries lesions to differentiate the outcome of post - operative sensitivity from the symptoms pulpal inflammation . in the present study , the restorations were performed without using anesthesia to inhibit pulpal motion . in most studies , the rubber dam was preferred as a protective measure against failure and to guarantee a low incidence of post - operative sensitivity in composite restorations . however , according to another clinical data , the use of anesthesia and rubber dam did not have any significant influence on post - operative sensitivity . cotton roll isolation was carefully used to obtain ideal clinical outcomes in the present study evaluating the cavities only limited by the occlusal surface . post - operative sensitivity might also be caused by penetrations of components of the adhesive systems into the pulp or by micro-/nanoleakage , allowing movement of the dentin liquid into those dentin areas where optimal adhesion and sealing of the dentin was accomplished . polymerization shrinkage , bulk filling technique , incomplete coating of the dentin surface with dental adhesives , and traumatic occlusion might also be responsible for post - operative sensitivity in posterior composite restorations . generally , posterior composite restorations are prone to higher post - operative sensitivity due to the etching procedure , polymerization shrinkage caused by the stress generated upon curing , and cuspal deflection related to different adhesive techniques . the curing procedure and intensity of the light device also affect post - operative sensitivity in terms of polymerization shrinkage . to eliminate these kinds of risks in this study , composite resins were applied using the incremental filling technique , and adequate light - curing was carefully performed . in the current study , due to their ease of preparation and lower variability in cavity boundaries , however , it has been demonstrated that in occlusal cavities , the c - factor is greater than 5 , thereby producing a higher concentration of stress because only one of the current 6 surfaces is free . concerning the c - factor , the highest polymerization stresses would be exhibited , which would cause higher post - operative sensitivity when occlusal cavities with 5 cavity walls are restored . however , the results of our study with its resulting low levels of post - operative sensitivity did not support this finding . occurrence of low post - operative pain sensation levels after different dentin bonding strategies used in the current study is in accordance with the findings of unemori et al . who studied the symptoms of post - operative sensitivity after resin composite restorations for all types of cavities performed by undergraduate students and found that only 11% of all teeth showed post - operative sensitivity . similar results were found by opdam et al . who reported post - operative sensitivity in 14% of class i resin composite restorations , and no sensitivity to cold was reported for self - etch adhesives . the use of a lining under the restorations to prevent the undesirable effects of restorative materials is still under discussion . it is reported that there was no significant difference in post - operative sensitivity when a liner or base had been used compared to restorations in which no lining was used . on the contrary , acid etchants and composite materials are reported to have no adverse effects on the dental pulp and that these restorations do not require a protective liner or base because the dentin - bonding materials function as a direct pulp - capping agent . in the present study , all of the restorations were performed without applying a liner or base material since the cavity depth standardized in the study did not require any pulp - capping procedure . a subjective assessment method ; a visual analogue scale ( values 0 to 10 ) providing effective statistical test evaluation was used to evaluate the intensity of post - operative sensitivity in the present study . vas provides more uniform instructions to participants , and the researcher avoids descriptors such as mild , moderate , and severe , which can be interpreted quite differently from one participant to another . the main problem with the three - component procedure has been unpredictability with regard to post - operative sensitivity . when three - step bonding agents are used meticulously and adequately placed , dentin desensitization will be assured , otherwise uncomfortable tooth sensitivity is likely to be a result of the poor sealing of dentinal tubules . technically , a less complicated material could be more suitable in undergraduate teaching programs where less experienced students are involved . a low frequency of post - operative sensitivity was reported for self - etching adhesives , which are less technique - sensitive than total - etch adhesives . self - etching adhesives cause less post - operative sensitivity regarding that the smear layer is not completely removed by acid etching and the de - mineralized dentin and exposed collagen - network can be completely infiltrated by resin to form a uniform hybrid layer and a reliable adhesion . in the current study , optibond fl applied by both the pdd and ds showed higher vas levels than protect bond after the application of cold stimuli . if the practitioner fails to control the moisture content of the dentin that is a major concern and difficult to standardize , post - operative sensitivity may arise due to the collapse of the collagen network . another reason is that the etching with phosphoric acid widens the dentinal tubule openings , and these may not be completely sealed by the adhesive resin . thus , the unsealed microporous zone could then permit the shift of hydraulic dentin fluid and penetration of microorganisms into dentin tubules , possibly resulting in a slower rate of resolution of post - operative sensitivity . protect bond is a simplified two - step antibacterial self - etching adhesive . due to its contact - active antibacterial monomer mdpb , it disinfects dentin and prevents the diffusion of new bacteria via microleakage . the previous version of this adhesive material without mdpb ; clearfil se bond , a self - etching primer , has been shown to produce a thin hybrid layer that is completely penetrated by the adhesive resin and proven to yield reliable results in terms of bonding effectiveness and durability . self - etching adhesives are not likely to result in a discrepancy between the depth of demineralization and the depth of resin infiltration because both processes occur simultaneously . primer of protect bond contains functional monomer 10-mdp dissolved in water and ethanol that results in a ph of approximately 2 . thus , smear plugs are fixed at the internal tubular walls , resulting in simultaneous demineralization and infiltration of enamel and dentin to form a continuum in the substrate incorporating the smear plug in the resin tag . in addition to its simplified bonding technique , protect bond eliminates rinsing and drying steps , which reduces the over - wetting and over - drying that can cause a negative effect on adhesion . it seems possible that the phosphoric acid used with optibond fl exerts higher osmotic pressure on the pulp dentin complex than the self - etching primer , which may explain the small difference in post - operative sensitivity . ibond is a single component self - etching adhesive containing glutaraldehyde as a desensitizer associated with its non - rinse acidic monomers . a blend of monomers ( udma and 4-meta ) in a high amount of solvents ( acetone and water ) is present in the formulation . the bonding effectiveness of mild two - step , self - etch adhesives seems quite durable in contrast to all - in - one products such as ib adhesives that produce less durable bonds . ibond does not contain hema in its formulation , and its complex blend of hydrophilic / hydrophobic ingredients , water , and a large amount of solvent makes these adhesives more prone to phase separation that leads to lower bond strength and mechanical properties . there is a common belief that the success of posterior composite restorations results from the skill of the operator as well as the material 's characteristics and placement techniques . to give an advanced oral health care to patients , it is important that all recent dental school graduates are skilled in a range of techniques , including the restoration of posterior teeth with resin - based composite materials . dental students may experience difficulty in the placement of posterior composite restorations and post - operative sensitivity if the procedure is not performed adequately in their under - graduate clinical practice . experts suggest that dental students should have a clear understanding of the basics and principles of the clinical application of adhesives . in this study , at all time intervals , post - operative sensitivity occurred in response to air and cold stimuli after the application of all materials applied by the pdd were lower than in restorations performed by ds . the lower vas scores ( for air stimuli ) following the restorations performed by the ds were obtained for ibond at baseline and during the following 6 months in response to air stimuli . the results demonstrated that the application complexity and the variability of the dentin bonding systems affected the post - operative sensitivity , which was low for all tested materials but depended to some degree on the experience and skill of the operator . the treatments performed by the dental student were conducted under the supervision of a staff member , which might have had a positive effect on the low rate of post - operative sensitivity . operators were told to closely follow the manufacturer 's instructions and to carefully perform the restorations . the post - operative sensitivity in all treatment groups was very low following applications by both kinds of operators who worked in a faculty clinic where application procedures are strictly followed according to the principles in the adhesive literature . the authors assume that the low post - operative sensitivity scores are due to the very careful application of the treatment procedures , the correct use of adhesive materials by following the manufacturer 's instructions , and clinical techniques that might depend on materials used . | objective : to evaluate the post - operative sensitivity of occlusal restorations using different dentin adhesives performed by an undergraduate and a post - doctorate dentist.materials and methods : one hundred and eighty - eight molar occlusal restorations were placed in 39 patients ( ages between 18 and 30 ) using 3 different kind of adhesive systems ; optibond fl ( obf ) , clearfil protect bond ( cpb ) , and ibond ( ib ) by a post - doctorate dentist or a fifth - year dental student according to the manufacturers instructions .
post - operative sensitivity to cold and air was evaluated using a visual analog scale ( vas ) after 24 hours , 30 , 90 , and 180 days .
data were analyzed using the mann - whitney u and friedman tests ( p < 0.05).results : post - operative sensitivity scores for obf and cpb were higher for the dental student ( p < 0.05 ) , while ib scores did not differ statistical significantly according to the operator ( p > 0.05).conclusion : operator skill and experience appears to play a role in determining the outcome of post - operative sensitivity of multi - step adhesive systems although the post - operative sensitivity was low .
it is suggested that the less experienced clinicians ( rather than experienced clinicians ) should better use the self - etching dentin bonding systems with reduced application steps to minimize the potential risk of post - operative sensitivity of dental adhesives . |
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there is strong epidemiological and genetic evidence that genetic factors play a major , though variable role , in its pathogenesis . migraine patients are characterized between attacks on various cns - evoked responses by a deficit of habituation [ 1 , 2 ] , which may have a familial character [ 35 ] . by contrast , other mild abnormalities have been identified that are unlikely to play a pathogenic role : a decreased safety factor at the neuromuscular junction on single fiber emg ( sfemg ) [ 79 ] and subclinical cerebellar hypermetria in the horizontal plane on opto - electronic analysis of upper arm - reaching movements . if this is correct , one may expect that in the same individual they be of a similar degree . we have therefore compared in the same migraine patient the sfemg and 3d - movement analysis . thirteen migraine patients ( ichd - ii ) were recruited from the headache clinic of the headache research unit in lige , belgium . eight patients suffering from typical aura with migraine headache ( mta ichd - ii code 1.2.1 ; three women and five men ; median age 28.5 years ; range 1367 ) and five patients with migraine without aura ( mo ichd - ii code 1.1 ; three women and two men ; median age 40.0 years ; range 3151 ) underwent both stimulation - sfemg and 3d - movement analysis . none of them had any other medical condition detectable by history and clinical examination ; none was taking drugs on a regular basis , nor had taken any drug within 3 days before the recordings . the recordings took place at least 3 days after and before an attack ( checked by telephone - interview ) . the study was conducted after approval of our institution s ethics committee and performed in accordance with the ethical standards of the 1964 declaration of helsinki , with the understanding and consent of each involved subject . a nicolet viking iv device ( nicolet biomedical , madison , wisconsin , usa ) was used for stimulation single fiber electromyography . single muscle fiber activity was recorded with 25-mm - long single fiber needles ( medelec neurodiagnostic accessories , ref : 16829 , witney , oxfordshire , uk ) , and the motor nerve was stimulated with nicolet teflon - insulated monopolar needles . we stimulated suprathreshold the motor branch of the radial nerve and assessed the variability in latency , i.e. the jitter ( fig . 1 ) , of single fiber action potentials in m. extensor digitorum communis ( edc ) of the right arm . off - line analyses of recordings were performed , and on average 18 artifact - free edc muscle fibers per patient were selected to assess their mean mcd . 1figures representing a normally ( on the top ) and an abnormally ( on the bottom ) jittered fibers figures representing a normally ( on the top ) and an abnormally ( on the bottom ) jittered fibers movements were recorded at 100 hz in three dimensions ( 3d ) using an infrared optoelectronic - tracking - system ( elite , milan , italy ) with a reflective marker attached to the tip of the index finger and another marker to the movement target . participants were seated with the target in the medio - sagittal plane on eye level . they were instructed to start with the right arm extended to the right , to touch the target with high precision ( but fast ) without any trunk movement , to go back to the starting position and to repeat above movements , in a given pace over 15 s ( one trial ) to result in 810 movements ( fig . 2 ) . the used cartesian coordinate system is head - fixed with a nasooccipital , a horizontal and a vertical axis and the origin in the target . since in our previous study abnormalities in migraineurs were most pronounced in the horizontal plane , we limited ourselves to an analysis of the mean deviation in the horizontal plane ( in millimeters ) , measured over four trials , each consisting of 810 arm movements . 2experimental setup for the movement task experimental setup for the movement task quantitative variables in each group of migraine patients ( mo and mta ) were expressed as medians . the spearman rank order correlation test was used to compare the values of the mean mcd on sfemg and the mean horizontal deviation on 3d - movement analysis . thirteen migraine patients ( ichd - ii ) were recruited from the headache clinic of the headache research unit in lige , belgium . eight patients suffering from typical aura with migraine headache ( mta ichd - ii code 1.2.1 ; three women and five men ; median age 28.5 years ; range 1367 ) and five patients with migraine without aura ( mo ichd - ii code 1.1 ; three women and two men ; median age 40.0 years ; range 3151 ) underwent both stimulation - sfemg and 3d - movement analysis . none of them had any other medical condition detectable by history and clinical examination ; none was taking drugs on a regular basis , nor had taken any drug within 3 days before the recordings . the recordings took place at least 3 days after and before an attack ( checked by telephone - interview ) . the study was conducted after approval of our institution s ethics committee and performed in accordance with the ethical standards of the 1964 declaration of helsinki , with the understanding and consent of each involved subject . a nicolet viking iv device ( nicolet biomedical , madison , wisconsin , usa ) was used for stimulation single fiber electromyography . single muscle fiber activity was recorded with 25-mm - long single fiber needles ( medelec neurodiagnostic accessories , ref : 16829 , witney , oxfordshire , uk ) , and the motor nerve was stimulated with nicolet teflon - insulated monopolar needles . we stimulated suprathreshold the motor branch of the radial nerve and assessed the variability in latency , i.e. the jitter ( fig . 1 ) , of single fiber action potentials in m. extensor digitorum communis ( edc ) of the right arm . stimulations off - line analyses of recordings were performed , and on average 18 artifact - free edc muscle fibers per patient were selected to assess their mean mcd . mean value of consecutive differences ( mcd ) of successive interpotential intervals , as usual in sfemg studies . 1figures representing a normally ( on the top ) and an abnormally ( on the bottom ) jittered fibers figures representing a normally ( on the top ) and an abnormally ( on the bottom ) jittered fibers movements were recorded at 100 hz in three dimensions ( 3d ) using an infrared optoelectronic - tracking - system ( elite , milan , italy ) with a reflective marker attached to the tip of the index finger and another marker to the movement target . participants were seated with the target in the medio - sagittal plane on eye level . they were instructed to start with the right arm extended to the right , to touch the target with high precision ( but fast ) without any trunk movement , to go back to the starting position and to repeat above movements , in a given pace over 15 s ( one trial ) to result in 810 movements ( fig . 2 ) . the used cartesian coordinate system is head - fixed with a nasooccipital , a horizontal and a vertical axis and the origin in the target . since in our previous study abnormalities in migraineurs were most pronounced in the horizontal plane , we limited ourselves to an analysis of the mean deviation in the horizontal plane ( in millimeters ) , measured over four trials , each consisting of 810 arm movements . 2experimental setup for the movement task experimental setup for the movement task quantitative variables in each group of migraine patients ( mo and mta ) were expressed as medians . the spearman rank order correlation test was used to compare the values of the mean mcd on sfemg and the mean horizontal deviation on 3d - movement analysis . on sfemg , the median value for mean mcd was not significantly different between mo ( 16.05 s ; range 9.5022.93 ) and mta ( 18.91 s ; range 11.5024.55 ) . the median value of mean horizontal deviation was 4.01 mm ( range 3.25 to 18.12 ) in mo and 10.74 mm ( range 1.6017.03 ) in mta , a nonsignificant difference . in mo by contrast , in mta , both variables were positively correlated ( r = 0.71 , p = 0.046 ) ( fig . 3 ) . 3scatter plot relating mean mcd on sfemg ( x axis , s ) and mean horizontal deviation on 3d analysis of a reaching arm movement ( y axis , mm ) , and linear regression lines . migraine without aura patients ( mo ) : squares , dashed line . migraine with aura patients ( mta ) : triangles , continuous line scatter plot relating mean mcd on sfemg ( x axis , s ) and mean horizontal deviation on 3d analysis of a reaching arm movement ( y axis , mm ) , and linear regression lines . our within - patient analysis shows that , in migraine with aura , the mean mcd on sfemg increases significantly with the degree of horizontal deviation in a visually guided reaching movement . this suggests that , in subgroups of migraine patients , neuromuscular transmission ( nmt ) performance and control of ballistic movements by the lateral cerebellum are similarly influenced by a common biochemical and/or neural mechanism . although no gene mutations have been identified till now , the genetic load is thought to be higher in the pathogenesis of migraine with aura than that without aura . in familial hemiplegic migraine 1 ( fhm1 ) , mutations have been found in the cacna1a gene , which codifies for the main subunit of p / q - type ca channels , heavily concentrated at the neuromuscular junction and in the cerebellum [ 1518 ] . thus , we initially hypothesized that the nmj and cerebellar abnormalities found in some migraine patients might be due to dysfunctioning ca channels . however , sfemg studies were normal in fhm1 , and mutations in the cacna1a gene are not found in patients suffering from the common forms of migraine , with or without aura . some evidence that the cacna1a gene may be involved in migraine with typical aura , and the facts that nmt impairment can be found in episodic ataxia type 2 , an allelic disorder of fhm1 , and normalizes after treatment with acetazolamide are still in favor of a possible involvement of cachannels , particularly in migraine with aura . other proteins implicated in migraine pathophysiology may constitute a common link between neuromuscular junctions ( nmj ) and the cerebellum . this is unlikely for the alpha-2 subunit of the na - k atpase , of which the gene atp1a2 is mutated in fhm2 , because the alpha-2 isoform is found at intracellular membranes and not at the nmj , where the major role is played by the alpha-3 isoform . by contrast , a different group of calcium channels , the r - type , influence neuromuscular transmission and can compensate for dysfunctioning p / q channels . moreover , they seem to play a role in cerebellar functions ; thus , their activity may influence in parallel nmj and cerebellar performances , similarly to what we found in the subgroup of migraineurs with aura . the cacna1e gene , which codes for the alpha-1e subunit of r - type channels , is precisely located on locus 1q31 , for which significant linkage was found in the common forms of migraine , and a single nucleotide polymorphism was more frequent in subgroups of patients affected by migraine with aura . in conclusion , the correlation between nmj and cerebellar performances that we have found in migraine with aura patients might be due to a common genetically determined molecular mechanism , possibly influencing ion channels functions , but this has still to be proven by appropriate genetic studies . | in previous studies , we described subclinical abnormalities of neuromuscular transmission and cerebellar functions in migraineurs .
the aim of this study was to search if these two functions are correlated in the same patient .
thirteen migraineurs [ five without aura ( mo ) and eight with aura ( ma ) ] underwent both stimulation - sfemg and 3d - movement analysis . single fiber emg ( sfemg ) results were expressed as the mean value of consecutive differences
( mean mcd ) .
precision of arm - reaching movements ( measured with an infrared optoelectronic tracking system ) was expressed as the average deviation in the horizontal plane .
median values of mean mcd and mean horizontal deviation were not different between mo and ma .
however , in ma , but not in mo , both variables were positively correlated .
thus , we conclude that neuromuscular transmission and cerebellar functions are correlated in the same patient when affected by migraine with aura .
we suggest that this correlation might be due to a common molecular abnormality . |
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the main goals of managing burns are to accelerate tissue repair and to prevent infection . infection is the most serious and fatal side effect of acute burns , which is produced by various microorganisms ; however , correct and on - time treatment is effective in curing the patient . topical antibiotic creams such as acetate mafenide and silver sulfadiazine are among the most common medicines used for the treatment of burns . silver destroys the cell wall of bacteria and allows sulfadiazine to act as an antibacterial agent , but wound healing can be affected by using this compound . acetate mafenide is a bacteriostatic drug which has bactericidal effect in high doses ; it is effective against approximately all pathogens colonized in burn wounds . however , these drugs may also have cytotoxic properties ; therefore , finding drugs with fewer side effects can be very helpful in treating this kind of patients . recently , some studies have investigated the use of honey to prevent infection and treat burns ; they used honey as a remedy for the prevention of infection in bedsores and burns . it has been reported that honey can facilitate removing necrotic tissues , increase the granulation and epithelialization speed , and reduce scars . the antimicrobial power of honey prevents the growth of bacteria on the moist environment of skin surface . the factors that make honey a useful remedy for burns are : acidic environment ( ph= 3.46.1 ) , high osmotic properties , avoids dressing from sticking to the wound , and decreases the dislodgement of granulation tissues at the time of dressing . since honey dressing reduces scar formation , it minimizes the need to graft . a number of enzymes such as invertase , amylase , and glucose oxidase and hydroxide peroxide are found in honey . the antibacterial quality of honey is attributed to the presence of oligosaccharides , glycopeptides , and peptides . sesame oil is also one among the agents that are currently used to treat burns ; it has shown different effects on wound healing . sesame oil was reported to be effective in reduction of cholesterol and blood glucose and it has antioxidant effect . olive oil was studied for its antioxidant effect and was found to have beneficial effects in cardiovascular diseases and cancers . in traditional medicine , olive oil has been considered to be effective in skin healing ; however , this issue has not been studied in advanced medicine . unfortunately , very few studies has been done on sesame oil , olive oil , and their effects in healing wounds , while some studies have shown their beneficial effects . combining honey with olive oil and sesame oil may increase their beneficial effects in healing wounds . also , this combination may reduce bacterial wound colonization ; however , no study has been conducted to examine this idea . nowadays , an ointment named olea , which is a combination of honey , olive oil , and sesame , is being used in iran for treatment of burns . this study aimed to examine the effect of this ointment on tissue repair and development of granulation tissues in patients with burns wounds . this study was approved by the ethical committee of kurdistan university of medical sciences ( project no . : 84/42 ) and was registered in iran registry of clinical trial ( rct registration code : irct2014020516490n1 ) . the inclusion criteria for patients enrolled in the study were : having second - degree burns ( burn depth of 0.25.0 mm ) ; burns covering 40% or less of their body surface area ; aged between 15 and 55 years ; gave consent to participate in the study ; referred durng the first 24 h of injury ; and had negative ulcer culture on admission . patients with underlying conditions such as diabetes , chronic renal or hepatic diseases , and those with simultaneous burns , trauma , and skin lacerations were excluded . the sample included 30 available patients in tohid hospital ( in sanandaj ) who were divided into two groups using simple randomized method and table of random numbers ; the chance of entering the control group was twice as that of the case group . the first group consisted of 10 patients who were treated using olea ointment ( manufactured in farateb , yazd , iran ) . olea ointment contains 33.4% honey , 33.3% olive oil , and 33.3% sesame oil . after washing the wound with normal saline solution , 35 mm thick layer of olea ointment was applied over the wound and closed dressing was performed every day until the patients were discharged , by one of the researchers . the second group consisted of 20 patients ; they were treated using about 1.5-mm - thick layer of acetate mafenide ointment ( 8.5% ; manufactured by sina daro pharmaceutical company , tehran , iran ) every 12 h , which is a broad - spectrum bacteriostatic ointment against gram - negative bacteria and is easily accessible . patients and researchers were aware about the treatment groups , but the microbiologist and pathologist were blinded to the treatment groups . to assess the outcomes , the burn wounds were evaluated daily after a week of intervention by a pathologist and a microbiologist for the formation of granulation tissues , debridement ( using scalpel ) , and wound culture results . if they had positive culture , they were excluded from the study and treated by routine treatment for bacterial strains . however , the excluded patients were entered in the analysis . using fisher exact test , the collected data were analyzed with spss 16 software to compare the qualitative variables between the two groups . the average time of granulation tissue formation was compared between the two groups using log - rank test . the mean age of participants was 24.8 ( 11.9 ) years . of the total number of participants , 21 ( 70% ) were male , 17 ( 56.7% ) were single , and 8 patients ( 26.7% ) were illiterate . concerning the cause of burn , the most common cause was direct fire or oil burns , which was observed in 26 patients ( 85% ) . six patients ( 60% ) in olea group and 11 patients ( 55% ) in acetate mafenide group had deep partial thickness ( second - degree ) burns . no statistically significant difference was observed between the two study groups regarding these variables [ table 1 ] . comparison of the basic characteristics of the participants in olea and acetate mafenide groups results of the study showed that none of the patients in the olea group needed to use a scalpel for debridement and debridement was performed by simple showers ; however , 13 patients ( 65% ) in the acetate mafenide group needed debridement , and the difference was statistically significant ( p = 0.001 ) . one patient ( 10% ) in the olea group and 19 patients ( 95% ) in the acetate mafenide group had positive cultures 7 days after intervention ; the difference was statistically significant ( p < 0.001 ) . the most common strains found in cultures were pseudomonas , staphylococcus aerogenes , enterobacter , and escherichia coli . as shown in figure 1 , the median time of formation of granulation tissue in the olea and acetate mafenide groups was 12 days ( range : 10.313.6 ) and 17 days ( range : 13.320.6 ) , respectively ( p < 0.001 ) . eight patients ( 80% ) from olea group and 16 patients ( 80% ) from acetate mafenide had formed granulation tissue by the end of the study , and no statistically significant differences was observed between the two groups ( p = 1 ) [ table 2 ] . comparing the duration of granulation tissue formation in both groups during the study period . the mean time of granulation tissue formation in the olea and acetate mafenide groups was 12 days ( 10.313.6 ) and 17 days ( 13.320.6 ) , respectively ( p < 0.001 ) comparison of frequency ( percent ) of main outcomes between olea and acetate mafenide groups results of this study showed that olea ointment was effective in facilitating debridement and preventing wound infection ; besides , treatment with topical olea ointment accelerated tissue repair . in our study , treatment with olea led to easy debridement . a study found that when honey is used in the process of dressing , necrotic tissue is easily detached from the wound , the wet environment generated by honey prevents the dressing from sticking to the wound , and consequently , the patient experiences less tissue damage , pain , and bleeding during dressing changes . when dressing does not stick to the wound , the newly formed tissue will not detach , and therefore , healing is accelerated and there is less pain . furthermore , using substances such as olive oil , which contains hypafix , can prevent damages caused by dressing and can facilitate washing wounds . several clinical researches have shown that using honey ointment for dressing burn is more effective than topical antibiotic ointments used in healing wounds and can reduce inflammation and swelling . also , honey ointment is effective in the formation of granulation tissue and in epithelialization . in our study , the antibacterial quality of honey is attributed to its moist and dense environment that prevents bacterial growth ; besides , honey contains some enzymes which can prevent the growth of bacteria and fungi . based on the results of a study , during the first week of treatment , 85% of patients treated with olea had sterile wounds while only 30% of patients treated with other methods had sterile wounds . also , due to its high acidity attributed to the high levels of enzymes , honey can also directly affect bacteria . however , unlike other antibacterial drugs , it does not cause tissue damage and does not have any side effects . according to a study , the average duration of tissue repair was 10.4 days in the honey group and was 16.2 days in the group treated with potatoes ; in another study , the average duration of tissue repair in two groups treated with honey and silver sulfadiazine was 15.4 and 17.2 days , respectively . based on the results of another research , the duration of tissue repair in the group treated with honey was half of that in the group treated with silver sulfadiazine ( 18.1 vs 32.7 days ) . kogan et al . showed in their study that sesame oil alone has no beneficial effects on healing burn wounds in animal samples . in another study , honey also has anti - inflammatory effects ; therefore , it can accelerate the tissue repair process as inflammation can prevent tissue repair . sesame seeds contain antioxidants and other substances that can prevent cellular damage and facilitate tissue repair . a systematic review also indicated that honey can accelerate the process and duration of tissue repair . in another study , 80% of patients treated with honey had tissue repair after 7 days , while it was only 52% in mafenide acetate groups . it seems that dressing with honey has advantages such as early wound epithelialization , reduced time taken for the wounds to test culture negative , and earlier pain relief . in our study , it seems that using honey ointment can reduce the costs of debridement surgeries and can even reduce the need to do graft , because the increased speed of granulation and epithelialization can increase the speed of healing and , consequently , will lead to minimal scarring . sesame and olive contain substances that can inhibit lipid peroxidation process , improve the supply of blood to collagen tissues , increase the fibril collagen longevity , and reduce cell damages . all these materials have special compounds that can be helpful to improve scar formation by burns ; however , in gurfinkel et al . also , we could not evaluate all forms of burn wounds and did not evaluate the scar of wound after healing . we suggest other studies to be conducted with larger sample size to evaluate the other effects of olea ointment . future studies should address the effectiveness of the olea on all forms of burn wounds . topical olea ointment is useful for treating burns , and it can prevent infections , accelerate tissue repair , and facilitate debridement . therefore , using this ointment is recommended for the treatment of grade ii burns . | background : the main goals in treating burns are to accelerate tissue renovation and prevent infection .
topical antibiotics are used in the treatment of burns , but they can cause side effects . recently , a traditional ointment ( olea ) has been used in iran in the treatment of burns .
this study examines the effect of topical honey ointment in healing of burn patients.materials and methods : in this randomized controlled trial ( rct ) , 30 hospitalized patients selected by conventional sampling ( 10 in olea group and 20 in acetate mafenide ointment group ) were evaluated .
inclusion criteria were : having second - degree burns and body surface area equal to or < 40% .
one group was treated using topical olea ointment and the other with acetate mafenide ointment ( 8.5% ) .
chi - square , fisher exact test , and kaplan meier were used .
significance level was considered as p < 0.05.results:none of the patients in the olea group needed surgery for debridement , while in the second group , 13 patients ( 65% ) needed debridement ( p = 0.001 ) . in the olea group , 1 patient ( 10% ) and in the second group , 19 patients ( 95% ) had positive cultures after 7 days ( p < 0.001 ) .
the mean time of granulation tissue formation in the olea group was 12 days ( 10.313.6 ) and in the other group , it was 17 days ( 13.320.6 ) ( p < 0.001).conclusions : olea ointment is a useful treatment for burns , and it can prevent infections , accelerate tissue repair , and facilitate debridement . therefore , using this ointment is recommended for the treatment of burns . |
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it occurs in half of patients before the age of 20 years and in a quarter before the age of 8 years . the two sexes are equally affected and there are no apparent differences in rates of occurrence according to skin type or race . nonsegmental vitiligo is the most common form of disease , accounting for 85 - 90% of cases overall . the initial cause of nonsegmental vitiligo is still debated but appears to involve immunologic factors , oxidative stress , or a sympathetic neurogenic disturbance . current treatment modalities aim to stimulate melanocyte proliferation or interfere with neural / inflammatory factors affecting melanocyte structure or function ; however , no single treatment method has been found that is consistently effective with relatively few side effects . topical corticosteroids are generally the first line of therapy in both children and adults with vitiligo . for generalized symmetrical types of vitiligo , topical clobetasol used over 2 - 6 months repigments vitiligo to some degree . recent guidelines recommend in children and adults with recent onset of vitiligo , that single treatment with a potent or very potent topical steroid should be considered for a trial period of no more than 2 months . topical corticosteroid preparations are categorized from class i to vii according to potency determined by the vasoconstrictor assay . with the introduction of super high potency adrenal insufficiency is a hormone deficiency syndrome attributable to primary adrenal diseases or caused by a wide variety of pituitary - hypothalamic disorders . in general , high potency topical corticosteroids ( class i ) such as clobetasol propionate and halobetasol propionate can produce a reversible laboratory adrenocortical insufficiency , while lower potency corticosteroids generally do not . as little as 2 g per day of clobetasol propionate 0.05% can cause a decrease in morning cortisol after a few days only . the reactivity of hypothalamic - pituitary - adrenal ( hpa ) axis can be assessed with acth test and wherein plasma cortisol levels are measured prior to and after a bolus administration of 250 g of synthetic -1 - 24-adrenocorticotropic hormone . administration of 250 g cosyntropin could be a pharmacological rather than a physiological stimulus , losing sensitivity for detecting adrenocortical failure . more sensitive and accurate , the low dose 1 g acth test is as simple and safe as the standard 250 g test . as an initial assessment of hpa function , this test has proved to be much more robust than high dose test , yet it retains all advantages of the latter ( sensitivity : 93.1% , specificity : 90% , and accuracy : 90% ) . in a meta - analysis of 12 studies ( 635 subjects ) , a basal cortisol less than 5 g / dl ( 138 nmol / l ) best predicted hypothalamic - pituitary - adrenal insufficiency ( hpai ) , while other meta - analysis of 11 studies ( 589 subjects ) found that values less than 16 g / dl ( 440 nmol / l ) at 30 min best predicted hpai . to determine if adrenocortical insufficiency occurs secondary to the use of class i ( high potency ) topical steroids in patients with vitiligo and intact cutaneous barrier ; to establish the role of measuring plasma cortisol levels by performing the acth test before , during , and after treatment with high potency topical steroids ; and finally to determine other side effects and response to treatment . to determine if adrenocortical insufficiency occurs secondary to the use of class i ( high potency ) topical steroids in patients with vitiligo and intact cutaneous barrier ; to establish the role of measuring plasma cortisol levels by performing the acth test before , during , and after treatment with high potency topical steroids ; and finally to determine other side effects and response to treatment . this was a prospective , randomized , double - blinded and single crossover study in which patients of 18-years or older with localized or disseminated vitiligo and with an affected body surface area of 20% or less were included . we excluded patients with segmental vitiligo , those with history use of steroids or any other medications within the past year that could interfere with steroid or cortisol metabolism , metabolic or autoimmune diseases , adrenal insufficiency , hiv , psychiatric disorders , pregnancy , lactation , granulomatous diseases , neoplasms , and panhypopituitarism . after the screening visit , where a complete physical exam and determination of the affected body surface area ( 1 hand with fingers by the palmar side = 1% body surface area ) were performed , informed consent was signed , and then the 1 g acth test was performed before starting treatment and again on weeks 6 and 12 . for this test , a disposable 21 g infusion kit ( terumo corporation , tokyo ind ) was used to draw venous blood samples and also to infuse synthetic 1 - 24 acth ( cortrosyn , amphastar ) . on each test , blood samples were obtained before ( basal ) and after 30 min of the infusion of acth . commercial electrochemiluminescence kits ( elecsys 2010 , roche hitachi ) were used to determine plasma cortisol levels . all samples were processed by duplicate , with intra- and inter - assay coefficients of variation determined for low , mid , and high values up to the moment when measures were performed . after having results from the screening visit and if normal , patients were randomized and assigned into group 1 ( clobetasol propionate 0.05% cream during 12 weeks ) or group 2 ( placebo during 6 weeks and then clobetasol propionate 0.05% cream during 6 weeks ) . treatment was manufactured and prepared into plastic jars that contained 50 g of steroid or placebo . patients were instructed to apply the treatment once a day with fingertip unit only for vitiligo areas of the neck , trunk , and extremities ; and once a day , on an on - and - off weekly basis on the face , axilla , genital area , and groins during 12 weeks , with a maximum of one jar ( 50 g ) per week . after week 6 , patients from group 2 were crossed over to start treatment with clobetasol propionate 0.05% cream for the remaining 6 weeks . all patients were appointed for medical follow - up every three weeks until the completion of the study ( week 12 ) ; where complete physical exam , determination of the affected body surface area in percentage , follow - up pictures , adverse effects , and amount of treatment used per week were recorded . an analytic weight was used to calculate the amount of grams used per week , that is , by weighing all the used returned jars . the amount of cream leftovers then was substracted from the original weight on every jar . adrenocortical insufficiency was considered for any baseline cortisol level less than 5 g / dl or less than 16 g / dl after the 30 min i.v . response to treatment was evaluated by percentage of repigmentation achieved at week 12 ( or the last follow - up visit ) from baseline as follows : worsening of the affected body surface area , no repigmentation , mild repigmentation ( < 25% ) , moderate repigmentation ( 25 - 50% ) , good repigmentation ( 51 - 75% ) , and very good repigmentation ( > 75% ) . response to treatment was considered if 25% or more repigmentation of the total affected body surface area was achieved . analysis of variance ( anova ) was performed to compare intragroup cortisol levels at different time points : baseline , weeks 6 , and 12 in both groups . the mann - whitney test was also performed for non - normally distributed continuous data at baseline , weeks 6 , and 12 in order to compare intergroup cortisol levels . chi - square test was used for categorical variables and to determine response to treatment . this study was approved and based in accordance with the ethical standards of our institution 's ethical and investigation committee on human experimentation and with the declaration of helsinki ( 1975 ) , as revised in 2000 . a total of 22 males and 22 females ( n = 44 ) were included . in each group mean ages for group 1 and 2 were 35.05 ( range : 18 - 59 years ) and 35.82 ( range : 18 - 57 years ) , respectively . in both groups , the mean affected body surface area was 8.5% ( range : 1 - 18 ) for group 1 and 7.64 ( range : 1 - 20 ) for group 2 . mean weekly dose in both groups were similar , 23.38 g in group 1 and 24.92 in group 2 [ table 1 ] . one patient from each group voluntarily retired from the study at week 9 . after determining cortisol levels and comparing the intergroup values , basal and 30 min cortisol levels were higher in group 1 except at week 12 . the statistical analysis between both groups at week 0 ( p = 0.078 ) , week 6 ( p = 0.542 ) , and week 12 ( p = 0.91 ) was not significant . the intragroup values in group 1 showed progressive decreasing cortisol levels from week 0 ( basal = 18.59 , 30 min = 27.25 ) to week 6 ( basal = 15.82 , 30 min = 24.83 ) and week 12 ( basal = 15.51 , 30 min = 24.62 ) , respectively ; while also decrease in cortisol levels occurred in group 2 but only from week 0 ( basal = 15.79 , 30 min = 25.44 ) to week 6 ( basal = 14.89 , 30-min = 23.65 ) , but not on 12 week ( basal = 15.82 , 30-min = 26.72 ) where surprisingly cortisol reached its top level . the statistical analyses in both groups for basal and 30-min cortisol levels were also not significant [ tables 2 and 3 ] . none of the 44 patients enrolled in the study fulfilled the criteria for adrenocortical insufficiency , including two patients that retired at week 9 . plasma cortisol levels at 0 min ( basal ) plasma cortisol levels at 30 min after examining treatment response or failure , our data demonstrated that there were more patients who responded to treatment in group 1 than in group 2 ( 7 vs 1 , respectively ) . by the same way , there were more treatment failures in group 2 than in group 1 ( 21 vs 15 , respectively ) . the statistical analysis between both groups was significant ( p = 0.019 ) [ table 4 ] . treatment response vs failure a total of 57 adverse effects were present in 28 patients ( 63.63% ) , being most from group 1 ( 36 ) . it is important to emphasize that five cases of acne , two of contact dermatitis , and one of foliculitis developed in group 2 after being crossed over and started on clobetasol . for statistical analysis and comparison of adverse effects between groups , patients form group 2 were further subdivided into side effects that occurred during weeks 0 - 6 ( placebo ) and during weeks 7 - 12 ( clobetasol ) . the only statistical significant adverse effects found were acne ( p = 0.035 ) , telangiectasias ( p = 0.043 ) , and xerosis ( p < 0.001 ) [ table 5 ] . the first reports about adverse effects of topical corticosteroids became available in 1955 after the use of fludrocortisone . the local side effects consist of atrophy , striae , purpura , acne , and telangiectases . less commonly ; local hypertrichosis , hypopigmentation , glaucoma , and allergic contact dermatitis occured . the potential systemic side effects of their use include diabetes , hypertension , and hpa axis suppression . allenby et al . , reported adrenal suppression in as many as 64% of adult patients with lichen planus , atopic dermatitis , and psoriasis ( altered skin barrier ) when using 50 g or more of potent class i topical corticosteroids ( clobetasol propionate ) for greater than 2 weeks . on the contrary , similar serum cortisol levels were achieved in both patients with lichen planus ( altered skin barrier ) and healthy volunteers treated for 3 weeks with 500 mg of a 0.05% fluocinonide gel ( class ii ) three times a day for 3 weeks . additionally , wester et al . , found no significant difference in the percentage of hydrocortisone ( class vii ) absorbed in psoriatic human skin , as compared with healthy skin . this can be explained by the different steroid potency and vehicles used , and also because of higher doses and altered cutaneous barrier in most of these studies , as compared with our study where a strict maximum dose of 50 g per week and intact cutaneous barrier were considered . these seem reasonable explanations for the results found in our study where no adrenocortical alterations were found for up to 12 weeks while on steroid treatment , and the importance of the acth test . we also consider that further studies are needed if the dose of 50 g per week is going to be used for longer than 12 weeks . nevertheless , the risk of adrenocortical insufficiency in children can be possible and dependable on steroid potency , frequency of application , affected body surface area , and barrier function of the skin . , in a retrospective study , where abnormal cortisol levels were found in 29% of pediatric patients with vitiligo ( intact skin barrier ) and topical steroids ( moderate to high potency , 1 - 4 applications per day , daily and on - and - off regimens for 3 - 8 weeks ) . the children with head and neck involvement were 8.26 times more likely to have an abnormal cortisol level compared with children with other affected areas . in our study we also included patients with head and neck involvement , and again no adrenocortical alterations were found . despite its proven strength , it should be emphasized that the 1 g acth stimulation remains a screening test . however , it can replace the standard high dose acth stimulation in screening for adrenal insuffciency . by knowing the accuracy and precision of the 1 g acth test and that no significant statistical differences were found in both inter- and intragroup cortisol values , we consider this test as nonessential in adults , but only if intact cutaneous barrier is present and the maximum steroid dose is not exceeded . regarding local adverse effects , cutaneous atrophy has been considered the most common side effect , but in our study we found that the most common was pruritus followed by acne and xerosis . this can be evidenced by decreased formation of lipid lamellar bodies and delayed barrier recovery with increased transepidermal water loss . although it was not frequent ( only three cases ) , but with statistical significance , the risk of developing telangiectasias has to be considered as steroids stimulate human dermal microvascular endothelial cells , being characterized by an abnormal dilatation of capillary vessels and arterioles . non - mycotic cutaneous infections were the most common infections ( foliculitis and herpes ) , with lower rates as compared to other studies ( 6.8 vs 16 - 43% ) . , reported repigmentation rates of 64% , but many combination regimens and more than single day application treatments were included . the studies of clayton et al . , and kandil et al . , showed that the use of a highly potent ( clobetasol ) or potent ( betamethasone ) topical steroid can repigment vitiligo , but only in a small proportion of cases . clayton found 15 - 25% repigmentation in 10 of 23 subjects ( ages not stated ) and > 75% in two of 23 ( the other 11 showed no response ) , while kandil et al . , found 90 - 100% repigmentation in six of 23 subjects ( ages not given for all but one was aged 12 years ) and 25 - 90% in three ( with six showing westerhof et al . , in probably the best controlled study to date of a topical treatment , compared topical fluticasone alone or combined with ultraviolet a ( uva ) photothereapy in 135 adults . they found that the potent topical steroid fluticasone used alone for 9 months induced mean repigmentation of only 9% ( compared with uva alone of 8% ) whereas the combination of fluticasone and uva induced mean repigmentation of 31% . although in our study , response to treatment was present in 8 of 44 patients ( 18% ) , we believe that this finding was due to the short period of treatment ( 6 or 12 weeks ) and also because a single day application regimen was used . if patients with any percent of repigmentation ( 17 of 44 ) are included , the percentage of response increases to 38.63% ( vs 64% , 52% , 39% , and 9% ) . with this taken into consideration , we also believe that topical steroids could be an acceptable treatment option with the regimen employed in this study , but for longer periods of time or in combination with other treatment modalities , even the actual guidelines describe the evidence that very potent or potent topical corticosteroids can repigment vitiligo in adults . it also has to be considered that the studies supporting this statement have been poorly conducted and side - effects are common if treatment lasts for more than a few weeks . as the guideline suggests , treatment with a potent or very potent topical steroid should be considered for a trial period of no more than 2 months in children and adults with recent onset of vitiligo . in the present study , no adrenocortical insufficiency was found secondary to the use of class i topical steroids in adult patients with vitiligo and intact cutaneous barrier with a maximum dose of 50 g per week for 12 weeks , this being to our knowledge the first study that clearly proves it and for such duration of treatment . the suggested dose of topical steroids demonstrated to be safe in adults , lacking the capacity to induce significant adrenocortical alterations if we take as a reference cortisol levels before , during , and after treatment and also against placebo . if the maximum dose ( 50 g per week ) of high potency topical steroids in adults is not exceeded , it is not necessary to perform the acth test . even class i topical steroid treatment has shown to be an acceptable option for vitiligo , it seems to be incomplete as a single therapy as presented in our study , making combined therapy a better option . treatment for 12 weeks has a better response than treatment for 6 weeks , although local adverse effects are possible . | background : topical corticosteroids are used as first line of therapy for vitiligo , although side effects such as adrenal insufficiency are possible.objectives:to establish the role of acth test before , during , and after treatment with high potency topical steroids ; to determine if adrenal insufficiency occurs secondary to the use of high potency topical steroids in patients with vitiligo and intact cutaneous barrier ; and also to determine response to treatment and side effects.materials and methods : forty - four adults with non - segmental vitiligo affecting 20% or less of the body surface area were included and randomized to receive topical clobetasol propionate 0.05% cream ( group 1 ) or placebo ( group 2 ) for 12 weeks , with a maximum dose of 50 g per week .
the placebo group was crossed over after week 6 and started on clobetasol until completion of the study .
serum cortisol levels with the 1 g acth test were determined at baseline and on weeks 6 and 12.results:no adrenal insufficiency was detected nor statistical significance was achieved when comparing cortisol levels between and within the groups at baseline and weeks 6 and 12 .
group 1 had a better response to therapy but with more side effects.conclusions:doses of 50 g or less per week of clobetasol during a period of 12 weeks are safe on adult vitiligo patients , although local side effects are possible .
repigmentation rates were incomplete with single steroid therapy , making combined therapy a better option . |
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the name curcumin has been used for different unrelated dyestuffs . today , the name is used more or less exclusively for 1,7-bis(4-hydroxy-3-methoxyphenyl)- 1,6-heptadiene-3,5-dione , the yellow dye from curcuma longa ( turmeric ) and other curcuma plants.1,2 the name curcuma is based on the oriental name of the plant , which refers to its similarity with saffron ( see the hebrew word for saffron :
) .3 approximately 2000 years ago , plinius described a plant with similar features which is considered to be turmeric ( est et per se indica herba , quae cypira vocatur , zingiberis effigie ; commanducata croci vim reddit.4 [ furthermore , an indian plant exists which is called cypira and which has a similar appearance as ginger ; while chewing it constitutes the effect of saffron ] ) . hybridization of c. longa with other species results in marked differences in the curcumin content between different plants.5c . this family contains several plants which are widely used as spices and/or officinal plants , for example aframomum spec . ( alligator pepper , false cardamom ) , alpinia officinarum ( chinese ginger ) , amomum subulatum ( hill cardamom ) , boesenbergia rotunda ( chinese keys ) , c. aromatica ( wild turmeric ) , c. xanthorrhiza ( temulawak ) , c. zedoaria ( zedoary ) , eletteria cardamomum ( cardamom ) , kaempferia galangal ( galangal ) , zingiber officinale ( ginger ) , and z. mioga ( japanese ginger ) . recently , the complete transcriptome of c. longa rhizomes was analyzed.6 for a long time , the lipid - soluble yellow compound which can be isolated from the curcuma plants was used for cooking , cosmetics , and textile dying.7 in the 13th century , turmeric was introduced into europe by arab traders.8 today , turmeric and the isolated curcumin are used as colorant in many food products . the color of curcumin depends on the ph and the presence or absence of other substances like boron ( fig . 1 ) . with boron , curcumin forms rosocyanine , a reddish color ( fig . 1 ) which can be used for the detection and colorimetric quantification of boron.9 multiple biological effects have been documented for curcumin . the anti - inflammatory activity might be mediated in part by the strong antioxidant activity of its caffeic acid moiety.12 curcumin influences multiple signaling pathways.13,14 in addition to the anti - cancer related activities described below , curcumin is used for the treatment of a plethora of other diseases including pulmonary , neurological , liver , metabolic , and autoimmune diseases.15 in the last decades , there has been growing interest in curcumin and curcumin derivatives for treatment or prevention of cancer as indicated by the increasing number of curcumin - related publications in the last years ( fig . the basic biological activities of curcumin has been summarized in an excellent review.16 early observations suggested that curcumin can inhibit growth of tumor cells,17 can inhibit chemical carcinogenesis,18,19 and can be used for the treatment of patients with cancer.20 since then , few clinical trials suggest that curcumin might be a drug of interest for treatment of cancer21,22 or for the prevention of different forms of cancer.23,24 it has been shown that curcurmin and curcumin derivatives alone or in combination with other drugs increase cell death in a wide variety of tumor cells , including brain tumors,2528 sarcoma,2935 breast cancer , 4046 ovarian cancer,4749 testicular cancer,50 prostate cancer,5153 pancreatic cancer,54,55 liver cancer,56 biliary cancer,57 gastric cancer,58,59 colorectal cancer,60,61 lung cancer,6265 mesothelioma,66 renal cancer,37 bladder cancer,67 esophageal cancer,6871 head and neck cancer,7275 and lymphoma / leukemia7686 ( table 1 ) . activation of apoptosis by curcumin is accompanied by modulation of multiple signaling pathways ( hedgehog pathway , extracellular signal - regulated kinase ( erk ) pathway , wingless ( wnt ) pathway , janus kinase / signal transducer and activator of transcription ( jak / stat ) pathway , notch pathway , nuclear factor of kappa light polypeptide gene enhancer in b - cells ( nfkb ) pathway ; table 1 ) . depending on the cell type investigated , activation or inhibition of certain signaling pathways can occur . the erk pathway , for example , is activated by curcumin in monocytic leukemia cells77 whereas curcumin down - regulates erk in breast cancer cells.43 in both cases , deregulation of this signaling pathway promotes cell death . curcumin can up - regulate pro - apoptotic components of the extrinsic apoptosis pathway ( fas , fas ligand , tumor necrosis factor ( tfr ) receptors or tnf - related apoptosis inducing ligand ( trail ) receptors).30,31 on the other hand , curcumin down - regulates anti - apoptotic factors ( b cell leukemia / lymphoma 2 ( bcl2 ) , bcl2 like 1 ( bcl2l1 ) , x - linked inhibitor of apoptosis ( xiap ) , survivin).25,27,33,41,56,57,58,67,79 in addition , curcumin has been shown to inhibit telomerase which reverses immortalization of cancer cells.25 it seems that curcumin is able to induce apoptosis in cancer cells without pronounced cytotoxic effects on healthy cells . by inhibition of multi - drug resistance transporters , curcumin can overcome chemotherapy resistance of tumor cells.64,87 the inhibition of these transporters can explain the depletion of side populations in tumor cells after treatment with curcumin.88 if curcumin increases the chemotherapy sensitivity of this putative stem cell population , a combination with conventional chemotherapy might be able to kill the stem cell population . antagonistic effects of curcumin and cytotoxic drugs have been observed , however.8991 these observations indicate that the combination of curcumin with other drugs should be carefully evaluated in appropriate models in vitro or in vivo before these combinations are tested in patients . another interesting activity of curcumin is the inhibition of tumor cell motility , invasion , and metastasis.26,42,92100 this activity is mediated in part by the down - regulation of matrix- metalloproteinases . a key step in tumor metastasis is the process of epithelial mesenchymal transition ( emt ) usually accompanied by loss of epithelial cadherin ( cdh1 ) expression . inhibition of emt and up- regulation of cdh1 in tumor cells by curcumin has been described.101,102 one inductor of cell motility in cancer cells is lysophosphatidic acid ( lpa ) . lpa receptors are often expressed in tumor cells and co - expression of lpa receptors and lpa - producing phospholipases in tumor cells have been observed.103 curcumin can also inhibit lpa - induced cancer cell motility.101 taken together , these observations indicate that curcumin does not only inhibit tumor growth but also metastasis formation . however , enhanced metastasis has been described in a lewis lung carcinoma model.104 in addition to the death inducing activities of curcumin on established tumor cells , curcurmin and curcumin derivatives can inhibit or delay tumor formation in different tumor models.92,105113 curcumin significantly inhibited the formation of preneoplastic lesions in animal models for chemical colon carcinogenesis . curcumin inhibited ultraviolet radiation - induced or chemically induced skin carcinogenesis in mice.106,107 furthermore , curcumin inhibited the development of gastrointestinal tumors,108,109 mammary tumors,110 and hepatocellular tumors111,112 in models for chemical carcinogenesis . the anti - inflammatory activity of curcumin seems to be responsible at least partially for such cancer preventive activities.113 in addition to the direct activities on tumor cells and pre - malignant cells , the inhibition of the growth of carcinogenic microorganisms such as helicobacter pylori may partially account for tumor prevention by curcumin.114 very infrequently , enhanced tumor formation in experimental models has been described.115 such observations indicate that the biology of the used experimental models has not been fully elucidated and that further investigations are required in order to understand the effects of curcumin in the different models . avian reticuloendotheliosis defines a group of diseases that are observed in turkeys , broiler chickens , ducks , among other birds . the causative agent of reticuloendotheliosis is a gamma - retrovirus that transforms diverse avian cells . analysis of the t strain of this virus ( reticuloendotheliosis virus strain t ; rev - t ) led to the identification of the rel oncogene.116118 thereafter , the human rel homologue was found on chromosome 2.119 rel is a member of a gene family that includes in the human genome at least 5 members : rel , rela,120,121 relb,122 nuclear factor of kappa light polypeptide gene enhancer in b - cells 1 ( nfkb1),123 and nfkb2 ( fig . 3 ) and usually one member of the nfkb1/2 subfamily forms a heterodimer with a member of the rel subfamily . the nfkb pathway is a highly conserved signaling pathway in eukaryotes . members of the nfkb family are involved in chromosomal rearrangements that have been detected in cancer cells and gene fusions involving rel125,126 or nfkb2124,127 have been described . one example is hodgkin s lymphoma ( hl ) , one of the most frequent lymphomas in western countries . 128 the etiology of hl is unclear , but immunological and molecular properties suggest that in most cases hl cells are derived from b cells.129131 hl cells have a characteristic gene expression profile that discriminates these cells from other normal and transformed hematopoietic cells.132 with the combination of radio- and chemotherapy the majority of patients , with hl can be cured . however , the established therapy is associated with the induction of secondary malignancies , cardiac toxicities , and treatment - related infertility.133135 in addition , up to 10% of patients still can not be cured with current therapy regimes , which represent a significant number.136,137 thus , there is a clear demand to search for new treatment options and also for optimization of current treatment strategies by identifying potential treatment resistance mechanisms . an involvement of the nfkb pathway in hl has been suggested by the high expression of nfkb family members in hl.138 mutations in the nfkb inhibitors nfkb inhibitor alpha ( nfkbia ) and tumor necros factor alpha - induced protein 3 ( tnfaip3 ) have been observed in a high percentage of hl.139,140 curcumin inhibits nfkb activity in different cell types ( table 1 ) and the inhibition of the nfkb pathway can be seen by decreased expression of nfkb . the activity of nfkb is regulated by inhibitors that bind nfkb and release nfkb only after degradation . curcumin can inhibit nfkb activity indirectly by stabilization of nfkbia.40 in addition to constitutive activation of nfkb , in some tumors nfkb expression and activation is induced by treatment with cytotoxic drugs or cytokines . curcumin derivatives , for example , have been shown to decrease the tfr - induced expression of nfkb in melanoma and neuroblastoma cells.40 in some cases the anti - nfkb activity of curcumin requires the presence of additional treatment elements . in pancreatic tumor cells , for example , curcumin inhibits dna binding - activity of nfkb only in combination with aspirin and sufuraphane.54 as the final consequence of nfkb inhibition , expression of nfkb target genes is suppressed . these target genes include anti - apoptotic factors and the suppression of these factors can increase tumor cell death . in addition , the crosstalk between nfkb and other signaling pathways can lead to complex alterations of the phenotype of tumor cells after inhibition of nfkb . the activation of the nfkb pathway in hl makes curcumin an interesting drug for the treatment of hl . indeed , curcumin induces cell death in hl cells . as shown in figure 4a , incubation with curcumin leads to decreased cell viability in all hl cell lines tested whereas normal peripheral blood mononuclear cells ( pbmc ) are not affected by the used concentration of curcumin . similar results were found by mackenzie et al who showed that curcumin leads to cell cycle arrest in g2-m and reduced cell viability in hodgkin and reed - sternberg cells.143 in order to determine whether the combination of curcumin with conventional chemotherapy might have synergistic effects , hl cells were incubated either without curcumin or with a low dose ( 1.25 m ) of curcumin and varying concentration of cisplatin . the results are shown in figure 4b . with the used concentration of curcumin , only marginal effects on cell viability were observed . in contrast , the combination of curcumin with cisplatin had a strong cytotoxic effect on the cells . however , inhibition of the fanconi anemia ( fa ) pathway might be an important factor for the synergistic effect of cisplatin and curcumin on hl cells . ubiquitination of the fa complementation group d2 ( fancd2 ) protein in response to dna damage is an important step during repair of cross - links in the dna , caused by cisplatin . chirnomas et al144 showed that curcumin inhibits the fa pathway by inhibition of ubiquituation of fancd2 . this inhibition sensitized ovarian and breast tumor cells for cisplatin.144 it remains to be shown whether similar mechanisms are responsible for increased cisplatin sensitivity of curcumin treated hl cells . the hl cell lines used in our study outlined above were established from patients with refractory disease and are not perfect models for the situation in most patients in vivo . however , the increased cell death of these highly chemotherapy resistant cell lines after treatment with curcurmin seems to be encouraging . curcumin shares the nfkb - inhibitory activity with another interesting dietary phytochemical : capsaicin.145 curcumin can bind the vanilloid receptor trpv1 ( transient receptor potential cation channel , subfamily v , member 1 ) , the same receptor that is activated by capsaicin.145 however , curcumin binding to trpv1 did not result in the same signaling . trpv1 is not the only receptor for curcumin and interestingly , curcumin can bind the aryl hydrocarbon receptor ahr.146 many carcinogenic polycyclic aromatic hydrocarbons are carcinogens only after in vivo activation . cytochrome p450 family member a1 ( cyp1a1 ) is an enzyme which can catalyze this activation . it was shown that curcumin can inhibit the cyp4a1 inducing activity of the ahr.147 binding to ahr and competition with aryl hydrocarbon derivatives might explain the chemopreventive activity of curcumin.147 probably more important for the cancer cell death inducing activity of curcumin is the third curcumin receptor , the vitamin d receptor.148,149 anti - neoplastic activities of vitamin d have been described , but epidemiological data also suggests that for some cancer types sunlight can reduce cancer risk independent from vitamin d.150 curcumin increases apoptosis and differentiation of vitamin d - treated tumor cells.151,152 direct binding of curcumin to the vitamin d receptor ( vdr ) was demonstrated.148 this binding allows vdrs to heterodimerize with the retinoic x receptor and translocate to the nucleus . the complex can then activate gene transcription of vitamin d target genes.148 recently , high expression of the vdr has been observed in hl biopsies.153 hl cell lines also express high levels of vdr ( fig . lymphoma cells can synthesize vitamin d154 and increased vitamin d levels have been repeatedly reported in hl patients.155 in addition , seasonal fluctuations of the prognosis of hl patients have been observed and discussed as being caused by fluctuations of endogenous vitamin d levels.156 vitamin d can probably activate the nfkb pathway,157,158 but in most systems , vitamin d inhibits nfkb activation.159162 in jurkat cells with constitutive expression of vdr , no inhibition of nfkb by vitamin d was observed,163 suggesting that some tumor cells have inactivated this pathway . interestingly , nfkb can inhibit vdr signaling.164 this cross - inhibition might explain the simultaneous high constitutive expression of vdr and activated nfkb in lymphoma cells . stimulation of vdr by curcumin might shift this balance to the inhibitory activity of vdr . curcumin can reduce the number of myeloid - derived suppressor cells,165 an important immuno - suppressive cell population in tumors . tumor patients , especially hl patients , often have a tumor - mediated dysfunction of the immune system . experimental data suggest that curcumin is able to restore the immune function in tumor - bearing hosts.166 on the other hand , induction of regulatory t cells might be increased in the presence of curcumin,167 and the direct induction of apoptosis in activated t cells has been described.168 as an anti- inflammatory agent , curcumin can suppress t cells.169,170 such effects , however , might negatively affect anti - cancer immune responses . the balance between immunostimulation and -suppression might be dependent on the concentration of curcumin.171 in the case of hl , the immune system is a double - edged sword . on the one hand , cytotoxic t cells can kill hl cells ; on the other hand , t cells can provide survival signals for hl cells . the very low number of tumor cells and the presence of a high background of normal cells in hl indicate the importance of the interaction between tumor cells and the stroma . whether curcumin can shift the balance between tumor promoting activities and anti - tumor activities of t cells in hl into the direction of tumor destruction must be analyzed . hl is a disease with features of chronic inflammation and the accumulation of stroma cells by hl derived cytokines is an important factor for hl biology.172,173 the anti - inflammatory activities of curcumin may be able to counteract this inflammatory stimulus . another interesting activity of curcumin is the suppression of histone deacetylases ( hdac).174,175 reports describe the inhibition of histon acetyltrasferases by curcumin without hdac inhibition.176,177 in addition , curcumin has dna methylation inhibiting activity.178 epigenetic regulation of gene expression by dna methylation or histone deacetylation plays an important role in hl biology . treatment of hl cells with hdac inhibitors can increase the sensitivity of hl cells to cytotoxic drugs.179 treatment of hl cells with dna methylation inhibitors can increase the expression of tumor antigens which might increase the recognition by the immune system.180,181 however , increased expression of factors involved in chemotherapy resistance might also occur after the treatment of hl cells with such drugs.181 interestingly , curcumin has been shown to inhibit transcription initiated by human immunodeficiency virus ( hiv ) type 1 long terminal repeats ( ltr).182 the activation of ltrs from endogenous retroviruses ( erv ) in hl has been demonstrated . this activation can lead to the aberrant expression of oncogenes in hl.183 whether curcumin can modulate the activity of erv promoters in tumor cells is under investigation . if erv activity is essentially involved in the pathogenesis of hl , such erv inhibition might reverse the malignant phenotype of the tumor cells . a high percentage of hl carries copies of the epstein barr virus ( ebv ) in the tumor cells . activation of the nfkb pathway by ebv seems to be involved in the pathogenesis of ebv positive hl . curcumin can inhibit b cell immortalization by ebv.184,185 ebv - immortalized b cells undergo pronounced apoptosis in the presence of curcumin which may inhibit the outgrowth of cell lines.185 in addition , it seems that oxidative stress promotes b cell immortalization by ebv.186 the anti - oxidant activity of curcumin might inhibit this effect in a similar way as it was shown for vitamin e.186 whether this activity can interfere with the ebv driven hl pathogenesis requires further investigations . for example , hl cells express the anti - apoptotic bcl - xl isoform of the b cell leukemia/ lymphoma 2 ( bcl2)-like 1 ( bcl2l1 ) oncogene.187,188 recently , a link between expression of bcl - xl and survival of hl patients has been suggested.189 curcumin can down - regulate bcl - xl.190 furthermore , curcumin is a topoisomerase ii inhibitor with a similar activity as etoposide , a drug which is used for treatment of hl.191 in addition to the nfkb pathway , curcumin inhibits the stat pathway ( table 1 ) . this pathway is similarly important for hl cell proliferation and survival as the nfkb pathway and inhibitors of this pathway can induce apoptosis in hl cells.143,189,192200 constitutive activation of the stat pathway by rearrangements of the jak2 have been described in hl.201 taken together , curcumin targets several pathways in hl cells . the scarcity of the tumor cells in the tumors hampers the analysis of larger numbers of ex vivo isolated living cells . the number of established hl cell lines is very low and these cell lines are not perfectly representative for the tumor cells in vivo . whether curcumin or curcumin derivatives with better bio - availability can be used for the treatment of hl patients requires further investigation . a number of microarray experiments of curcumin treated cells and animals have been published ( table 2 ) . these experiments indicate that the specific effects of curcumin on gene expression are dependent on the investigated model . these activities include the shift from pro- inflammatory to anti - inflammatory gene expression signatures , the shift from anti - apoptotic to pro- apoptotic signatures , cell cycle inhibition , and down - regulation of pro - invasive factors ( e.g. , metal - loproteinases ) . such experiments might help to identify the relevant pathways which are involved in the biological effects of curcumin in the different models and may lead to the identification of new therapeutic targets . the bio - availability of curcumin is limited by the rapid metabolism and the low water solubility of this phytoceutical . curcumin is rapidly conjugated with sulfate or glucuronic acid in the gut and liver.230 in addition , reduction to tetrahydrocurcumin and hexahydrocurcumin occurs.230 the resulting curcumin sulfate , curcumin glucuronide , and reduced curcumin derivatives can be found in the feces of curcumin treated patients.231 the curcumin metabolites have some biologic activities , for example tetrahydrocurcumin can inhibit the activity of multi - drug resistance ( mdr ) transporters.232 however , enhanced excretion of curcumin metabolites and the shortening of the half life of curcumin after conjugation are not desired . inhibition of glucuronidation by piperine ( the alkaloid from black pepper ) has been shown to increase the bio - availability of curcumin.233 in addition to inhibition of glucuronidation , piperine can inhibit the activity of mdr transporters.234 on the other hand , increased expression of these trans porters after exposure to piperine has been observed.235 together with the inhibitory effect of curcumin on mdr transporter expression and function,88,236 inhibition of mdr transporter function by piperine might counteract the effect of piperine on mdr transporter expression . in order to increase the solubility of curcumin , conjugation of curcumin with proteins or cyclodextrins have been tested.236,237 in addition , different types of nanoparticles , liposomes , and self- assembling polymers have been used for encapsulation of curcumin.238240 such formulations might finally overcome the bio - availability problem of curcumin . another interesting question that remains to be addressed is whether the effects of turmeric are only mediated by curcumin or whether additional turmeric ingredients are involved . early studies suggest that curcumin - free turmeric extracts also have cancer preventing activities.241,242 indeed , differences between turmeric and curcumin in the regulation of pro - inflammatory genes have been described.243 the characterization of the factors that are responsible for biological activities of curcumin - free turmeric requires further investigations . | curcumin , a phytochemical isolated from curcuma plants which are used as coloring ingredient for the preparation of curry powder , has several activities which suggest that it might be an interesting drug for the treatment or prevention of cancer .
curcumin targets different pathways which are involved in the malignant phenotype of tumor cells , including the nuclear factor kappa b ( nfkb ) pathway .
this pathway is deregulated in multiple tumor entities , including hodgkin s lymphoma ( hl ) . indeed
, curcumin can inhibit growth of hl cell lines and increases the sensitivity of these cells for cisplatin . in this review
we summarize curcumin activities with special focus on possible activities against hl cells . |
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papillary thyroid carcinoma ( ptc ) is the most common malignant tumor among all thyroid cancers , accounting for an estimated 75 - 85% of thyroid cancers . in india and worldwide the national cancer registry programme has reported the thyroid gland as the leading site of cancer accounting for 1.5% of all cancers in men and 3.3% in women . ptc is more common in females and the peak age group is 30 - 50 years , with some variations in different populations . such age - specific data are not available for our population . in the last few years , we have noticed it to be occurring more often in younger patients . this study was conducted to find out the epidemiological pattern of this tumor in our population and to observe and analyze the shift in the peak age for this tumor , if present . in contrast to the high incidence , death from ptc is rare and most patients respond well to surgery and targeted therapy with radioactive iodine . ptc in the younger age group has a better prognosis as compared to the middle age group . the established diagnostic criteria of ptc on fine - needle aspiration cytology ( fnac ) and histopathology are characteristic . fnac is the first - line diagnostic test for the evaluation of swellings of the thyroid gland . it is an efficient method of selecting patients who require surgical treatment owing to its simplicity , low cost , high sensitivity , and specificity . despite these advantages there is a worrisome possibility of false positive and false negative results on cytology , which can have a serious effect on patient care . the most common reasons for false - positive diagnosis are the presence of similar features in various other diseases of the thyroid gland . a false negative diagnosis could have been given in cases where the characteristic features of papillary carcinoma are absent or these features are present less frequently in cytology smears . another aim of our study was to analyze the correlation between the cytological diagnosis and histopathological ( hp ) diagnosis of ptc . this would allow the pathologist to be more vigilant when dealing with such cases in order to avoid misdiagnosis . this is a retrospective analysis conducted between 2009 and 2014 at our institution and it was approved by the institution 's ethical committee . the information gathered were the age and sex of the patient , and cytology and histopathology diagnoses so as to study the age distribution of this tumor and correlation between the cytology and histopathology reports . an attempt was made to find out if there was any frequent cause of misdiagnosis . in the database , there were a total of 72 cases reported as ptc on cytology or histopathology . the cytological diagnosis of ptc was given in 53 cases and suspicious for ptc in 10 cases . all the 53 cases were confirmed to be ptc on hpe . out of 10 cases reported as suspicious for ptc on cytology , six cases were confirmed on hpe . in four cases , the cytological diagnosis did not correlate with the hpe diagnosis amounting to false positives of 5.56% [ figure 1 ] . the hpe diagnosis in these cases was adenomatous goiter in two cases , hashimoto 's thyroiditis in one case , and nodular goiter in one case [ table 1 and figure 2a c ] . histopathology correlation among the cases analyzed details of false positive cases on cytology cytology of cases reported as suspicious of papillary carcinoma : ( a ) showing nuclear grooves ( arrows ) . ( mgg stain , 100 ) ( b ) showing intranuclear cytoplasmic inclusions ( arrows ) . ( mgg stain , 100 ) ( c ) showing nuclear grooves ( black arrows ) and intranuclear cytoplasmic inclusions ( yellow arrow ) . ( mgg stain , 40 ) the hpe diagnosis of ptc was given in 68 cases , out of which in nine cases the cytology reports differed . the cytology diagnosis in seven out of these nine cases was follicular neoplasm , in one case cystic lesion of the thyroid gland , and hashimoto 's thyroiditis in one case . the hpe diagnosis for these nine cases was a follicular variant of ptc in seven cases , cystic ptc in one case , and micropapillary carcinoma arising in hashimoto 's thyroiditis in one case [ table 2 and figure 1 ] . details of false negative cases on cytology the sensitivity ( true positive / true positive + false negative ) of fnac for detecting ptc was 86.7% and positive predictive value ( true positive / true positive + false positive ) was 93.6% [ table 3 ] . the false positive rate , specificity , and negative predictive values could not be calculated as the study included only ptc cases and there were no true negative cases . cytohistopathology correlation the age and sex distribution of ptc was calculated for 68 cases and was confirmed to be ptc on histopathology . the peak incidence was noted in the age range of 31 - 40 years among females and 41 - 60 years among males . age distribution of papillary carcinoma of thyroid in our study age distribution of the cases in the present series thyroid diseases are among the most common endocrine disorders worldwide . in india too , there is a significant burden of thyroid diseases . papillary carcinoma is the most common type of thyroid malignancy . according to surveillance , epidemiology , and end results ( seer ) stat fact sheets , the number of estimated new cases of thyroid cancer in 2014 was 62,980 and it accounted for 3.8% of all the new cancer cases with the estimated deaths amounting to 1,890 . in india , the national cancer registry programme has reported thyroid as a leading site of cancer accounting for 1.5% of all cancers in men and 3.3% in women . the reported female : male ratio varies between 2:1 and 4:1 . in our study , the indian council of medical research established the national cancer registry programme , which has collected the data of more than 300,000 cancer patients between 1984 and 1993 . among these patients , the national cancer registry programme ( ncrp ) noted 5,614 cases of thyroid cancer , and this included 3,617 females and 2,007 males . most papillary carcinomas are diagnosed in patients in the third to fifth decades of life . carcangiu et al . from italy noted the peak incidence in the age group of 31 - 40 years . according to the cancer research statistics of the uk , age - specific incidence rates in males rise gradually from around the age of 1 - 14 years . the rates in females rise sharply from around the age of 10 - 14 years , reaching a peak at the age of 35 - 39 years and then remaining high . incidence rates are higher for females than for males for most age groups . in our study also , the peak age group was 31 - 40 years in females and 41 - 60 years in males . the age - specific incidence in females remained relatively high till the age of 70 years . unlike the uk cancer society , we noted a decline in incidence after 70 years of age , which was probably because of a lesser life expectancy in our indian population . according to seer , between 2007 and 2011 , the peak age for thyroid cancer was 45 - 54 years with 24.2% cases . the age group with patients less than 20 years accounted for only 1.8% of the cases . in the present series , which included only ptc , there were seven cases who were less than 20 years of age accounting for 10% of the total cases . six of the seven patients were in the 11 - 20 years age group and only one patient was less than 10 years of age ( 7 years at the time of diagnosis ) . this indicates a much higher incidence of this tumor in our younger population as compared to the american population . however , our incidence in < 20 years age group appears to be similar to the publication from italy . a trend toward an increase in pediatric thyroid cancer has been noticed by other researchers as well . the incidence of clinically palpable thyroid nodules in children is estimated to be around 1 - 1.5% . when compared to adults , children have four times greater risk of malignancy when a thyroid nodule is diagnosed . despite this , pediatric thyroid cancers have a much better prognosis than their adult counterparts with an almost 100% disease - free survival . it is simple , reliable , time - saving , minimally invasive , and cost - effective . one of the major advantages is that fnac can be done as an outpatient procedure . the diagnostic value and accuracy fnac in the evaluation of thyroid nodules is well - established . fnac is relied upon to distinguish benign from neoplastic or malignant thyroid nodules and therefore , has led to a dramatic decrease in thyroid surgeries . however , fine - needle aspiration has some limitations such as specimen inadequacy , sampling techniques , worrisome histologic alterations following fine - needle aspiration of the thyroid ( whafft ) changes , and false negative and false positive results . histopathological examination ( hpe ) of surgically excised thyroid gland swelling is the most accurate way to determine the pathology . in the present study , cytology diagnosis clearly correlated with histopathology diagnosis in 53 out of 72 cases . in 10 cases , suspicious for papillary carcinoma ; these cases were also included in the papillary carcinoma group on cytology in this study because all the cases suspicious for papillary carcinoma of the thyroid gland are considered as papillary carcinoma as far as treatment is concerned . out of 10 cases of suspicious for papillary carcinoma , six cases were confirmed as papillary carcinoma in hp . in the remaining four cases , the cytological diagnosis did not correlate with hp , resulting in false positives of 5.56% . the false positive rate , specificity , and negative predictive values could not be calculated as the study included only ptc cases and there were no true negative cases . the hp diagnoses in these four cases were adenomatous goiter in two cases , hashimoto 's thyroiditis in one case , and nodular goiter in another . it is noteworthy that all the cases , which were reported as papillary carcinoma on cytology proved to be so on hp . this indicates that when sampling was adequate and there was presence of frequent characteristic features on cytology , the diagnosis was confirmatory , whereas four out of 10 lesions that were reported as this emphasizes the pitfall of fnac in rendering confirmed diagnosis in cases where there is a paucity of characteristic features of ptc . the cytohistological correlation including the suspicious cases came to 81.94% . the literature review for similar studies has reported a correlation ranging from 72% to 88% . although cytologic diagnosis of papillary carcinoma has a high specificity , it is the most common false positive diagnosis when compared to other thyroid malignancies because of the presence of papillary carcinoma - like features in other lesions as well . the percentage of false positive cytological diagnosis in our study was 5.56% as compared to the literature percentage ranging from 5.25% to 11.60% in various studies . the reason accountable for false positive diagnosis in our study and also in the literature was the presence of features diagnostic of ptc , which can also be seen in benign conditions of the thyroid gland . there is evidence in the literature that the characteristic features of ptc such as nuclear grooves , nuclear pseudoinclusions , and many more features can be seen in benign conditions of the thyroid gland such as adenomatous goiter , hashimoto 's thyroiditis , nodular goiter , and follicular neoplasm . however , studies have stated that the occurrence of these features in benign lesions is very less as compared to ptc . in our study figures 2a and c show nuclear grooves on the cytology of cases , which were found to be adenomatous goiter and hashimoto 's thyroiditis on histopathology , respectively . similarly , figures 2b and c show intranuclear cytoplasmic inclusions on the cytology of cases , which were found to be nodular goiter and hashimoto 's thyroiditis on histopathology , respectively . owing to the presence of these characteristic features of ptc suspicious of ptc on cytology . however , all the four cases showed the presence of nuclear grooves and intranuclear cytoplasmic inclusions in less number of cells and in addition , the other characteristic features of ptc such as metaplastic cytoplasm , psammoma bodies , and three - dimensional fragments were absent . hence , the awareness of pathologists in this context and strict adherence to adequacy criteria can reduce the false positive rate . the false negative fnac results may occur because of sampling error or misinterpretation of cytology , and are of great concern because they indicate the potential to miss a malignant lesion . , nine cases with a diagnosis of ptc on hpe had been misdiagnosed on cytology . out of these , for seven cases a diagnosis of follicular neoplasm had been rendered on cytology and the corresponding hpe diagnosis was a follicular variant of ptc ( fvptc ) . out of the remaining two cases , one was hashimoto 's thyroiditis and the other was cystic lesion of the thyroid gland . the false negative rate in the present study was 13.2% as compared to 4 - 19% reported in other comparable studies . studies in the past have highlighted the fact that in fvptc , the nuclear features essential for a cytologic diagnosis of ptc are infrequent . one of the most common and clinically significant pitfalls in the assessment of an aspirate of a follicular lesion is the failure to recognize the nuclear features of the fvptc , the most common variant of papillary carcinoma . this indicates that the sensitivity of fna in establishing a diagnosis of fvptc is low . these concerns in the literature are further affirmed in our observation study where fvptc cases accounted for the high false negative rate ( seven out of nine false negative cases ) . from this , we infer that the presence of infrequent nuclear features with a follicular pattern in cytology should alert the pathologist to consider the diagnosis of fvptc , which is a major diagnostic challenge . false negative diagnosis can also occur due to inadequate sampling or due to the aspiration of fluid in cystic lesions of the thyroid gland with an underlying malignancy , as happened in one of our cases ( reported as cystic lesion on cytology ) . among all the thyroid cancers sampling of this hemorrhagic fluid with sparse tumor cells may result in false interpretation as a benign cyst . in the present case , suggestion for surgical excision had been given by the pathologist in order to rule out an underlying malignancy , which led to timely surgery and saved the patient of undue complications of ptc . although fine - needle biopsy is the best predictor of malignancy in either cystic or solid thyroid lesions , it is slightly less reliable when a thyroid lesion is fluid - filled rather than solid . a close follow - up or thyroid lobectomy for diagnosis should be strongly considered in these patients even when fna cytologic finding is interpreted as benign . any recurrent cystic lesion should raise a strong suspicion for malignancy and should be treated accordingly . one of the other false negative cases was reported as hashimoto 's thyroiditis on cytology . the term papillary microcarcinoma is used when the papillary carcinoma is an incidental finding and measures less than 1 cm in diameter . papillary microcarcinoma arising in hashimoto 's thyroiditis or in any other benign lesion has high chances of being missed because fnac is a blind procedure and as a result of which the aspirated sample may not be fully representative of the existing lesion . papillary microcarcinomas of the thyroid ( i.e. , papillary carcinomas < 1 cm in size ) are frequent ; autopsy surveys have revealed a prevalence of up to 30% in high - risk groups . however , their clinical impact is limited in the vast majority of cases and do not cause a significant reduction in the survival . in our study , we found that fnac is a reliable method of screening thyroid nodules for ptc with a sensitivity as high as 86.7% and a positive predictive value of 93.6% . however , there are some studies in the literature reporting a sensitivity of as low as 55.3% and a false negative as low as 44.7% . our results are comparable to other reports in the literature . the likely reason reported for lower sensitivity in a series of mistry et al . was a combination of operator variability , low number ( 56 cases ) , and diagnostic difficulty of using fnac in certain thyroid pathologies . in our series of 72 cases where fnacs were conducted by trained personnel and cytology smears studied by an experienced pathologist , ptc is the most common thyroid malignancy and its incidence seems to be on a rise . there appears to be an increase in its incidence in the younger population , causing an epidemiological shift in the age distribution . fnac serves as a gold standard and an indispensable tool for early diagnosis . however , fnac has pitfalls that lead to false positive and false negative diagnosis . false positive diagnosis can be caused due to the presence of characteristic features of ptc in benign conditions of the thyroid gland . false negative diagnosis occurs as a result of inadequate sampling and the presence of infrequent nuclear features of ptc , which can cause diagnostic dilemma such as in fvptc . cystic ptc is another diagnostic challenge and can be missed on fna . therefore , despite a benign cytology clinically suspicious lesions should have close clinical follow - up and should probably be excised . papillary microcarcinoma is the most common form of ptc but it is an incidental diagnosis as most of the cases are missed owing to its small size . awareness of pathologists regarding these pitfalls of fnac and meticulous examination of smears with strict adherence to the diagnostic criteria of ptc can reduce false positive and false negative diagnoses and thus , provide better patient care . no financial support of any form was obtained by any of the authors in relation to this study . no financial support of any form was obtained by any of the authors in relation to this study . | background : the role of fine - needle aspiration cytology ( fnac ) in thyroid nodules has been well - studied but there is a paucity of studies solely involving papillary thyroid carcinoma ( ptc ) .
the diagnostic criteria for ptc are established but still there is a worrisome possibility of false positive and false - negative results , which can have a serious impact on patient care .
this article correlates the cytological and histological diagnosis of ptc with an eventual aim of analyzing the cause of false positive and false negative results in order to prevent misdiagnosis .
an increase in the incidence of ptc in the younger population has been noted.aims:to analyze the epidemiological trends and cytohistological correlation of ptc and evaluate the discordant cases and pitfalls of fnac.materials and methods : seventy - two cases who had undergone both fnac and histopathological examination ( hpe ) of the thyroid gland were selected .
age and sex distribution as well as cytohistological correlation were done for all the cases.results:cytohistological correlation was 81.94% .
false positives were 5.56% and the false negative rate was 13.2% .
sensitivity was 86.7% and the positive predictive value was 93.6% .
the peak age was 31 - 40 years among females and 41 - 60 years among males .
seven of our patients were < 20 years of age ( 10%).conclusion : fnac is an indispensible tool for the early diagnosis of ptc .
however , certain conditions of the thyroid gland can cause diagnostic dilemma .
awareness of pathologists regarding these pitfalls can prevent misdiagnosis and provide better patient care .
increasing the incidence of ptc with a more striking increase in the younger population makes early diagnosis all the more important owing to better prognosis in this age group . |
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l929 cells were maintained in minimum essential medium
eagle ( sigma ) with 5% fetal bovine serum and penicillin / streptomycin . mefs and 293 t cells were maintained in dulbecco 's modified
eagle 's medium with 10% fetal bovine serum and penicillin / streptomycin . mefs and 293 t
cells were transiently transfected by fugene 6 ( roche applied science ) . for
the preparation of cell extracts , cells were lysed with lysis buffer ( 50
mm tris - hcl , ph 7.5 , 150 mm nacl , 1 mm edta ,
1% nonidet p-40 , 0.1 mg / ml leupeptin , 1 mm phenylmethylsulfonyl
fluoride , and 1 mm sodium orthovanadate ) and centrifuged at 245,000
g for 10 min . the supernatant was used for sds - page and
electrophoretic mobility shift assays ( emsas ) . oligonucleotides oligonucleotides ( 800 bp ) corresponding to
the coding sequence of green fluorescent protein were amplified by pcr using
t7-primer ( 5-cgtaatacgactcactataggggatatcagcaaaggagaagaactttt-3 )
and t3-primer
( 5-gcaattaaccctcactaaagggaggcctagggagaagacagtgagctc-3 ) . long
dsrna ( ds800 ) was prepared by annealing complementary strands separately
synthesized by in vitro transcription using the ampliscribe t7 flash
transcription kit ( epicentre biotechnologies ) and the ampliscribe sp6 high
yield transcription kit ( epicentre biotechnologies ) . the annealed dsrna was
treated with s1 nuclease ( takara bio ) to generate a blunt end and alkaline
phosphatase ( takara bio ) to remove 5-phosphate . p - ds800
was prepared by labeling the 800-bp dsrna using t4 polynucleotide kinase and
[ -p]atp . reporter assay mefs were transfected with p55-c1bluc , prltk ,
and expression plasmids for rig - i mutants or mda5 mutants . cells were split
into two aliquots , stimulated with rna ( 5-pppgg25 )
( 6 ) or poly(i - c ) transfection
or sendai virus ( sev ) infection for 12 h , and harvested at 48 h after dna
transfection . l929 cells were transfected similar to mefs but were stimulated
by newcastle disease virus infection . the
rna transfection and poly(i - c ) transfection were performed using
lipofectamine rnaimax ( invitrogen ) . the luciferase assay was performed
with a dual - luciferase reporter assay system ( promega ) . luciferase activity
was normalized using renilla luciferase activity ( prltk ) as a
reference . quantitative pcr assay plasmid constructs p-55c1bluc and prltk , pef - flag - rig - i ,
pef - flag - rig - icard , pef - flag - mda5 , and pef - flag - mda5card were described
previously ( 14 ) . data on snps
for rig - i and mda5 were obtained from the ncbi
( www.ncbi.nlm.nih.gov )
and hapmap databases . the expression plasmids for rig - i mutants
( pef - flag - rig - ir7c , pef - flag - rig - is144f , pef - flag - rig - is183i ,
pef - flag - rig - it260p , pef - flag - rig - ii406 t , pef - flag - rig - id580e ,
pef - flag - rig - if789l , and pef - flag - rig - icards183i ) and mda5 mutants
( pef - flag - mda5t260s , pef - flag - mda5l274i , pef - flag - mda5k351e ,
pef - flag - mda5i442v , pef - flag - mda5h460r , pef - flag - mda5e627 * ,
pef - flag - mda5h843r , pef - flag - mda5i923v , pef - flag - mda5a946 t , and
pef - flag - mda5d1014e ) were generated using a geneeditor in vitro
site - directed mutagenesis system ( promega ) . antibodies and immunoblotting the anti - flag ( m2 ; sigma )
antibody is a commercial product . sds - page and immunoblotting were performed
as described previously
( 14 ) . emsa293 t cells ( 1 10/6-cm dish ) were
transfected with 1 g of expression vector . at 24 h after transfection ,
cell extract was prepared and mixed with anti - flag beads ( sigma ) to adsorb
flag - tagged proteins . the beads were washed , and bound protein was eluted with
flag peptide ( sigma ) . construction of rig - i mutants and their biological activities in mefs
derived from rig - i knock - out mice nssnps were selected from
nucleotide sequence polymorphisms of human rig - i reported in databases and
introduced into the rig - i expression vector by site - directed mutagenesis . mefs transiently transfected with these
vectors , the wild type and seven human rig - i mutants were expressed at
comparable levels ( fig . these results indicate that none of the amino acid
substitutions significantly affect the synthesis and/or stability of rig - i . the signaling function of the mutants was analyzed by temporarily
complementing the function of rig - i in rig - i mefs
using a virus - responsive luciferase reporter gene
( fig . although
the transfection of 5-ppprna did not activate the reporter gene
significantly in rig - i mefs
( fig . 2a ,
vector ) , the ectopic expression of wt rig - i restored the
responsiveness to the ligand . all the mutant constructs exhibited functional
complementation except the r7c , s144f , and s183i mutants , which exhibited a
reduced response to 5-ppprna , particularly the s183i mutant , which
exhibited a severe defect . because the activity of s144f was not reproducible ,
we did not investigate this mutant further . it has been shown that rig - i
senses sev and activates the ifn promoter . therefore , the ability of the
mutants to respond to this viral inducer was tested
( fig . sev
efficiently activated the reporter gene when wt rig - i was expressed ; however ,
s183i was virtually inactive and r7c exhibited partial activity . we further confirmed the effect of
the r7c and s183i mutations by monitoring the expression of endogenous mouse
ifn- mrna ( fig .
2c ) . the results clearly demonstrate that s183i is barely
active and that r7c is partially active . the above results strongly suggest
that ser-183 is critical for rig - i to sense transfected 5-ppprna as
well as sev - derived pamps . ser-183 resides within the second card , and this
prompted us to explore the impact of the s183i substitution on the signaling
function of the isolated rig - i card . unlike that of the full - length rig - i ,
overexpression of the truncated rig - i ( 1229 ) , which encompasses the two
repeats of card , constitutively activated the reporter p-55c1b without a viral
stimulus ( fig . rig - i ( 1229 ) with s183i failed to activate the reporter gene .
in these cells , levels of rig - i ( 1229 ) with or without the s183i
mutation were comparable , suggesting that ser-183 is critical for the
signaling function but does not affect protein levels of rig - i card
( fig . it was
reported that human rig - i undergoes ubiquitination at lys-172 in the second
card , and this process is essential for rig - i signaling
( 15 ) . to compare the mutation
at ser-183 , we generated a k172r mutant and tested its activity
( fig . 3 , c and
d ) . surprisingly , the mutant exhibited virus - induced
signaling activity comparable with the wt , suggesting that the ubiquitination
of lys-172 plays a minor role in the regulation of rig - i and that the
phenotype of the s183i mutant is unlikely due to a failure of
ubiquitination . a and
b , rig - i mef cells were transiently
transfected with p-55cibluc together with empty vector ( vector ) or
the indicated constructs . the cells were subjected to a dual - luciferase assay
after stimulation with 5-ppp - ssrna ( 12 h ) ( a ) or sev ( 12 h )
( b ) . the relative firefly luciferase activity , normalized to the
renilla luciferase activity , is shown . error bars show the
sds for triplicate transfections . mock , mock - treated . c ,
rig - i mefs were transfected with empty vector
( vector ) or expression vectors for wt rig - i or mutants as indicated
( the total amount of plasmid was kept at 6gby adding empty vector ) . to
observe the dose response cells were mock - treated or transfected with
5-ppp - ssrna for 12 h , and ifn- mrna was quantified by
quantitative pcr by using the applied biosystems primer set for mouse
interferon-1 : mm00439546_s1 . a and
b , rig - i mef cells were transiently
transfected with p-55cibluc together with empty vector ( vector ) or
the indicated constructs . the cells were subjected to a dual - luciferase assay
after stimulation with 5-ppp - ssrna ( 12 h ) ( a ) or sev ( 12 h )
( b ) . the relative firefly luciferase activity , normalized to the
renilla luciferase activity , is shown . error bars show the
sds for triplicate transfections . mock , mock - treated . c ,
rig - i mefs were transfected with empty vector
( vector ) or expression vectors for wt rig - i or mutants as indicated
( the total amount of plasmid was kept at 6gby adding empty vector ) . to
observe the dose response , cells were mock - treated or transfected with
5-ppp - ssrna for 12 h , and ifn- mrna was quantified by
quantitative pcr by using the applied biosystems primer set for mouse
interferon-1 : mm00439546_s1 . a ,
rig - i mef cells were transfected with reporter genes
together with empty vector ( vector ) or plasmid expressing flag - tagged
wt rig - i , rig - i card ( the n - terminal region , amino acid 1229 ) , or rig - i
card s183i . after transfection ( 24 h ) , the cells were subjected to a
dual - luciferase assay . b , each protein expressed in
rig - i mef cells was detected by immunoblotting using
an anti - flag antibody . c ,
reporter assay of the k172r mutant was performed as in
fig . rig - i k172r and wt rig - i
were expressed at comparable levels . e , empty vector or expression
vectors for full - length rig - i with the t55i or s183i mutation were introduced
into l929 cells ( the total amount of plasmid was kept at 9 g by adding
empty vector ) and infected with newcastle disease virus , and reporter activity
was analyzed as in panel a. to observe the dose response , cells
received 1 , 5 , or 9 g of the expression plasmid for t55i and s183i as
indicated . a ,
rig - i mef cells were transfected with reporter genes
together with empty vector ( vector ) or plasmid expressing flag - tagged
wt rig - i , rig - i card ( the n - terminal region , amino acid 1229 ) , or rig - i
card s183i . after transfection ( 24 h ) , the cells were subjected to a
dual - luciferase assay . b , each protein expressed in
rig - i mef cells was detected by immunoblotting using
an anti - flag antibody . c ,
reporter assay of the k172r mutant was performed as in
fig . rig - i k172r and wt rig - i
were expressed at comparable levels . e , empty vector or expression
vectors for full - length rig - i with the t55i or s183i mutation were introduced
into l929 cells ( the total amount of plasmid was kept at 9 g by adding
empty vector ) and infected with newcastle disease virus , and reporter activity
was analyzed as in panel a. to observe the dose response , cells
received 1 , 5 , or 9 g of the expression plasmid for t55i and s183i as
indicated . it is known that human rig - i with the amino acid substitution t55i acts as
a dominant inhibitor ( 7 ) . this
mutation within the first card inactivates the signaling function of the
isolated tandem cards . l929 cells were transfected with control vector or rig - i
mutants and then activated by infection of newcastle disease virus . cells
transfected with the vector exhibited ifn promoter activity due to
endogenous rig - i ( fig . expression of t55i as well as s183i significantly
reduced the promoter activity in a dose - dependent manner , suggesting that
s183i causes a dominant negative phenotype similar to t55i . construction of mda5 mutants and their biological activities in mefs
derived from mda5 knock - out mouse next , the biological activity of
human mda5 mutants was analyzed similarly using mda5-deficient mefs . ten nssnps including a946 t identified in familial t1d
( 16 ) were introduced into the
mda5 expression vector ( fig . the wild type and mutants were expressed in
mda5 mefs at comparable levels
( fig . 4b ) , showing
that the mutations , including the e627 * truncation , did not affect mda5
protein levels . the biological activity of the mutants was assayed similarly
to that of the rig - i mutants . as a mda5 agonist , a commercial poly(i - c ) with
an average length of 2 kbp wild - type mda5 clearly
conferred responsiveness to the poly(i - c )
( fig . eight mda5 mutants ,
including a946 t , which was implicated in human t1d
( 16 ) , exhibited complementing
activity comparable with the wild type . the phenotypes of these mutations were
further tested by transient expression in mda5 mefs
and monitoring endogenous ifn- mrna
( fig . 5b ) . although
mda5 is absent , intracellular poly(i - c ) induced ifn- gene
expression in the control cells ( vector ) . this is presumably due to
the activation of rig - i by a short poly(i - c ) present in the preparation we
used . unlike the reporter assay , which monitors only transfected cells , this
quantitative pcr assay detects ifn- transcripts from both
transfected and non - transfected cells , increasing the background signal . irrespective of the background , overexpression of wt mda5 resulted in an
enhancement of ifn- expression by poly(i - c ) , and this induction
was not observed with e627 * and i923v . the e627 * mutant lacks a part of the
helicase domain and the entire ctd in which ile-923 resides . figure 4.mda5 nssnp mutants and their expression in mefs . a ,
schematic representation of wt mda5 and its nssnps . b , flag - tagged mda5 snps were produced in
mda5 mefs and detected by immunoblotting using an
anti - flag antibody . mda5 nssnp mutants and their expression in mefs . a ,
schematic representation of wt mda5 and its nssnps . b , flag - tagged mda5 snps were produced in
mda5 mefs and detected by immunoblotting using an
anti - flag antibody . vector , empty vector ; mda5 full ,
full - length mda5 . because in the case of rig - i , the ctd determines rna recognition
specificity , we investigated the rna binding activity of these mutants by emsa
using p - labeled poly(i - c ) . wild - type and recombinant mda5
proteins were expressed in 293 t cells and purified . the recombinant proteins
were virtually free of other cellular proteins as analyzed by coomassie
brilliant blue staining ( fig . wild - type mda5 clearly formed a complex with
poly(i - c ) ( fig . i923v and a946 t
exhibited activity to bind poly(i - c ) as strongly as the wild type under these
conditions . 5e ) and confirmed that mda5 wt and the i923v mutant bind
to dsrna in a comparable fashion . these results suggest that the e627 * mutant
is biologically inactive due to its failure to recognize its agonist . human rig - i polymorphism we identified s183i as a loss of
function mutation of rig - i . this serine residue is conserved in human , monkey ,
cow , and pig rig - i . the mutation apparently inactivates the tandem card , which
relays signals downstream . furthermore , s183i exhibits a dominant inhibitory
phenotype , suggesting that individuals retaining this mutation as a
heterozygote would exhibit hypersensitivity to viral infections . it has been
shown that rig - i mutants with a card deletion ( rig - ic ) , card point mutation
( t55i ) , and atp - binding site mutation ( rig - i k270a ) all function as a dominant
inhibitor ( 3 ,
7 ) . it has been reported that
the repression domain present in the c - terminal region of rig - i and lgp2
dominantly suppresses the activation of rig - i in trans through
interaction with card and the helicase loop region
( 7 ) . because the repression
domain encompasses the rna - binding domain of rig - i
( 6 ) , one mechanism is likely
competition of rna binding with wt rna . however , the rna binding - deficient
rig - i mutants , k888a / k907a and k858a / k861a
( 6 ) , functioned as a dominant
negative inhibitor.3
interestingly , lgp2 mutants , k643e and k651e , which correspond to lys-888 and
lys-907 of rig - i , lost rna binding activity but retain repression function
( 17 ) . a ,
mda5 mefs were transfected with reporter genes
together with the indicated constructs as in
fig . 2a . after
stimulation with poly(i - c ) ( 12 h ) , the cells were subjected to a
dual - luciferase assay . b , mda5 mefs were transfected with
expression vectors for wt mda5 or e627 * or ile-923 mutants ( the total amount
of plasmid was kept at 6 g by adding empty vector ) . to observe the dose
response , cells were mock - treated or transfected with poly(i - c ) for 12 h , and
ifn- mrna was quantified by quantitative pcr as in
fig . 2c . c ,
293 t cells were transfected with empty vector , wt mda5 , e627 * , i923v , or
a946 t , and the produced proteins were purified using anti - flag
( experimental procedures ) . the purified proteins were separated
by sds - page and stained by coomassie brilliant blue . d , emsa of the
purified mda5 proteins ( 500 ng ) using p - labeled poly(i - c ) as a
probe . emsa
was performed using 500 , 300 , and 100 ng of mda5 and ile-923 protein . a ,
mda5 mefs were transfected with reporter genes
together with the indicated constructs as in
fig . 2a . after
stimulation with poly(i - c ) ( 12 h ) , the cells were subjected to a
dual - luciferase assay . b , mda5 mefs were transfected with
expression vectors for wt mda5 or e627 * or ile-923 mutants ( the total amount
of plasmid was kept at 6 g by adding empty vector ) . to observe the dose
response cells were mock - treated or transfected with poly(i - c ) for 12 h , and
ifn- mrna was quantified by quantitative pcr as in
fig . 2c . c ,
293 t cells were transfected with empty vector , wt mda5 , e627 * , i923v , or
a946 t , and the produced proteins were purified using anti - flag
( experimental procedures ) . the purified proteins were separated
by sds - page and stained by coomassie brilliant blue . d , emsa of the
purified mda5 proteins ( 500 ng ) using p - labeled poly(i - c ) as a
probe . emsa
was performed using 500 , 300 , and 100 ng of mda5 and ile-923 protein . one proposed function of the second card is to conjugate to ubiquitin ( at
lys-172 ) , which may be essential for signaling activity . however , human rig - i
with k172r , which is resistant to ubiquitination , did not affect phenotype
( fig . moreover , the corresponding amino acid in mouse rig - i
is glutamine , and the corresponding position of mda5 is glutamic acid ( human ,
monkey , mouse , cow , and pig ) , suggesting that the ubiquitination of lys-172
has a minor impact on signaling activity . the precise function of the second
card is not clear , but a comparative study of ser-183 and wt rig - i will
elucidate the molecular function of this residue . human mda5 polymorphism we identified two loss of function
mutations , e627 * and i923v , in human mda5 . each of these mutations is actually
present in the human population
( 13 ) . it is worth
investigating the phenotype of the homozygote because mda5 knock - out
mice clearly exhibit hypersusceptibility to the family
picornaviridae , genus cardiovirus . e627 * , which lacks ctd and a part
of the helicase domain , lost its dsrna binding activity ; hence there was no
signaling activity . although the i923v mutation occurs in the ctd , this mutant
exhibited intact dsrna binding , suggesting a novel function of ile-923 other
than the recognition of rna . it is worth noting that this isoleucine is
conserved in human , monkey , mouse , cow , and pig mda5 . it is tempting to
speculate that ile-923 participates in an interaction with some other domain
of mda5 or other unknown regulatory protein(s ) . a new report by nejentsev et al .
( 13 ) describing the
relationship between susceptibility to t1d and mda5 polymorphism was
published . the report describes that four rare mutations ( two mutations in the
exon and two mutations in the intron ) in the human mda5 gene are
associated with protection against t1d . our analysis included the two exon
mutations ( e627 * and i923v ) , which exhibited a loss of function phenotype . the
genetic analysis included another rare nssnp , h460r , shown to be independent
of t1d resistance . on
the other hand , a946 t , which was suggested to associate with t1d in previous
reports ( 13 ,
16 ) , did not exhibit a loss of
function phenotype . ( 13 ) suggested that the
association of a946r with t1d was due to the effect of another nssnp , r843h , a
combination of these mutations might confer loss of function on mda5 . in
summary , our analysis strongly suggests that loss of function mutations of
mda5 have a causative role in resistance to t1d . although t1d has a complex
pathology , these findings may provide a new strategy for establishing an
animal model for t1d . the human genome encodes multiple sensors , including tlrs and rlrs for
viral pamps . some of these may act redundantly to secure defense against viral
infections . in the case of rlrs , rig - i and mda5 detect a distinct spectrum of
viruses , as suggested from the phenotype of respective knock - out mice . there
is variation within human populations in susceptibility to a particular viral
infection . we argue for a possible contribution of the genetic diversity of
rlrs , including those identified in the current investigation , to
susceptibility . in addition to the impact of the infection itself , secondary
effects such as autoimmunity , which is remotely triggered by certain viral
infections , may be influenced at least in part through the functional
diversity of rlrs . in summary , our results suggest a critical relationship
between rlr polymorphisms and diseases including viral infections and
autoimmunity . | retinoic acid - inducible gene i ( rig - i ) and melanoma
differentiation - associated gene 5 ( mda5 ) are essential for detecting viral rna
and triggering antiviral responses , including production of type i interferon .
we analyzed the phenotype of non - synonymous mutants of human rig - i and mda5
reported in databases by functional complementation in cell cultures .
of seven
missense mutations of rig - i , s183i , which occurs within the second caspase
recruitment domain repeat , inactivated this domain and conferred a dominant
inhibitory function .
of 10 mutants of mda5 , two exhibited loss of function .
a
nonsense mutation , e627 * , resulted in deletion of the c - terminal region and
double - stranded rna ( dsrna ) binding activity .
another loss of function
mutation , i923v , which occurs within the c - terminal domain , did not affect
dsrna binding activity , suggesting a novel and essential role for this residue
in the signaling .
remarkably , these mutations are implicated in resistance to
type i diabetes .
however , the a946 t mutation of mda5 , which has been
implicated in type i diabetes by previous genetic analyses , affected neither
dsrna binding nor ifn gene activation .
these results provide new
insights into the structure - function relationship of rig - i - like receptors as
well as into human rig - i - like receptor polymorphisms , antiviral innate
immunity , and autoimmune diseases . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
different reasons might be responsible for delayed or nonunion healing . in the clinical setting , nonunion increases the cost of care , necessitates additional surgeries , and results in a prolonged period of convalescence , which is associated with increased mortality . the incidence of hemorrhagic shock following long bone fractures is common among severely injured patients . the inflammatory response after trauma may also represent a significant factor in bone healing by regulating enchondral bone formation and bone remodeling [ 4 , 5 ] . therefore , the immune response after hemorrhagic shock involves a complex interaction characterized by the release of diverse inflammatory mediators ( e.g. , cytokines ) and a vast array of immune competent cells into organ tissue . however , the results of experimental studies investigating fracture healing in models with additional trauma are controversial . some studies identified salutary effects of an additional traumatic insult [ 6 , 7 ] , whereas others observed a negative influence of trauma - hemorrhage on fracture healing . however , these studies utilized significantly different hemorrhagic shock protocols , which may contribute to the controversial results . we investigated the effect of hemorrhagic shock on fracture healing in a standardized mouse model of a closed femoral fracture in combination with pressure controlled hemorrhagic shock in order to test the hypothesis that hemorrhage impairs bone healing . all animal studies were carried out in accordance with the german animal welfare legislation , and experiments were permitted by the local government of north rhine - westphalia , germany . male c57/bl6 mice weighing 25.0 2.0 g were obtained from charles river laboratories ( sulzfeld , germany ) . all mice were kept by a 12-hour light - dark cycle and a constant room temperature . all procedures were performed under deep anesthesia with 50 mg / kg intraperitoneally injected phenobarbital and inhaled isoflurane . animals were randomly assigned to one of two experimental groups , with each group consisting of 14 animals . animals of the control group received a femoral fracture on the right side and implantation of the catheter into the left femoral artery but did not sustain blood loss ( group fx ) , while the experimental group included animals with a right sided femoral fracture and pressure controlled hemorrhagic shock ( group fxhs ) . the left femoral artery was cannulated with polyethylene tubing ( becton dickinson , sparks , md ) . blood pressure was measured via the arterial line using a blood pressure analyzer ( micro - med , louisville , ky , usa ) . animals were bled through an arterial catheter to a mean arterial blood pressure of 35 5 mmhg within 5 min , which was then maintained for 90 minutes . afterwards , the animals were resuscitated with two times the shed blood volume using ringer 's solution ( berlin - chemie - ag , berlin , germany ) . after removing the catheter , blunt dissection was used to expose the femoral notch and a 27 g needle was introduced into the intramedullary canal through the proximal metaphyseal zone as described by bonnarens and einhorn . 50 l of blood was taken two hours before and six hours after fracture induction by retrobulbar venous puncture . concentrations of tnf , il-6 , mcp-1 , and kc were measured by bio - plex pro assays ( biorad , hercules , ca , usa ) according to the manufacturer 's instructions . animals were anesthetized with isoflurane 21 days after fracture and euthanasia was performed by cervical dislocation . the femoral bones were harvested and seven of each group were fixated in formalin for histological examination . both femora of the other seven mice were frozen for ct examination and mechanical testing . tissue samples were decalcified in 12.5% edta ( schweizerhall chemie ag , basel switzerland ) with 1.25% sodium hydroxide ( fluka ) as confirmed by radiography and embedded in paraffin . sections of 46 micrometers were cut longitudinally through the center of the medullary canal using a leica microtome . they were placed onto histobond microscope slides ( marienfeld , germany ) and left overnight at 37c . the sections were pretreated with trap - buffer ( 40 mm sodium acetate ( merck ) , 200 mm sodium tartrate dihydrate ( merck ) in aqua dest . , ph5 ) for 10 minutes followed by incubation in a staining - solution ( 19,4% ( w / v ) naphthol as - phosphate mix ( sigma ) , 116,5% ( w / v ) fast red violet lb salt ( sigma ) , triton - x100 ( sigma ) 1% ( v / v ) , and 1,9% ( v / v ) n - n - dimethylformamide ( sigma ) solved in trap - buffer ) at 37c for 2 hours . after washing in aqua dest . , the slides were counterstained with methyl green . the analyses were performed by 2 blinded examiners ( rb and mk ) who were unaware of the treatment . the number of trap - positive cells and cartilage segments in these fields were counted . the bone quality was assessed using the score system : score 1 , only connective tissue and cartilage within the callus area ; score 2 , woven bone with some islands of cartilage and connective tissue ; score 3 , woven bone without any cartilage at the callus area ; score 4 , woven and lamellar bone at the callus area ; score 5 , only lamellar bone at the callus area . the entire callus region in each sample was micro - ct scanned ( vivact 40 , scanco medical , brttisellen , switzerland ) with an isotropic voxel size of 25 m ( 55 kvp , 145 a , 200 ms integration time , 500 projections on 180 1024 ccd detector array , and cone - beam reconstruction ) . the accuracy of the scanner was monitored weekly for density measurements using hydroxyapatite ( ha ) phantoms with densities of 0 , 100 , 200 , 400 , and 800 mg ha / cm and monthly for geometry measurements using aluminum rods with a known volume of 0.104 mm . in the reconstructed micro - ct images , a semiautomated contouring method was used to outline the inner and outer boundaries of the callus structure . the volume within the contour was then isolated , and the volumetric bone mineral density ( bmd , bone mineral content divided by the entire analyzed volume ) was determined from the grayscale image ( image processing language v5.15 , scanco medical switzerland ) . the images were gaussian filtered ( = 0.8 , supp = 1 ) and thresholded ( 19% of maximal grayscale value ) resulting in binarized images containing only bone and background . the bone volume ratio ( bv / tv ) was then determined as the ratio of bone tissue divided by the entire volume and the tissue mineral density ( tmd ) as the bone mineral content within the bone voxels divided by the total volume of bone voxels . the tensile strength of mouse femora was analyzed using a universal testing machine ( z2.5 , zwick , ulm ) with a 200 n load cell . the alligator clips were attached to cardan joints and the construct was placed into the universal testing machine . the cardan joints were used to prevent angular mounting and to ensure straight tensile load along the femoral shaft . the mechanical testing was performed with no initial load and a constant feed rate of 1 mm / min . nonnormally distributed parameters were tested using the kruskal - wallis test . the null hypothesis was rejected for p < 0.05 . overall 32 animals were included in this study . two mice died during haemorrhagic shock , one mouse was euthanized due to failure of osteosynthesis , and one mouse died on day 10 . we measured the proinflammatory cytokines il-6 , mcp-1 , kc , and tnf- at baseline and 6 hours after operation . each cytokine demonstrated significant increases in group fxhs compared to the fx group and to the baseline values ( figures 1(a)1(d ) ) . il-6 showed no significant difference between the baseline value and fx group ( 156.6 versus 340.4 pg / ml ; p = 0.21 ) but a nearly fivefold increase between fx and fxhs ( 340.4 versus 1577.3 pg / ml ; p < 0.001 ) ( figure 1(a ) ) . the increase in kc serum levels in group fxhs was also nearly fourfold in comparison to group fx ( 280.0 versus 1018.7 pg / ml ; p = 0.002 ) ( figure 1(b ) ) . mcp-1 and tnf- also demonstrated a significant difference between the fxhs and fx groups ( mcp-1 : 336.2 versus 818.1 ; p = 0.002 , tnf- : 469.4 versus 738.5 ; p = 0.001 ) ( figures 1(c ) and 1(d ) ) . blinded histological evaluation by two independent experts using the above - mentioned score from 1 to 5 showed a significant decreased bone quality in the fxhs group ( 2.9 versus 1.8 ; p = 0.001 ) . in parallel we could identify more cartilage segments in the callus region ( 0.9 versus 2.1 ; p = 0.002 ) and the number of osteoclasts was significantly decreased in fxhs ( 6.2 versus 4.2 ; p = 0.001 ) ( figures 2(a)2(c ) ) compared to fractures without hs ( fx ) . micro - ct analysis ( figure 4 ) showed no significant differences in bmd or bv / tv between fx and fxhs . while not statistically significant , there was a trend towards reduced tmd in the fxhs group compared to the fx group ( p = 0.098 ) ( figure 3 ) . as expected , the tensile strength of the fractured femora did not reach the strength of the uninjured opposite side . however , comparison of the relative failure strength ( fractured right femur / uninjured left femur ) between groups fxhs und fx showed no significant difference ( 0.58 versus 0.56 ; p = 0.71 ) ( figure 5 ) . we analyzed hemorrhagic shock - related differences in fracture healing in a femoral fracture model in mice . our main findings are as follows.we could observe a typical inflammatory response after fxhs whereas fx alone caused no significant increase in inflammatory cytokines.histological examination three weeks after fracture showed significantly decreased number of osteoclasts and reduced bone quality after hemorrhagic shock . in parallel , ct analyses showed a trend towards decreased mineral density in the fracture region after hemorrhagic shock . despite these microscopic findings , the mechanical properties of the fractured femora were not affected by hemorrhagic shock . we could observe a typical inflammatory response after fxhs whereas fx alone caused no significant increase in inflammatory cytokines . histological examination three weeks after fracture showed significantly decreased number of osteoclasts and reduced bone quality after hemorrhagic shock . in parallel , ct analyses showed a trend towards decreased mineral density in the fracture region after hemorrhagic shock . despite these microscopic findings , the mechanical properties of the fractured femora were not affected by hemorrhagic shock . three major factors are crucial for sufficient fracture healing : stability , blood supply , and the presence of osteogenic mediators . it is known that interfragmentary shearing forces and movement can delay fracture healing or lead to nonunions [ 1113 ] . a sufficient blood supply is predicated on the integrity of the vascular system and on the circulating blood volume . disorders in angiogenesis have a negative influence on fracture healing especially in the early stages after fracture [ 14 , 15 ] . reported no changes in the blood supply in the fracture region in rats after hemorrhagic shock . furthermore , the mechanical properties of the fracture were not affected four weeks after injury . these results confirmed the results of lucas et al . who showed an osteogenic effect of hemorrhagic shock in rats . additionally , a study in goats revealed no differences in mechanical properties , bone formation rate , and callus remodeling after hemorrhagic shock . recent studies have focused on osteoimmunology , specifically the importance of cellular and molecular interactions between the immune system and the bone . in addition , t - lymphocytes and cytokines seem to hold specific relevance in fracture healing in the context of osteogenic mediators [ 18 , 19 ] . the proinflammatory cytokine il-6 is involved in the regulation of the differentiation of osteoclast progenitor cell into mature osteoclasts [ 20 , 21 ] . on the other hand , il-6 can suppress chondrocyte proliferation and decrease the differentiation of growth plate chondrocytes , although this role of il-6 appears to be controversial [ 22 , 23 ] . tnf was shown to increase the serum calcium of mice and to stimulate new osteoclast formation and bone resorption , whereas it is potently proapoptotic for osteoblasts . previously , studies on fracture healing after hemorrhagic shock did not focus on the systemic inflammatory response in their models . the chosen durations of shock were relatively short in comparison to most established experimental shock models . this shortened time of depressed blood pressure might result in a lower immunological reaction [ 28 , 29 ] . therefore , it might be speculated that the positive effects on fracture healing were mostly related to improved blood flow properties after resuscitation with colloids or crystalloids . in contrast to the aforementioned studies , wichmann et al . reported increased osteocyte necrosis adjacent to the fracture site and a decrease of plasma calcitonin levels after hemorrhagic shock when compared to a closed fracture without associated hemorrhagic shock . as expected , we measured a significant increase in inflammatory cytokines after the shock period comparable to preceding studies . similar to two studies which used comparable shock models [ 8 , 30 ] , we found an impaired fracture healing response after hemorrhagic shock . we demonstrated a decrease in bone quality scoring and an increased number of cartilage islands within the callus region three weeks after hemorrhage . this impairment may to some extent be explained by the reduced number of osteoclasts after shock . they are responsible for the resorption of damaged and necrotic tissue to prepare the field for the reparative phase . nevertheless , the initial stage of enchondral fracture repair does not necessarily depend on osteoclasts , although they may contribute to vascular invasion and early enchondral ossification . in the further stages of healing , the inhibition of osteoclasts therefore , the reduced number of osteoclasts after hemorrhagic shock might be responsible for delayed clearance of necrotic tissue as well as impaired remodeling of callus . this suggestion is in line with another recent study which showed a decreased rate of osteoclasts 8 days after hemorrhagic shock whereas the differentiation of osteoblasts was unaffected . in both histological and ct analyses , we observed bridging of the fracture site with bone in both groups . this is in line with manigrasso and o'connor who reported that callus volume decreases after 14 days . hemorrhagic shock did not appear to have any effect on bmd or bv / tv within the callus region measured by ct analyses . however , there was a trend towards decreased tmd in the shock group , indicating that the bone formed in this model may have an inferior quality of mineralization . with a voxel size of 25 m , the grey value of the bone surface voxels will be subject to partial volume effects . it is therefore difficult to ascertain whether the tmd values observed here are accurate depictions of the state of mineralization of the tissue . future studies in this area should apply a smaller voxel size ( < 12 m ) in order to produce a more accurate calculation of tmd . a crucial aspect of healing is that new tissue in the fracture zone must provide sufficient strength to the injured limb in order to regain function . to investigate the stability of the callus zone , we chose the distraction - to - failure model as a mode of testing because the geometry of the fracture is variable . despite stabilization of the fracture , the analysis of the mechanical properties of inhomogeneous and porous materials like bone is easily affected by the distribution of the load throughout the specimen [ 35 , 36 ] . the three point bending test for example has maximum load at only one point of the specimen . to eliminate measurement artifacts caused by variable femur thickness and shape , we chose tensile testing to homogeneously strain the femur cross - section . at three weeks , the maximum force at failure of the hs group was equivalent to the sham group . the decreased failure strength of the fracture zone in comparison to the uninjured bone is consistent with a previous study , which indicated that significant increases in structural or material strength did not occur until 6 to 12 weeks after fracture . in our model , we are not able to report any information about the stiffness of the fracture region . therefore , stiffness differences between sham and hs groups are a topic for future studies . additionally , we performed a selective examination three weeks after fracture , which is during the remodeling phase . therefore , this time point might have been too delayed to measure significant differences in mechanical stability despite histological evidence of impaired fracture healing . another study that assessed the influence of simvastatin on fracture healing might support this assumption . simvastatin - related increases in callus volume could be measured only two weeks after fracture . at three weeks , they noted no differences in mechanical strength or callus volume between the control and drug - treated group . therefore , potential differences in mechanical properties as well as callus volume and mineralization should be analyzed in further studies at earlier time points . in conclusion , our results suggest an impaired maturation of the fracture callous three weeks after hemorrhagic shock . changes in the number of osteoclasts may play an important role in this bone healing delay . | impaired fracture healing can occur in severely injured patients with hemorrhagic shock due to decreased soft tissue perfusion after trauma .
we investigated the effects of fracture healing in a standardized pressure controlled hemorrhagic shock model in mice , to test the hypothesis that bleeding is relevant in the bone healing response .
male c57/bl6 mice were subjected to a closed femoral shaft fracture stabilized by intramedullary nailing .
one group was additionally subjected to pressure controlled hemorrhagic shock ( hs , mean arterial pressure ( map ) of 35 mmhg for 90 minutes ) .
serum cytokines ( il-6 , kc , mcp-1 , and tnf- ) were analyzed 6 hours after shock .
fracture healing was assessed 21 days after fracture .
hemorrhagic shock is associated with a significant increase in serum inflammatory cytokines in the early phase .
histologic analysis demonstrated a significantly decreased number of osteoclasts , a decrease in bone quality , and more cartilage islands after hemorrhagic shock .
ct analysis showed a trend towards decreased bone tissue mineral density in the hs group .
mechanical testing revealed no difference in tensile failure .
our results suggest a delay in fracture healing after hemorrhagic shock .
this may be due to significantly diminished osteoclast recruitment .
the exact mechanisms should be studied further , particularly during earlier stages of fracture healing . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
spinal stenosis is a narrowing of the neural canal and foramina , resulting in the compression of the neurologic structures with resultant symptoms . the first description may have very well been by portal of france in 1803 , and details have been popularized by verbiest and classified by arnoldi et al based on the pathology of the stenosis.1
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4 in 1957 , brain and wilkinson described stenosis in both the cervical and lumbar regions , a condition that would later be termed tandem spinal stenosis ( tss ) by dagi et al.5
6 teng and papatheodorou in 1964 further expanded on the topic , observing significant spinal stenosis occurring in both the cervical and lumbar regions of the spine.7 patients typically present with both upper and lower motor neuron dysfunction , including progressive gait disturbance and neurologic claudication . spinal decompression procedures for isolated spinal stenosis are common in the adult population with relatively good outcomes noted in the literature.8
9 however , there is limited literature addressing the surgical management of symptomatic spinal stenosis in multiple regions of the spine . in particular , triple - region spinal stenosis ( trss ) , or stenosis in the cervical , thoracic , and lumbar spine , is not well described . to date , 15 articles were identified on the topic of tss . the prevalence of this condition as well as the optimal surgical management are not clear . the available literature demonstrates a wide variation in the prevalence , ranging from 0.9% to 25%.10
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16 due to the potential risk and morbidity of simultaneous surgery , four available publications recommended a staged decompression approach for tss.6
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16 the purpose of this study is to review the available literature and to present a unique case of trss treated with simultaneous tandem decompression surgery . a 50-year - old male commercial truck and bus driver had a history of a work - related ground - level fall 10 years prior to presentation complicated by chronic back pain . upon clinic presentation , he reported a 2-month history of progressive gait instability , worsening leg weakness , difficulty walking , numbness in both of his forearms , numbness in his left c7 dermatome , difficulty picking up objects , and pain in his lumbar , thoracic , and lower cervical spine . his examination was remarkable for unsteady gait , inability to stand on a single leg , limited lumbar flexion and extension , global weakness in multiple upper and lower extremity muscle groups , numbness in the upper and lower extremity regions , and a positive hoffman sign in the left hand . magnetic resonance imaging of the cervical , thoracic , and lumbar spine revealed severe c5t1 stenosis , severe t9t11 stenosis , and severe l4l5 stenosis ( figs . 1 , 2 , and 3 ) . ( a ) sagittal and ( b ) axial magnetic resonance imaging showing severe cervical stenosis . ( a ) sagittal and ( b ) axial magnetic resonance imaging showing severe thoracic stenosis . ( a ) sagittal and ( b ) axial magnetic resonance imaging showing severe lumbar stenosis . the patient was diagnosed with progressive myelopathy and severe lumbar spinal stenosis with evidence of symptomatology from all three spinal regions . due to his history of progressive and worsening symptoms , he was found to be an appropriate surgical candidate , and surgical intervention was offered . prior to the surgery , the patient underwent presurgical cardiac risk stratification by the cardiology team for left heart strain found on electrocardiogram . the patient had a history of long - standing hypertension and severe left ventricular hypertrophy and was found to be low risk for any cardiac event in the perioperative period . a mayfield tong head - holding device was applied , and the spine was prepped and draped from the occiput to the sacrum . via three separate incisions , the following procedures were performed : posterior cervical c5c7 laminectomy and c4t1 instrumented posterolateral fusion using local autogenous bone graft and multilevel cervical polyaxial lateral mass screws , t1 pedicle screws , 3.5-mm rods , and one crosslink t9t11 laminectomy and decompression of the spinal cord and t8t12 posterior instrumented fusion using local autogenous bone graft and left - sided t8t12 unilateral instrumentation consisting of titanium pedicle screws and a unilateral rod l4l5 bilateral lumbar decompression ( partial laminectomy , partial facetectomy , and bilateral foraminotomy ) the surgery was performed in succession by a single attending surgeon and surgical assistant with closure then performed simultaneously ( figs . 4 and 5 ) . at each level , a fenestrated active drain device was placed prophylactically and sewn into the skin . closure was achieved with absorbable polyglactin sutures for the fascia and subcutaneous tissues and staples for the skin . the patient tolerated the procedure well with no changes noted on somatosensory evoked potentials or motor evoked potential monitoring . the estimated blood loss ( ebl ) for the procedure was 600 ml total ( 250 ml cervical , 250 ml thoracic , 100 ml lumbar ) . he was discharged to inpatient rehabilitation on postoperative day 7 and discharged home on postoperative day 11 . at 6-month follow - up 6 , 7 , and 8) , gait was markedly improved , and motor function returned to normal in both upper and lower extremities . ( a ) anteroposterior and ( b ) lateral cervical spine radiographs at postoperative 6 months . ( a ) anteroposterior and ( b ) lateral thoracic spine radiographs at postoperative 6 months . ( a ) anteroposterior and ( b ) lateral lumbar spine radiographs at postoperative 2 weeks . a 50-year - old male commercial truck and bus driver had a history of a work - related ground - level fall 10 years prior to presentation complicated by chronic back pain . upon clinic presentation , he reported a 2-month history of progressive gait instability , worsening leg weakness , difficulty walking , numbness in both of his forearms , numbness in his left c7 dermatome , difficulty picking up objects , and pain in his lumbar , thoracic , and lower cervical spine . his examination was remarkable for unsteady gait , inability to stand on a single leg , limited lumbar flexion and extension , global weakness in multiple upper and lower extremity muscle groups , numbness in the upper and lower extremity regions , and a positive hoffman sign in the left hand . magnetic resonance imaging of the cervical , thoracic , and lumbar spine revealed severe c5t1 stenosis , severe t9t11 stenosis , and severe l4l5 stenosis ( figs . 1 , 2 , and 3 ) . ( a ) sagittal and ( b ) axial magnetic resonance imaging showing severe cervical stenosis . ( a ) sagittal and ( b ) axial magnetic resonance imaging showing severe thoracic stenosis . ( a ) sagittal and ( b ) axial magnetic resonance imaging showing severe lumbar stenosis . the patient was diagnosed with progressive myelopathy and severe lumbar spinal stenosis with evidence of symptomatology from all three spinal regions . due to his history of progressive and worsening symptoms , he was found to be an appropriate surgical candidate , and surgical intervention was offered . prior to the surgery , the patient underwent presurgical cardiac risk stratification by the cardiology team for left heart strain found on electrocardiogram . the patient had a history of long - standing hypertension and severe left ventricular hypertrophy and was found to be low risk for any cardiac event in the perioperative period . a mayfield tong head - holding device was applied , and the spine was prepped and draped from the occiput to the sacrum . via three separate incisions , the following procedures were performed : posterior cervical c5c7 laminectomy and c4t1 instrumented posterolateral fusion using local autogenous bone graft and multilevel cervical polyaxial lateral mass screws , t1 pedicle screws , 3.5-mm rods , and one crosslink t9t11 laminectomy and decompression of the spinal cord and t8t12 posterior instrumented fusion using local autogenous bone graft and left - sided t8t12 unilateral instrumentation consisting of titanium pedicle screws and a unilateral rod l4l5 bilateral lumbar decompression ( partial laminectomy , partial facetectomy , and bilateral foraminotomy ) the surgery was performed in succession by a single attending surgeon and surgical assistant with closure then performed simultaneously ( figs . 4 and 5 ) . at each level , closure was achieved with absorbable polyglactin sutures for the fascia and subcutaneous tissues and staples for the skin . the patient tolerated the procedure well with no changes noted on somatosensory evoked potentials or motor evoked potential monitoring . the estimated blood loss ( ebl ) for the procedure was 600 ml total ( 250 ml cervical , 250 ml thoracic , 100 ml lumbar ) . the total operative time was 3.5 hours . he was discharged to inpatient rehabilitation on postoperative day 7 and discharged home on postoperative day 11 . at 6-month follow - up , 6 , 7 , and 8) , gait was markedly improved , and motor function returned to normal in both upper and lower extremities . ( a ) anteroposterior and ( b ) lateral cervical spine radiographs at postoperative 6 months . ( a ) anteroposterior and ( b ) lateral thoracic spine radiographs at postoperative 6 months .
( a ) anteroposterior and ( b ) lateral lumbar spine radiographs at postoperative 2 weeks . the medline database was searched using the key words tandem stenosis , tandem spine , triple region , and cervical , thoracic , and lumbar stenosis . a total of 15 articles were identified as having been published on the topic of treatment of tandem stenosis . several of the articles did not include patients who were treated surgically ; only 10 of the 15 published articles included patients who underwent surgical management for tss . five publications included patients who were treated with a staged surgical approach , and six publications included small numbers of patients who had a one - staged or simultaneous surgical approach . all of the published studies were either commentaries or retrospective reviews containing small numbers of patients with multiple surgeons . authors have previously described degenerative spine conditions that may confuse clinical presentations by having symptoms from two different spine regions . laban and green suggested that when there is a combination of symptomatic spinal stenosis from different regions , the surgical decision - making process can be confusing.12 the authors suggested that symptoms of one region usually predominate , and only after one is treated does the second become more evident . since tss was first described , estimates of its frequency have varied widely , ranging from as low as 0.9% to as high as 28%.10
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13 several studies have attempted to accurately define the incidence of tss.12
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17 lee et al examined cervical and lumbar cadaveric specimens of 440 skeletally mature spines and found the prevalence of tandem stenosis between 0.9% and 5.4% . the authors defined spinal stenosis as an absolute midsagittal canal diameter less than 12 mm present in at least one spinal level.17 laban and green performed a 10-year retrospective review of 460,964 hospital admissions at a large community teaching hospital to identify the frequency of tss.12 the authors identified a base population of only 54 patients with tss , with a frequency rate for all ages of 12 per 100,000 admissions . more recently , in a cadaveric study of 1,072 adult skeletons , bajwa et al attempted to determine whether tandem stenosis is related to congenital narrowing in specific regions of the spine.14
15 the authors reported the incidence of tandem cervical and lumbar stenosis to be 2% , with tandem cervical and thoracic stenosis at only 1% . in general , the best estimates of the incidence of tandem stenosis in the general population appear to be 1 to 2% , and symptomatic tandem stenosis is much less . within certain patient populations , however , specifically patients with symptomatic stenosis in one region , the prevalence of tandem stenosis can be higher.10
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few studies in the literature address the surgical management of tss , with only 10 being identified at the time of this manuscript writing.6
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23 each of these studies consists of small numbers of patients and are of a retrospective nature . our report is unique as it describes a safe and simultaneous surgery for all three regions of the stenosis . with regards to these studies , dagi et al performed a retrospective review of 19 patients with tss with a mean follow - up of 22 months . the authors recommended decompression of both the cervical and lumbar regions , but suggested the more symptomatic level be performed first.6 an excellent outcome was seen in 5 patients ( 26% ) , 4 improved ( 21% ) , 5 deteriorated despite initial improvement ( 26% ) , and 1 was unchanged . there was 1 postoperative death and 3 patients could not be found for follow - up . the postoperative improvement correlated inversely with symptom duration , and the authors stressed the importance of an early diagnosis and timely surgical intervention . hsieh et al reported an overall incidence of 7.6% of tss in a series of 158 patients who had surgery for degenerative spinal stenosis.16 the authors ' surgical preference in the treatment of these 12 patients diagnosed with tss required that cervical surgery be addressed first if signs of upper motor neuron or upper extremity symptoms were predominant . for patients with predominant lower extremity symptoms but no upper motor neuron exam findings , lumbar surgery was performed first . in this series , the majority ( 67% ) of the patients received cervical surgery first . the average follow - up was 32 months , and good or excellent clinical outcomes were demonstrated in 67% of patients . in 7 of the 12 patients with tss , ossification of the posterior longitudinal ligament ( opll ) was noted ( 58% ) , which the authors felt might be a predominant factor found in tss . epstein et al felt that the correct order for operative treatment in tss depended on the severity of myelopathy and radiculopathy.11 patients requiring cervical surgery first had stenosis with a cervical spinal canal of 10 mm or less in anteroposterior diameter . patients requiring lumbar surgery first had a lumbar spinal canal between 11 and 13 mm in diameter and first presented with symptomatic lumbar radiculopathy , with a significant portion ( 50% ) having intermittent neurogenic claudication . in this series , cervical decompression often improved lumbar symptoms with the resolution of pain , spasticity , and sensory deficits of myelopathic origin . latent findings of severe claudication due to lumbar spinal stenosis were not relieved by cervical decompression and increased in severity . significant improvement was shown in 90% of the patients in this series , with latent severe lumbar symptoms treated with staged multilevel lumbar decompressive surgery . aydogan et al also performed a retrospective study on tss between 1998 and 2004.10 eight adult patients were diagnosed with tss in a series of 230 patients who underwent surgery for spinal stenosis for an overall incidence of 3.4% . three patients first received cervical surgery and five patients first received lumbar surgery based on main symptomatology and physical exam findings . the japanese orthopaedic association ( joa ) scores of all patients improved from an average of 8.1 preoperatively to an average of 12.7 points at final follow - up with a mean follow - up of 35 months . the oswestry disability index ( odi ) scores improved from a mean of 58.1 to 19.3 , and all patients had good or excellent results without neurologic deterioration . similar to other authors , the researchers felt that the order of decompression surgery should be based on the clinical symptoms and exam findings . all surgeries were staged , with the second surgery performed between 2 weeks to 2 months after the first surgery in an attempt to avoid higher complication rates . simultaneous surgery for tss has not been addressed extensively in the literature , with only six published studies identified.18
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23 limits of these studies include the small numbers of patients and the retrospective nature of a relatively uncommon condition . in their case report , naderi and mertol described two patients with symptomatic tss in which simultaneous decompressive surgery was performed.22 the total surgical time for these patients was 130 and 150 minutes , and the authors reported neurologic and gait improvement postoperatively . no outcome measures were utilized in this case report , but the authors recommended simultaneous surgery as an alternative approach for patients with symptomatic tss . kikuike et al performed a retrospective review of elderly patients ( average age 71 years ) with tss who received single - stage combined cervical and lumbar decompression with average follow - up of 5 years.20 the authors utilized clinical outcomes with joa - b and joa - c scores and activities of daily life prior to surgery , 6 months postoperatively , and at final follow - up . the joa - b and joa - c scores and activities of daily life improved significantly 6 months after surgery , but ultimately deteriorated with additional follow - up in 7 patients ( 41% ) . complications involving other parts of the body significantly influenced clinical deterioration at final follow - up , including lower extremity fractures , knee osteoarthritis , malignant tumor diagnosis , and cerebral infarction . twelve patients ( 70% ) were satisfied with their surgical outcome at the final follow - up . the authors felt that reasons other than the spinal pathology affected symptom deterioration at final follow - up , and that single - stage combined cervical and lumbar decompression surgery was safe for elderly patients . in 2011 , eskander et al published a retrospective study on the outcomes after simultaneous decompression of the cervical and lumbar spine versus staged operations in 43 patients.19 twenty - one patients , or nearly half ( 49% ) , underwent simultaneous decompression of both the cervical and lumbar spine , and 22 patients ( 51% ) underwent staged decompression of the cervical spine followed by decompression of the lumbar spine at a later date . there was no significant difference between the groups in regards to major or minor complications and joa and odi scores at 7-year follow - up . several factors increased the risk of minor complications , including age above 68 years , ebl 400 ml or more , and operative time 150 minutes or more . the authors concluded that tss can be managed by either surgical intervention or simultaneous or staged decompression , but that patient age , blood loss , and operative time should be considered when planning surgical strategies to help minimize postoperative complications . chen et al reported a retrospective series of 15 patients found to have combined cervical and thoracic opll or ossification of the ligament flavum , in which the authors termed tandem ossification . the patients were treated with a single - stage combined decompression of the cervical and thoracic spine.18 the authors reported significant overall improvements in joa score and nurick classification at minimum follow - up of 2 years , but noted that satisfaction was tied to perioperative complications and disease progression . krishnan et al reported a retrospective series of 53 patients undergoing single - stage decompression for tss , the largest series yet reported.21 the authors noted improvement in modified joa and odi scores . they found that operating room time of < 150 minutes and ebl < 400 ml lessened complications and improved scores . they stressed patient selection , noting significant improvement in odi and nurick grade in the group younger than 60 years . the authors recommended single - stage surgery in this younger group , but staged surgery in patients over 60 years of age . hong and liu described a case of multiregional spinal stenosis involving opll in a 36-year - old man in which one - stage cervical and thoracic decompression and fusion surgery was performed.23 the patient did well initially with upper cervical laminectomy and instrumented fusion , lower cervical laminoplasty , and upper thoracic laminectomy and instrumented fusion . however , 10 months postoperatively , severe tandem mid- and lower thoracic stenosis due to opll required further surgical intervention . the postoperative course was complicated by epidural hematoma and paraplegia that required additional surgery with some eventual return of neurologic function at 3.5-year follow - up . the authors stressed the importance of reducing kyphosis , performing an adequate decompression , and using increased implant density to prevent kyphosis and further spinal cord injury . we have reported a patient with symptomatic stenosis in three separate regions of the spine . the thoracic and lumbar regions demonstrated stenosis due to the typical degenerative cascade involving disk degeneration with bulging and facet and ligamentum flavum hypertrophy . we demonstrate that simultaneous surgery for trss appears to be an effective treatment option but may be associated with prolonged hospital stay after the surgery . there is a paucity of literature describing tss and successful management . to our knowledge , this report is the first of symptomatic trss treated with simultaneous surgery in three different regions of the spine . symptomatic triple - region spinal stenosis treated with simultaneous surgery : case report and review of the literature this case report was selected by evidence - based spine - care journal ( ebsj)global spine journal ( gsj ) due to the clinical concerns raised in dr . cheung 's discussion : the difficulty in making a clinical diagnosis of symptomatic spinal stenosis in the setting of concurrent upper and lower motor neuron compression . most surgeons would agree that most patients who have complaints of diffuse back pain probably suffer from a somatization disorder . similarly , nonfocal upper and lower extremity complaints as presented in this case report may lead to a deferral of neuroimaging investigation and be managed by prolonged rehabilitative care and even exploration of psychological confounders . in our era of increasing use of algorithm - driven medicine , simple findings such as back pain with clear radicular symptoms will be managed with a carefully choreographed sequence of diagnostic and management steps . patients with complex multifocal spine disease like the case presented by dr . schaffer and colleagues how should we as spine professionals in charge of offering our patients best possible responsible care respond to such a conundrum ? should we perhaps lower our threshold for ordering neuroimaging studies in patients with certain stigmata for multifocal stenosis or disk disease ? can we come up with better questions or physical tests to screen our patients better ? clearly patients with conditions such as severe congenital stenosis in one region of the spine are probably reasonable candidates for consideration for coordinated neuroimaging of the remainder of their spinal columns . similarly , patients with opll or pervasive advanced disk degeneration or hypertrophic osteoarthropathy would likely benefit from neural axis imaging prior to making a detailed surgical plan . at present , our diagnostic algorithm lacks such clarity and routinely leads to contentious approval procedures for more expanded neuroimaging requests . as we are still trying to sort out the threshold for imaging screening for patients with possible multiregional spine disease , the other question of management either contemporaneously , as done by schaffer et al , or staged in separate surgeries remains completely unanswered at the present time . the magnitude of performing three spine surgeries at the same setting in three different parts of the body of one patient is a major logistics and physiologic undertaking in all regards and likely requires considerably greater investments in postoperative rehabilitation . any complication in one area would probably have undesirable consequences in the other surgical sites . this question is similar to the option of performing bilateral hip or knee replacement surgeries a surgical option that has yet to be resolved well over a decade after its introduction . ebsj - gsj hopes that its readership will find the discussions of diagnostic and treatment challenges for patients with multiregional symptomatic spinal stenosis to be interesting and worthy of further discussion . | study design case report .
objectives symptomatic triple - region spinal stenosis ( trss ) , defined as spinal stenosis in three different regions of the spine , is extremely rare . to our knowledge ,
treatment with simultaneous decompressive surgery is not described in the literature .
we report a case of a patient with trss who was treated successfully with simultaneous decompressive surgery in three separate regions of the spine .
methods a 50-year - old man presented with combined progressive cervical and thoracic myelopathy along with severe lumbar spinal claudication and radiculopathy .
he underwent simultaneous decompressive surgery in all three regions of his spine and concomitant instrumented fusion in the cervical and thoracic regions .
results estimated blood loss for the procedure was 600 ml total ( 250 ml cervical , 250 ml thoracic , 100 ml lumbar ) and operative time was 3.5 hours .
no changes were noted on intraoperative monitoring .
the postoperative course was uncomplicated .
the patient was discharged to inpatient rehabilitation on postoperative day ( pod ) 7 and discharged home on pod 11 . at 6-month follow - up , his gait and motor function was improved and returned to normal in all extremities .
he remains partially disabled due to chronic back pain .
conclusions this report is the first of symptomatic trss treated with simultaneous surgery in three different regions of the spine .
simultaneous triple region stenosis surgery appears to be an effective treatment option for this rare condition , but may be associated with prolonged hospital stay after surgery . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
fatty liver is a major health problem in high yielding dairy cows in early lactation [ 1 , 2 ] . obese cows have a greater decrease in feed intake during peripartal period and , therefore , have a more severe negative energy balance during early lactation . nonesterifed fatty acids ( nefa ) that are released from the stored triacylglycerol ( tag ) in the adipose tissue are readily taken up by the liver where they provide energy for liver function . if more nefa arrive at the liver , that needed for energy purposes , the excess may be oxidized incompletely and generate ketone bodies as well as converted to tag for deposition . under normal conditions , tag are secreted from the liver as very - low - density lipoproteins ( vldl ) . however , obesity in dry cows is not always associated with fatty liver indicating that there are risk factors other than obesity that predispose the cow to development of fatty liver . inadequate endocrine adjustments during initiation of lactation may play a role in fatty liver pathogenesis . in particular , the thyroid gland may play a role since the intensity of metabolic activity , metabolism of carbohydrates and lipids as well as lactogenesis are determined by hormones released from that tissue . there are evidences in literature about strong relation between thyroid hormone status during the peripartal period ( 3 weeks before to 3 weeks after calving ) , when the homeostatic mechanisms are under the greatest challenges , and postpartal metabolic disorders in holstein - friesian dairy cows [ 6 , 7 ] . although there is inconsistency in literature data due to experimental procedure , it is generally observed that thyroid hormone concentrations decrease during the peripartal period due to the adaptation of organisam to negative energy balance and initiation of lactation [ 812 ] . if reductions are pronounced or not synchronized with other metabolic and endocrine adjustments , the decrease may cause a metabolic disturbance that leads to increased lipolysis of adipose tissue and triglyceride accumulation in parenchymal tissue , especially the liver . kapp et al . observed that decreased levels of thyroid hormones in blood were associated with reduced mitochondrial capacity to oxidize fatty acids which led to diffuse lipid infiltration of hepatocytes and pronounce hepatic function impairment . the hormonal status of cows in the far - off and mid dry period and its relationship to postpartal metabolic disorders , to our knowledge , have not been reported in the literature . with respect to fatty liver pathogenesis , identification of risk factors that are associated with homeorhetic mechanisms leading to metabolic disorders would provide a tool that could be used to better manage cows to prevent fatty liver or provide early warning signs that could be used to segregate cows and mark them for intensive interventional protocols after calving . this strategy requires an understanding of the point in time when adjustments of regulatory mechanisms break through physiological limits thus predisposing the cow to metabolic problems . the aim of this study was to monitor thyroid gland activity from 30 days before expected calving to 12 days postpartum in order to investigate its relationship with liver lipid content on day 12 postpartum . the objective of the work was to develop a predictor of fatty liver that could be used for diagnostic and interventional purposes . twenty one healthy dry cows ( 30 days before expected calving ) that had yielded 7000 liters of milk or more per annum in the previous lactation were chosen from the commercial dairy herd and placed on the study . ingredients and chemical compositions of the dry and early lactation cow diets are listed in tables 1 and 2 . three blood samples were taken by jugular venipuncture from each animal : the first 30 days before expected calving ( mid dry period ) , the second 2 days before expected calving ( late dry period ) , and the third 12 days after calving ( early lactation period ) . samples were obtained with a sterile needle into tubes and allowed to clot spontaneously ( approximately 15 minutes ) on ice . samples were subsequently centrifuged at 1,000 g for 20 minutes , and the serum was decanted and stored at 18c until analyzed . to compare hormone concentrations without influence of daily rhythms , samples were taken 4 to 6 hours after morning feeding . triiodthyronine ( t3 ) and thyroxine ( t4 ) concentrations in the blood serum were measured by radioimmunoassay kit ( inep - zemun , serbia ) validated for use with bovine serum . the mean intraassay coefficients of variation ( cv ) for duplicate samples were 4.1% and 3.5% for t4 and t3 , respectively . liver percutaneous biopsies were obtained using a biopsy instrument following the method of hojovcova and kacafirek . the biopsy was performed at the right 11th intercostal space , approximately 2 cm below the horizontal line through the tuber coxae . a medium - sized cannula ( ~6 mm o.d . and ~4 mm i.d ; 20.5 cm long ) surrounding a solid , retractable needle - pointed trocar was inserted through the muscle and peritoneum into the liver in the direction toward the left shoulder . after boring the cannula into the liver , a sample ( 3 to 5 cm long and 3 to 4 mm in diameter ) was obtained through creating a vacuum by drawing back the trocar and then flexing the tip of the cannula upward and gently pushing forward . the liver sample was expelled onto a clean wipe , blotted free of blood , and placed into storage vials that contained 10% phormaldehyde solution for fixation . liver tissue lipid content was determined at the pathomorphology department at the faculty of veterinary medicine , university of belgrade . for pathohistological determination of lipids lipid content in the hepatocytes was determined through computer image analysis ( software q win ) . cows were divided into three groups of equal size based on the degree of lipid accumulation in the liver : mild fatty liver ( < 20% fat , n = 7 ) , moderate fatty liver ( 20 to 30% fat , n = 7 ) , and severe fatty liver ( > 30% fat , n = 7 ) . normality was tested using the kolmogorov - smirnov test before statistical analysis . as all variables showed normal distribution , statistical analysis was performed using two - way anova , with time and the degree of lipid accumulation in the liver as fixed factors . where anova revealed a significant effect , tukey post hoc test was administered to identify differences between groups . analyzing data for all cows ( diagonally patterned bar , figure 1 ) , t4 and t3 concentrations showed a downward trend from day 30 to day 12 after calving ( figures 1(a ) and 1(b ) ) , while the t3/t4 ratio increased from day 30 to day 2 and decreased from day 2 to day 12 ( figure 1(c ) ) . our results are consistent with previous reports in the literature relative to the effects of time during the periparturient period on thyroid hormone concentrations in cows [ 812 ] . however , according to the results obtained by two - way anova with time and the degree of lipid accumulation in the liver as the fixed factors , time was a signifant factor for t4 only ( f = 6.34 , p < .01 , and f = 2.84 , p = .067 , f = 0.41 , p = .667 for t3 and t3/t4 ratio , resp . ) . t4 concentration 30 days before expected calving significantly differed from values on 2 days before calving and 12 days of lactation ( p < .05 and p < .01 , resp . ) . the t3/t4 ratio is a simple calculated weight units ratio that reflect thyroid gland function and the potential for action of these hormones on peripheral tissues . the thyroid gland produces mainly t4 , but also a small quantity of t3 . in the peripheral tissues the mostly inactive t4 undergoes extrathyroidal enzymatic activation by 5deiodinase ( 5d ) producing the much more potent t3 . according to pezzi et al . , type i 5d prevails in liver and is most active during the dry period , while the highly efficient type ii 5d predominates in mammary glands , and its activity is the highest during early lactation . according to content of lipids in liver at day 12 after calving , cows were divided into three groups : cows with mild fatty liver ( n = 7 ; 10.34 4.81% fat ) , cows with moderate fatty liver ( n = 7 ; 25.77 3.92% fat ) , and cows with severe fatty liver ( n = 7 ; 43.58 6.10% fat ) . concentrations of t3 , t4 as well as t3/t4 ratio were all dependent on the content of lipids in liver ( f = 26.82 , f = 63.42 , and f = 24.12 , resp . , . thus , t4 concentrations were significantly less for cows with severe fatty liver ( figure 1(a ) ) than for cows with mild ( p < .001 ) and moderate ( p < .001 ) fatty liver , and a slight but significant difference was also found between mild and moderate fatty liver ( p < .05 ) . furthermore , cows with severe fatty liver had significantly lower concentration of t3 than cows with mild ( p < .001 ) and moderate fatty liver ( p < .001 ) ( figure 1(b ) ) . the t3/t4 ratios ( figures 1(c ) ) for cows with severe fatty liver were significantly higher than for cows with mild ( p < .001 ) and moderate fatty liver ( p < .001 ) . interaction between time and the degree of liver lipids was insignificant for all parameters ( f = 2.54 , p = .055 , f = 1.29 , p = .287 , and f = 1.64 , p = .179 , for t3 , t4 , and t3/t4 ratio , resp . ) . correlation coefficients between determined parameters ( t4 , t3 , and t3/t4 ratio ) and the content of lipids in the liver of cows are shown in table 3 . this work demonstrates that dairy cows with severe fatty liver experience lower serum t4 concentrations before the peripartal period . additionaly , the results of this study provide evidence of a significant negative correlation between t4 concentrations and the degree of fatty accumulation in the liver by 12 days after parturition not only during late - dry ( r = 0.898 ; p < .001 ) and early lactation period ( r = 0.769 ; p < .001 ) , when lipid mobilization occurs , but also during mid dry period ( r = 0.633 ; p < 0.01 ) ( table 3 ) . low thyroid hormone ( t3 and t4 ) concentrations during the early lactation period in cows with severe fatty liver has already been established [ 811 ] . during the liver steatosis , fatty acids accumulate in the liver parenchyma , and it has been demonstrated that some fatty acids inhibit type - i liver 5d activity . that is probably the reason for low t3 concentrations during the late dry ( day 2 ) and early lactation ( day 12 ) periods in cows with severe fatty liver . additionally , a significant negative correlation exists between t3 concentrations and the degree of fat accumulation in liver during the late dry ( r = 0.680 ; p < .01 ) and early lactation ( r = 0.606 ; p < .01 ) periods , but not during the mid dry period ( table 3 ) . high t3/t4 ratios in cows with severe fatty liver during the mid dry period and a significant positive correlation with the degree of fat accumulation in liver ( r = 0.606 ; p < .01 ) may indicate a pronounced decline of t4 levels or involvement of mechanism that increases t3 concentration . according to pezzi et al . , t3 concentration increase could be due to increased 5d tissue activity . significant positive correlations between t3/t4 ratio and the degree of fat accumulation in liver exist until calving ( r = 0.772 ; p < .001 ) . since liver is the main site of deiodinase activity during the dry period , it can be hypothesized that cows that will be affected with severe fatty liver had enhanced metabolic activity in the liver during mid dry period which may compromise hepatocyte adaptability during the peripartal period . additionally , prevalence of cows with t4 concentrations below the lowest physiological value ( 4.2 g / dl , according to kaneco et al . ) , was significantly higher in cows with severe fatty liver during mid dry period than in cows with moderate and mild fatty liver ( p < .01 both ) , while during late dry period the difference was statistically significant only between cows with severe and mild fatty liver ( p < .01 ) . namely , all cows with severe fatty liver had t4 below cut off value , which indicates that they experienced hypothyroidsm before calving . only one cow in group with moderate fatty liver and no cow in group with mild fatty liver had t4 concentrations below 4.2 g / dl . in conclusion , we can affirm that cows , that were affected with severe fatty liver during early lactation , were in a hypothyroid state during the entire examining period , which means prior to development of the condition , too . the hypothyroid state during the mid dry period may be both a risk factor and an early indicator of fatty liver , while , during early lactation , it is important etiopathogenetic factor of fatty liver . according to our results , it may be suggested that low peripheral serum thyroid hormone status may provoke t3/t4 ratio increase . based on the avaible data from this experiment , no conclusion related to mechanism that provokes t3/t4 ratio increase | relationship between postpartal fatty liver and thyroid gland activity during the peripartal and mid dry periods was studied .
twenty one dry cows were chosen .
blood samples were obtained on days 30 , 2 , and + 12 related to calving and analized for thyroxine ( t4 ) and triiodothyronine ( t3 ) .
a t3/t4 ratio was calculated .
liver tissue samples were taken 12 d after calving and tested for the lipid content .
cows were divided into three groups : mild ( < 20% fat ) , moderate ( 20 to 30% ) , or severe fatty liver ( > 30% ) .
cows , that were affected with severe fatty liver , were hypothyroid prior to development of the condition due to lower t4 concentrations , and had significantly lower concentration of t3 and higher t3/t4 ratios than cows with mild and moderate fatty liver .
thus , hypothyroid state during mid - dry period may be an early indicator of postpartal fatty liver and may provoke t3/t4 ratio increase in this group of cows . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
for the past 50 years , the pressurized metered - dose inhaler ( pmdi ) and the nebulizer have been the primary means of delivering inhaled drugs to patients with asthma and copd . these devices can be used effectively , but are inefficient and may be difficult or cumbersome to use . some of the limitations of these standard inhaler devices are accentuated in patients with copd , especially if they are elderly or have severe disease . standard pmdis at best deposit 10%15% of the delivered dose in the lungs and most of the inhaled dose is deposited in the oropharynx . to perform optimally , pmdis require coordination of actuation with inhalation as well as a slow inspiratory flow rate and breath - holding ( wolff and niven 1994 ) . poor coordination of pmdi actuation with inhalation has been shown to result in lower lung deposition of drug and reduced lung function response ( newman et al 2001 ) . the pmdi breathing maneuver requires education and repeated instruction , and can be problematic for older patients with copd . addition of a spacer or holding chamber can decrease oropharyngeal drug deposition and improve lung delivery , but makes the system less portable . handling of a pmdi can also be more difficult if the patient has arthritis of the hands . while jet nebulizers may be popular with some patients with copd , they are time - consuming , very inefficient , and require cleaning . for routine use of treatment for ambulatory patients , standard jet nebulizers are not portable and have not been proven to offer additional efficacy in asthma or copd patients ( dolovich et al 2005 ) . other issues that may be prominent in an elderly copd population are the confusion that arises with prescription of multiple inhalers with different instructions for use and also the expense of newer inhaled drug device combinations . events of the past decade have stimulated development of novel drug formulations and delivery devices that have improved the efficiency , ease of use , and environmental impact of inhaled drug therapy . because of deleterious effects of chlorofluorocarbon ( cfc ) propellants on the ozone layer , the montreal protocol was developed by the united nations in 1987 , banning substances that deplete the ozone layer ( leach 1995 ) . this ban phased out the use of cfcs by 1996 , although pharmaceutical companies had exemptions . further developed countries will follow in the next few years . while the contribution of cfc inhaler propellants has a minute environmental impact development of novel inhaler devices has focused on three areas : pmdis with hydrofluoroalkane ( hfa ) propellants , dry powder inhalers ( dpis ) , and liquid multidose spray devices that do not require propellants . hfa - pmdis generate aerosols that exit at lower velocity and may contain smaller particles . this may improve lung deposition , but these devices still require good coordination and slow inhalation . dpis require high inspiratory flow rates and may not work effectively in a patient with severe copd . dpis also are associated with high oropharyngeal deposition ( fink 2000 ) and the drug powder can be sensitive to moisture . respimat soft mist inhaler ( boehringer ingelheim , ingelheim , germany ) is a novel , multidose , propellant - free , hand - held liquid inhaler that represents a new category of inhaler devices . soft mist is used to describe the mechanism of aerosol generation and the qualities of the aerosol cloud . the aerosol plume generated by respimat travels much slower and lasts much longer than aerosol clouds from other devices , properties that improve delivery and make this device a valuable addition to inhalers available for inhalation therapy to copd patients . respimat is a hand - held inhaler with a hinged cap that is similar in size to pmdis and some dpis ( figure 1 ) . medication is stored as a solution in the drug cartridge , an aluminum cylinder containing a double - walled , plastic , collapsible bag that contracts as the solution is used . the solution may be formulated with either ethanol or water , with benzalkonium chloride and ethylene diamine tetra - acetic acid ( edta ) added as preservatives . the amount of preservatives in each puff is extremely low : approximately 0.44 g for benzalkonium chloride and 2.2 g for edta . tests on used cartridges have shown that patient use does not cause bacterial contamination of the solution in the cartridge . the first marketed product delivers 120 actuations and has a dose indicator , which is a color - coded gauge marked in increments of 30 doses . this indicator gives an estimate of doses remaining but does not count individual doses . after 120 actuations have been delivered , a locking mechanism prevents further use by preventing twisting of the base so that no further doses can be actuated . twisting the base of the device 180 degrees compresses a spring and provides mechanical power to aerosolize the dose of drug and also transfers a metered dose of drug ( usually 1015 l ) from the drug cartridge through a capillary tube to the pump cylinder . when the dose - release button is pushed , the energy from the compressed spring forces the drug through a nozzle system called the the uniblock consists of a silicone wafer bonded to a glass plate and measures approximately 2 x 2.5 mm . channels are etched into the silicon wafer using a technique derived from microchip technology and these channels feed into the nozzle outlet . actuation of the respimat dose - release button utilizes the mechanical energy from the spring to force the metered drug solution through the channels in the uniblock , producing two fine jets of liquid at the outlet that converge at a predetermined angle to form the aerosol cloud ( spallek et al 2002 ) . this cloud contains an aerosol with a fine - particle fraction ( particles smaller than 5.8 m ) at least twice as high as most pmdis and dpis , which would allow a higher proportion of the emitted dose to be delivered to the lungs and less to the oropharynx . the fine particle fraction is higher for ethanolic formulations than for aqueous formulations , with a mass median aerodynamic diameter of 2.00.4 m for aqueous solutions and 1.00.3 m for ethanolic solutions ( zierenberg 1999 ) . the fine - particle fraction is approximately 66% for an aqueous drug solution and 81% for an ethanolic solution . moves more slowly and has a more prolonged duration than the aerosol cloud from a pmdi . hochrainer et al ( 2005 ) have compared respimat aerosol velocity and spray duration with that of cfc - pmdis and hfa - pmdis via video - recording ( figure 3 ) . the velocity of the aerosol from respimat was between one - sixth and one - tenth of that from the cfc - pmdis and two of the hfa - pmdis and approximately one - third of the velocity of the slower hfa - pmdis . the mean velocity of the aerosol cloud measured at a 10 cm distance from the nozzle was 0.8 m / s for respimat and 2.08.4 m / s for pmdis , while the mean duration was 1.5 s and 0.150.36 s , respectively . the combination of smaller particle size , lower velocity , and longer duration of the aerosol cloud implies that there would be improved coordination of inhalation with actuation , higher lung deposition , and lower oropharyngeal deposition compared with pmdis . after receiving a new respimat inhaler , a patient must insert the cartridge by removing the transparent base and pushing the cartridge into the inhaler until it clicks into place . the dose is loaded by holding the inhaler in an upright position and turning the base 180 degrees until it clicks . the inhaler requires priming prior to first use by actuating until an aerosol cloud is visible and then completing three more actuations . spray content uniformity measurements show priming actuations necessary to achieve 100% of target volume and also show spray uniformity over 120 doses without tail - off effect ( spalleck 2002 ) . priming with actuation of one dose is recommended after a week of no use , and full priming is recommended after 21 days of no use . instructions for inhalation direct the patient to breathe out slowly and deeply and close lips around the end of the mouthpiece while holding the inhaler in a horizontal position . the dose - release button should be pushed while the patient takes a slow , deep breath in through their mouth and a 10-second breath - hold is recommended . the dose can be administered to the patient with the inhaler held in any position . improved lung deposition of drug aerosols from the respimat has been demonstrated by several studies using radiolabeled drug particles and gamma scintigraphy ( newman et al 1996 , 1998 ; steed et al 1997 ) . this group of studies in healthy nonsmoking volunteers used either fenoterol , which was formulated in an aqueous medium , or flunisolide , formulated in 96% ethanol . in one study with flunisolide , the mean whole - lung deposition from respimat ( 40% of the metered dose ) was significantly higher than from the pmdi ( 15% ) or pmdi plus spacer ( 28% ) ( newman et al 1996 ) . a follow - up study using flunisolide and a final prototype respimat showed significantly more lung deposition with respimat compared with pmdi plus spacer ( 45 vs 26% ) ( newman et al 1998 ) . in this same study , mean whole - lung deposition of fenoterol was significantly greater when delivered by respimat ( 39% ) than via pmdi ( 11% ) or pmdi plus spacer ( 10% ) . in both of these studies in normal subjects , the oropharyngeal deposition was significantly lower with respimat than with pmdi ( approximately 40% vs 70% ) , but not as low as with pmdi plus spacer . a more recent investigation has compared lung deposition of inhaled steroids in mild to moderate asthmatic subjects using respimat , turbuhaler , and a pmdi ( pitcairn et al 2005 ) . respimat contained budesonide solution , the turbuhaler contained budesonide dry powder , and the pmdi contained beclomethasone dipropionate ; the turbuhaler inhalations were performed at either 60 or 30l / m . mean whole - lung deposition of drug from respimat was significantly greater than that from turbuhaler dpi at fast or slow inhaled flow rates , or from the pmdi . the deposition pattern within the lungs was more peripheral for the respimat than for the turbuhaler . these data in normal and asthmatic subjects would predict that lower doses of drugs via the respimat would provide equal efficacy to higher doses delivered via the pmdi or turbuhaler . currently , the respimat is marketed with a combination of ipratropium bromide ( ib ) and fenoterol hydrobromide ( fen ) ( berodual respimat ) and was launched in germany in january 2004 . five clinical studies have been performed with berodual respimat in patients with copd and in adults and children with asthma ( reviewed in kssner et al 2004 ) . a pivotal phase iii , multicenter , parallel - group study of 892 patients with moderate to severe copd compared ib / fen via respimat with a cfc - pmdi ( kilfeather et al 2004 ) . patients were randomized to one of five treatment arms : respimat containing ib 10 g / fen 25 g , respimat containing ib 20 g / fen50 g , pmdi containing ib 20 g/ fen50 g , respimat placebo , or pmdi placebo four times daily for 12 weeks . the primary efficacy endpoint was the average change from pre - dose in fev1 over the first hour after dose administration on day 85 of treatment and the study was powered for non - inferiority . the bronchodilator response to ib / fen on day 85 showed that the 20/50 g dose via respimat was not inferior to the 40/100 g dose via cfc - pmdi ( figure 5 ) . four trials of ib / fen via respimat have been conducted in patients with asthma ( thee in adults and on in children ) . two of these were proof - of - concept , phase ii , dose - ranging and cumulative dose studies in adults with asthma ( kunkel et al 2000 ; goldberg et al 2001 ) . in the dose - ranging study ( goldberg et al 2001 ) , five different doses of ib / fen ( from 5/12.5 g to 80/200 g ) were used and showed a log - linear dose response for average increase in fev1 up to 6 hours . responses to ib / fen doses of 5/12.5 and 10/25 g administered via respimat were closest or slightly superior to that for the ib / fen dose of 40/100 g delivered with cfc - pmdi . therapeutic equivalence could not be demonstrated statistically in this study due to higher - than - expected variability . the cumulative dose study in adults with asthma ( kunkel et al 2000 ) concluded that ib / fen delivered by respimat was as effective as the cfc - pmdi at half the cumulative dose . two phase iii studies were done comparing ib / fen via respimat and cfc - pmdi in adults and children with asthma ( vincken et al 2004 ; von berg et al 2004 ) . these studies concluded that delivery by respimat instead of a cfc - pmdi allows a two- to four - fold reduction in dosage of ib / fen while achieving similar efficacy . these trials in copd and asthmatic subjects provide evidence in favor of the deposition experiment predictions that ib / fen delivered by respimat can be given at a reduced dosage ( one - fourth to one - half ) compared with that given by cfc - pmdi without any loss of efficacy . the only other clinical trial published to date using respimat in copd patients was a cumulative dose study of ipratropium bromide compared with delivery via pmdi ( iacono et al 2000 ) . this was a three - period cross - over study in 36 patients in which two dosages of ib by respimat were used ( 10 and 20 g per puff ) compared with 20 g per puff via cfc - pmdi . the study found greater bronchodilation from half the cumulative dose of ib by respimat compared with the pmdi . there have been two phase ii published comparative trials using fen , given alone to asthmatic subjects via respimat and cfc - pmdi ( van noord et al 2000 ; vincken et al 2003 ) , and again showing the ability to use reduced doses with the respimat . there have also been clinical trials conducted examining clinical efficacy and safety of tiotropium bromide delivered by respimat in copd subjects , but these have not been published . safety analysis of the five clinical trials using ib/ fen in the respimat showed an adverse event profile similar to the cfc - pmdi . in one of the phase ii studies ( kunkel et al 2000 ) , the 320/800 g dose given with respimat was associated with a slightly higher incidence of headache , nervousness , and tremor than the same dose by pmdi . this may have been due to slightly increased 2-adrenergic stimulation and cholinergic blockade with the greater drug delivery to the lungs by the respimat . in the phase iii studies , the adverse - event profiles of the 10/25 and 20/50 g doses of ib / fen via respimat were comparable with that of the 40/100 g dose via cfc - pmdi , and the frequency of adverse events thought to be treatment - related was low across all groups . because of the use of the preservatives benzalkonium chloride and edta in bronchodilator preparations in the respimat , there have been questions about the possibility of paradoxical bronchoconstriction with respimat . this has been analyzed in both the phase ii and phase iii data sets for ib / fen and either drug alone , and incidence of paradoxical bronchoconstriction was not found to be increased compared with cfc - pmdi ( koehler et al 2004 ; hodder et al 2005 ) . for the copd and asthma patients in this analysis , there were no observed episodes of bronchospasm following administration of active drug or placebo via the respimat . the incidence of asymptomatic falls in fev1 > 15% from baseline after respimat use was 0%2.8% in the active treatment group and 0%7.6% in the placebo group and was no different than that measured after cfc p - mdi . many patients lack coordination for the split - second timing required between beginning a slow inhalation and activation of a pmdi ( mcfadden 1995 ; fink and rubin 2005 ) . asynchronous actuation can greatly reduce the amount of medication inhaled from a pmdi ( wilkes et al 2001 ) and can also reduce clinical effect of the inhaled drug ( mcfadden 1995 ; newman et al 2001 ) . one study showed that incidence of critical errors in pmdi use were significantly higher in copd patients ( 26% ) compared with asthma patients ( 13% ) ( melani et al 2004 ) . breath coordination can be addressed by use of breath - actuated pmdis , pmdis plus spacer devices , dpis , or nebulizers ( newman 2005 ) . newer hfa - pmdis create aerosols that exit at a lower velocity than cfc - pmdis and may have a smaller particle size , both factors which may help overcome problems with poor coordination . like the pmdi , respimat is also a press and breathe device and requires some coordination of actuation and inhalation . respimat demonstrates lower aerosol velocity and longer spray duration that either cfc or hfa pmdis , which should allow patients to coordinate actuation and breathing more easily . the deposition data show that respimat deposits significantly more drug in the lungs of asthmatic patients than the turbuhaler dpi or a cfc - pmdi in the setting of optimal inhalation technique . to date , no studies have been published that examine drug deposition from the respimat in subjects with copd or assess deposition and efficacy of respimat in patients with poor inhalation technique . in a clinical trial comparing ib / fen via respimat or an hfa - pmdi in 245 patients with copd , asthma or mixed disease , correct assembly , and inhaler technique were assessed after training at the beginning of each treatment period ( schurmann et al 2005 ) . patients were given up to five attempts to demonstrate satisfactory technique and were scored on seven different device handling and breathing tasks . at the beginning of each treatment period , 96.4% could demonstrate satisfactory inhaler technique with respimat by the final attempt , compared with 98% of patients with hfa - pmdi . the proportion of patients achieving a score of 7 at the first attempt was higher for hfa - pmdi ( 80% ) than for respimat ( 48% ) , perhaps due to the novelty of the respimat device . while patients required more training for good technique with respimat , patients retained good technique with respimat after 7 weeks ( 97% ) , compared with 94% in th hfa - pmdi group . most patients found the respimat easy to assemble and only one patient was unable to assemble the device . with the multitude of inhaler devices now available , there has been increased interest in the assessment of patient preference and satisfaction , as preference for a particular medication or inhaler device may be associated with improved adherence with therapeutic regimens . boehringer ingelheim developed and validated a patient satisfaction and preference questionnaire for inhalation devices to better assess satisfaction with the respimat compared with other devices ( kozma et al 2005 ) . the patient satisfaction and preference questionnaire ( pasapq ) was developed using published papers , focus groups , and expert opinion . the final questionnaire contains 14 satisfaction items , including a global satisfaction question , as well as one preference question and one question on willingness to use the device in the future . the satisfaction items were grouped into two domains : performance ( seven items ) and convenience ( six items ) . of all device satisfaction instruments that have been used in clinical trials , only the pasapq has a published validation and determination of minimally important difference , which is very important for discriminating the degree of difference that is clinically significant . the pasapq was used in a study specifically designed to examine preference for and satisfaction with inhaler devices in patients with copd , asthma , or mixed disease in a crossover study of ib / fen delivered via respimat vs hfa - pmdi ( schrmann 2005 ) . the questionnaire was administered after each 7-week treatment period and of the 201 out of 224 subjects expressing a preference , 81% preferred respimat . of the 44 patients who had concomitant diagnoses that might affect inhaler handling , such as eye problems or arthritis , 89% preferred respimat . patients were more willing to continue to use respimat and mean ratings for 13 of 15 items in the satisfaction questionnaire were significantly higher for respimat . there were no differences between the inhalers for efficacy measures such as peak expiratory flow , rescue inhaler use , and symptom scores . taking device preference and satisfaction into account when choosing an inhaler device may be associated with improved clinical outcomes , but this has not been proven to date . respimat represents a novel approach to the delivery of inhaled drugs and overcomes some of the limitations of pmdis , dpis , and nebulizers . it is portable , propellant - free , uses mechanical energy for actuation , and does not require cumbersome spacers or holding chambers . unlike some dpis , optimal aerosol generation does not depend on high inspiratory flow rates . respimat produces an aerosol with a greater fine - particle fraction than most pmdis , dpis , and nebulizers and the aerosol spray produced exits the inhaler more slowly and lasts for a longer time . this translates into higher lung drug deposition and lower oropharyngeal deposition than the pmdi and also turbuhaler dpi . for some drug formulations , therapeutic ratio could be improved with respimat , by offering higher lung deposition with lower oropharyngeal deposition and lower nominal dosing . it is also possible that drug delivery and efficacy will be improved with respimat in those patients who have difficulties in actuating and coordinating inhalation when using a pmdi , but the device still requires some degree of hand breath synchronization . in clinical trials in patients with copd , bronchodilator drugs delivered from respimat were equally effective at half of the dose delivered from a pmdi . in one study of inhaler preference , respimat was preferred over the pmdi by patients with copd and other obstructive lung diseases . it is not clear , however , if preference for the device will lead to improved adherence and clinical outcomes . currently , respimat is available in germany containing a combination of ipratropium bromide and fenoterol . as availability increases , respimat is a novel and valuable addition to the range of inhaler devices available to the patient with copd . breath coordination that may be a problem for patients when using a pmdi and does not require generation of high inspiratory flow rates required for some dpis . respimat also makes it possible to give lower nominal doses of delivered drugs without decreasing efficacy . a continuing challenge in this population of patients , however , is the expense and confusion engendered by use of multiple inhaler devices . | events of the past decade have stimulated development of new drug formulations and delivery devices that have improved the efficiency , ease of use , and environmental impact of inhaled drug therapy .
respimat soft mist inhaler is a novel , multidose , propellant - free , hand - held , liquid inhaler that represents a new category of inhaler devices .
the aerosol cloud generated by respimat contains a higher fraction of fine particles than most pressurized metered dose inhalers ( pmdis ) and dry powder inhalers ( dpis ) , and the aerosol spray exits the inhaler more slowly and for a longer duration than with pmdis .
this translates into higher lung drug deposition and lower oropharyngeal deposition , making it possible to give lower nominal doses of delivered drugs without lowering efficacy . in clinical trials in patients with copd , bronchodilator drugs delivered from respimat
were equally effective at half of the dose delivered from a pmdi .
in one study of inhaler preference , respimat was preferred over the pmdi by patients with copd and other obstructive lung diseases .
respimat is a valuable addition to the range of inhaler devices available to the patient with copd . |
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macular telangiectasia ( mactel ) , also known as idiopathic juxtafoveolar telangiectasia , is an uncommon bilateral disease that affects the juxtafoveolar region of the macula . mactel was first described by reese in 1956 , and was classified into three groups by gass and blodi in 1993 . mactel type 2 is bilateral and associated with minimal macular edema , deep hyperfluorescence on fluorescein angiography ( fa ) , loss of macular transparency , superficial white crystals , depletion of macular pigment , progressive foveal thinning and edema in the non - proliferative stages , and subretinal neovascularization ( srn ) in the proliferative stage . we report a patient who first presented with bilateral non - proliferative mactel type 2 , who showed a proliferative transformation in her left eye , and underwent intravitreal bevacizumab ( ivb ) treatment as - required with a beneficial outcome . a 47-year - old female was admitted to our clinic with a complaint of decreased vision in the right and left eye ( ou ) in december 2009 . biomicroscopic anterior segment examination was normal , and intraocular pressure was within normal limits ou . fa , showed a horse shoe shaped hyperfluorescence on the temporal fovea ou [ figure 1a ] . optical coherence tomography ( oct ) showed an internal limiting membrane ( ilm ) drape ou [ figure 2a ] . the central retinal thickness ( crt ) was 198 microns and 210 microns , in the right and left eyes respectively . based on the clinical and imaging findings the patient was diagnosed with mactel type 2 and advised to present for examinations every 2 months . four months later , the patient complained of decreased vision in the left eye ( os ) . on the examination , bcva was 20/100 os , and fundus examination revealed a grey reflex representing a srn beneath the fovea . fa showed a subfoveal classic choroidal neovascularization ( cnv ) associated with leakage ou [ figure 1b ] . oct showed a high reflective area located subfoveally , and associated with intraretinal and subretinal fluid collection , and the crt was 318 microns os [ figure 2b ] . the patient was diagnosed with proliferative mactel type 2 os , and underwent 3 consecutive monthly 1.25 mg ivb injections . after these injections , bcva increased to 20/25 , and both clinical and angiographic features showed significant improvement with minimal leakage on fa and absence of intra- or sub - retinal fluid on oct examination . follow - up examinations were scheduled monthly and treatment regimen of ivb injections as clinically required was planned . retreatment criteria were determined as ; loss of visual acuity of 1 line , and detection of any amount of intra- or subretinal fluid on oct . at month 12 , oct showed subretinal fluid with a crt of 259 microns os . the patient underwent another ivb injection os . at month 24 , the bcva decreased to 20/63 , and fa showed active leakage around the cnv os . after the fifth ivb injection , bcva improved to 20/40 os . at month 29 as bcva decreased to 20/126 and oct showed subretinal fluid , a sixth ivb injection was administered . at month 30 , visual acuity improved to 20/40 , and oct revealed a hyperreflective cnv scar located subfoveally , with a crt of 198 microns os [ figure 2d ] . the right eye remained stable with a visual acuity of 20/25 for 30 months , and did not show any signs of proliferative mactel type 2 . fluorescein angiography images of the patient ( a ) horse shoe shaped hyperfluorescence temporal to the fovea ( white arrow ) , ( b ) subfoveal classic choroidal neovascularization with active leakage ( white arrow ) . optical coherence images of the patient ( a ) internal limiting membrane drape ( white arrow ) at baseline , ( b ) hyperreflective subfoveal classic choroidal neovascularization ( white arrow ) , and subretinal fluid ( white star ) at the 4 month , ( c ) resolution of subretinal fluid and small hyperreflective scarring secondary to classic choroidal neovascularization , after the first three intravitreal bevacizumab injections ( white arrow ) , ( d ) small hyperreflective scarring secondary to classic choroidal neovascularization ( white arrow ) at the last follow - up visit . macular telangiectasia type 2 is a symmetrical disease that involves the temporal side of the fovea . several classifications are described according to clinical , fa , oct and fundus autofluorescence findings . mactel type 2 was divided into 5 stages by gass and blodi . in stage 1 , a grayish reflex temporal to the fovea may be present on clinical examination , fa is especially important at this stage , and in the late phases , fa shows hyperfluorescence in the perifoveal region . stage 2 is characterized by the loss of transparency of the retina temporal to the fovea and early staining at the level of outer capillary zone and late staining in the outer retina . in stage 3 , fa reveals capillary dilatation , staining and prominent right angled venues . stage 4 shows migrating rpe cells appearing as stellate foci along the right - angled venules . yannuzzi , combined the classification with oct findings , and wong added fundus autofluorescence and microperimetry findings to the classification . in spite of different classifications , the first stage is the non - proliferative stage , which is characterized by slow , progressive visual loss in association with outer retinal atrophy . the second stage is the proliferative stage , which is characterized by the presence of srn . several treatment options were studied for non - proliferative stage ; however , most were reported to be ineffective because of the degenerative and progressive nature of this disease . in contrast , the proliferative stage was successfully treated with photodynamic therapy , intravitreal anti - vascular endothelial growth factor ( vegf ) injections , and combination therapies . engelbrecht et al . , showed that the natural course of proliferative mactel type 2 resulted in very poor prognosis including visual acuity of 1/20 or less in 80% of cases . , showed that pdt was effective for stabilizing the vision in 5 of 7 eyes . however , improved vision was achieved only in 3 of 7 eyes , and there was no improvement in median visual acuity over a mean follow up time of 21 months . anti - vegf agents , have rapidly become the mainstay of therapy against any type of cnv in various diseases , including proliferative mactel type 2 . a case series of 6 eyes , reported that median visual acuity improved from 20/200 to 20/100 , and the mean crt decreased from 263 microns to 201 microns over a mean follow up time of 4.2 months . these results were fairly good ; however , the follow - up time was very limited . roller et al . , also reported beneficial results of intravitreal bevacizumab in 9 patients . in their study , after a mean follow - up time of 17 months , and mean of 4.9 bevacizumab injections ( range 1 injection and 15 injections ) , the mean visual acuity improved 1.1 lines , and mean central macular thickness decreased by 48 microns . in addition , the visual acuity either remained stable or improved in 7 of 9 eyes ( 78% ) , and the cnv resolved completely in 8 of 9 eyes ( 89% ) after treatment . karagiannis et al . , showed that monthly injections of ranibizumab were effective in a patient with proliferative mactel type 2 . they reported that , the visual acuity improved from 1/20 to 3/10 after 12 monthly intravitreal ranibizumab injections . in our case , two different stages of the disease were observed during follow - up . in the first 4 months , the non - proliferative stage of the disease was seen which was followed by a proliferative stage over the following 26 months . the visual acuity decreased from 20/25 to 20/100 , and crt increased by 108 microns while the disease was transforming from the non - proliferative to the proliferative stage . after the intravitreal bevacizumab therapy for the proliferative phase , the visual acuity increased from 20/100 to 20/40 , and crt decreased from 318 microns to198 microns . the visual acuity improved by 4 lines with intravitreal bevacizumab therapy after the transformation of the disease . to our knowledge this is the first reported case of a patient who showed newly proliferative transformation of mactel type 2 , and was immediately treated with intravitreal bevacizumab injections on an as needed treatment regimen associated with beneficial results ( based on a pubmed and medline searches ) . in conclusion , non - proliferative mactel type 2 patients should be informed about the symptoms as decreased vision and metamorphopsia are associated with proliferative transformation , and followed carefully . intravitreal bevacizumab injections on an as needed treatment regimen may be effective in proliferative mactel type 2 . although investigation of large series is difficult due to the relative paucity of patients with proliferative mactel type 2 , further studies are required to evaluate the safety and efficacy of intravitreal anti - vegf agents in this group of patients . | the clinical and imaging findings and therapeutic outcomes of intravitreal bevacizumab injection in a patient with macular telangiectasia type 2 are described .
the patient first presented with the non - proliferative stage of the disease for 4 months , then the disease transformed to the proliferative stage . in the proliferative period ,
the patient was treated with intravitreal bevacizumab injections as - clinically warranted . over a follow up period lasting 26 months
, the patient received 6 intravitreal bevacizumab injections , the visual acuity improved from 20/100 to 20/40 , the central retinal thickness decreased from 318 microns to 198 microns .
this case implies that the patients with non - proliferative macular telangiectasia type 2 should be followed carefully for proliferative transformation , and intravitreal bevacizumab treatment seems to be effective for proliferative macular telangiectasia type 2 . |
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that was a type of phlebothrombosis complicated with portal hypertension and a reduction of the blood flow to the liver . owing to various symptoms in pvt , it is difficult to make an accurate diagnosis clinically.1 pvt causes portal hypertension and varix , and if severe , can cause variceal hemorrhage and ischemic bowel disease.1 the pathogenesis of pvt has not been identified clearly , but recent studies report that it is related to systemic causes such as protein c , protein s , and antithrombin iii deficiency disorder.2 however , there are very few studies of optimal treatment owing to the low incidence rate ( less than 0.004%).3 in korea , there are several reports of acute pvt caused by oral contraceptives and pancreatitis , but few cases of pvt caused by a deficiency of anticoagulant factors and reperfused with anticoagulation therapy.4 we report a case of pvt caused by protein c and s deficiency that ended in successful reperfusion with anticoagulation therapy . a 66-year - old man was admitted via the emergency department because of systemic myalgia and anorexia lasting for 2 weeks . he complained of high fever , nausea , and vomiting , and the results of liver function tests were abnormal . he had taken antihypertensive medication for 5 years and clopidogrel for 4 years owing to a history of transient ischemic attack . he had undergone subtotal gastrectomy for early gastric cancer 2 years ago . on admission , his vital signs were as follows : blood pressure , 130/70 mmhg ; resting heart rate , 70 beats per minute ; respiratory rate , 20 breaths per minute ; and body temperature , 37. his consciousness was clear but his conjunctivae were pale . he did not complain of tenderness or rebound tenderness of the abdomen , and there were no signs of hepatomegaly or splenomegaly . the laboratory findings were as follows : white blood cell count , 12,080/mm ( normal : 4,000 - 10,000/mm ) ; hemoglobin , 10.5 g / dl ( 13.0 - 17.0 g / dl ) ; hematocrit , 31.3% ( 39 - 52% ) ; platelet count , 351,000/mm ( 150,000 - 40,000/mm ) ; serum aspartate aminotransferase ( ast ) , 42 iu / l ( 0 - 37 iu / l ) ; serum alanine aminotransferase ( alt ) , 137 ( 0 - 41 iu / l ) ; serum alkaline phosphatase ( alp ) , 442 iu / l ( 39 - 117 iu / l ) ; gamma()-glutamyl transferase ( -gt ) , 255 iu / l ( 11 - 50 iu / l ) ; serum total bilirubin , 0.9 mg / dl ( 0.2 - 1.3 mg / dl ) ; total protein , 7.3 g / dl ( 6.7 - 8.3 g / dl ) ; albumin , 3.0 g / dl ( 3.5 - 5.2 g / dl ) ; and prothrombin time international normalized ratio ( pt inr ) , 1.21 ( 0.85 - 1.15 ) . abdominal ct scan showed complete occlusion of the intrahepatic portal vein by portal vein thrombi and small hepatic abscess around the occluded vessels ( fig . 1 ) . abdominal doppler ultrasound , esophagogastroduodenoscopy , and coagulation factor analysis were performed to identify the cause of the pvt . the abdominal doppler ultrasound showed complete portal vein occlusion but could not identify the surrounding microvessels , which implied a low probability of malignancy ( fig . coagulant factor viii and protein c factor were 261% ( normal : 60 - 140% ) and 41% ( normal : 60 - 140% ) , respectively . protein s factor showed a decrease of 14% ( normal : 60 - 140% ) . thus , the patient was diagnosed with pvt caused by a deficiency of protein c and s. administration of warfarin ( at a dose of 5 mg twice daily ) and subcutaneous injection of low molecular weight heparin ( enoxaparin , 1 mg / kg , twice daily ) was promptly started . at the time of discharge , the maintenance dose of warfarin was 2.5 mg per day ( target inr : 2 - 2.5 ) . the abdominal doppler ultrasound , performed just before discharge , revealed that the portal vein was still completely occluded by thrombi but decreased in diameter . the results of the laboratory tests conducted on an outpatient basis were as follows : inr , 1.90 ; protein c , 41% ; protein s , 14% ; ast , 61 iu / l ; alt , 54 iu / l ; alp , 56 iu / l ; and rgt , 215 iu / l . abdominal doppler ultrasound showed blood flow in the pvt ( fig . the results of the blood tests conducted 6 months later were as follows : serum inr , 1.71 ; ast , 57 iu / l ; alt , 46 iu / l ; alp , 113 iu / l ; and gt , 121 iu / l . on the abdominal doppler ultrasound performed at that time , the portal vein thrombi had disappeared and a smooth bloodstream was observed in the portal vein ( fig . 2c , d ) . pvt interrupts blood flow to the liver and causes portal hypertension.3 phlebothrombosis is caused by local , genetic , or acquired thrombophilic factors.5 local factors include local inflammatory lesions such as neonatal funiculitis , diverticulitis , appendicitis , pancreatitis , duodenal ulceration , tuberculous lymphadenitis , portal vein injury by surgical operations , thrombokinesis , pressure mass effect by an abdominal neoplasm , and portal hypertension by liver cirrhosis.5 denninger et al . reported that 6% to 11% of pvt is attributed to liver cirrhosis , and 35% is related to hepatocellular carcinoma.3 webb et al . reported that 40% of pvt is caused by intraperitoneal or systemic septicemia . in other reports , 7% of pvt was reported to be caused by chronic pancreatitis.6 primary myeloproliferative disorder , antiphospholipid antibody syndrome , paroxysmal nocturnal hemoglobinuria , oral contraceptives , pregnancy , childbirth , malignant tumor , and hypercysteinemia are reported as acquired thrombophilic factors.5 genetic thrombophilic factors can be divided into the frequent type , such as factor v leiden mutation and mutation iig20210 , in which pvt is rarely associated , and the rare type , such as protein c , s , and antithrombin iii , in which pvt is more likely to occur.5 clinically , pvt is classified as acute or chronic . however , it is difficult to identify when the symptoms begin and even the temporal criteria about chronicity have not yet been established.7 malkowski et al . classified pvt as acute or chronic according to symptom onset time before admission ( 60 days).7 acute pvt typically provokes stomachache , nausea , emesis , and fever . the symptoms can be more severe if mesenteric infarction provokes infarction of the bowel or liver . nonspecific symptoms such as diarrhea , anorexia , weight loss , and abdominal distention may develop.8 the symptoms of chronic pvt are similar to the acute ones , but such symptoms may be related to portal hypertension and splenomegaly.9 splenomegaly , anemia , leukopenia , thrombocytopenia , and variceal bleeding can be provoked.9 our case did not develop portal hypertension - related complications but showed a fever , stomachache , nausea , and emesis , and these symptoms are compatible with pvt . duplex , doppler ultrasound , and abdominal ct scan can be used to make a accurate diagnosis of pvt in the early stage , and abdominal mri can be another diagnostic tool.10 in particular , abdominal mri is known to be more useful than doppler ultrasound in identifying venous collateral development and cavernoma.10 in patients with pvt , local factors such as liver cirrhosis and pancreatitis should be suspected . if no local factor can be identified , it is necessary to investigate genetic or acquired thrombophilic factors.8 in our case , it was possible to infer that pvt was secondary to a liver abscess of the right lobe . however , it was more likely that the abscess was secondary to pvt for the following reasons . first , although massive thrombi obstructed the main and left portal vein , the abscess was restricted to the right lobe only . second , it took more than 1 year after abscess resolution for the cholestatic features on the liver function tests to be normalized . third , antibiotic therapy of only 3 weeks brought about a significant increase in main portal vein flow . regarding the treatment of pvt , long - term anticoagulant therapy and invasive methods such as surgical thrombectomy , local thrombolytic therapy , thrombolytic agentbased selective portography , and intrahepatic stent insertion via the jugular vein can be applied.9 in many cases , chronic pvt is accompanied by gastroesophageal varix , which can result in variceal bleeding during anticoagulation therapy.8 in the case of acute pvt , however , early anticoagulant therapy can prevent the spread of thrombi and promote blood reperfusion.9 sheen et al . applied anticoagulant therapy to 9 acute pvt patients with a target inr of 2 to 4 for 3 months . they reported that reperfusion was achieved in 78%.1 however , there are no clear guidelines on the choice of anticoagulant drugs , the period of administration , and even the need for anticoagulant therapy.7 in our case , we successfully treated our acute pvt patient with a 2-week subcutaneous injection of low molecular weight heparin and oral anticoagulant therapy . in conclusion , our case shows that pvt can be provoked by protein c and s deficiency and that the pvt can be recanalized by short - term low molecular heparin plus oral warfarin therapy . further study is needed to investigate the target inr , the period of administration , and the dosage and duration for relapse prevention in treatment with oral anticoagulants . | portal vein thrombosis ( pvt ) is a rare form of venous thrombosis that affects the hepatic portal vein flow , which can lead to portal hypertension .
treatment of pvt includes anticoagulants , thrombolysis , insertion of shunts , bypass surgery , and liver transplantation .
single anticoagulation therapy is not regarded as a curative treatment but can be associated with a reduction in new thrombotic episodes .
we experienced a case of acute total occlusion of pvt provoked by protein c and s deficiency syndrome .
pvt was completely recanalized with oral anticoagulant therapy following low molecular weight heparin therapy . |
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patients with primary oag and healthy subjects included in this study were recruited from the diagnostic innovations in glaucoma study ( digs ) and african descent and glaucoma evaluation study ( adages ) . the multicenter adages includes participants from the hamilton glaucoma center at the department of ophthalmology , university of california - san diego ; the new york eye and ear infirmary ; and the department of ophthalmology , university of alabama at birmingham . the diagnostic innovations in glaucoma study includes participants recruited at the university of california - san diego . all the methods adhered to the tenets of the declaration of helsinki and to the health insurance portability and accountability act . the institutional review boards at the university of california - san diego , new york eye and ear infirmary , and university of alabama at birmingham approved the methods . the african descent and glaucoma evaluation study and digs are registered as cohort clinical trials ( http://www.clinicaltrials.gov , identifiers nct00221923 and nct00221897 , september 14 , 2005 ) . methodological details of digs and adages have been described previously . in brief , for glaucoma subjects , inclusion criteria were 20/40 or better best - corrected visual acuity at baseline , spherical refraction within 5.0 diopters ( d ) , cylinder correction within 3.0 d , open angles on gonioscopy , and at least two consecutive and reliable standard automated perimetry vf examinations with either a pattern standard deviation ( psd ) or a glaucoma hemifield test ( ght ) result outside the 99% normal limits . exclusion criteria were eyes with coexisting retinal disease and eyes with nonglaucomatous optic neuropathy . for healthy subjects , inclusion criteria were 20/40 or better best - corrected visual acuity , spherical refraction within 5.0 d , cylinder correction within 3.0 d , iop < 22 mm hg with no history of elevated iop , and at least two reliable normal vfs , defined as a psd within 95% confidence limits and a ght result within normal limits . all subjects underwent an annual comprehensive ophthalmologic examination including review of medical history , best - corrected visual acuity , slit - lamp biomicroscopy , dilated funduscopic examination , and stereoscopic optic disc photography . semiannual examination included intraocular pressure ( iop ) , sd - oct imaging ( sd - oct circular scan , sd - oct onh cube scan , sd - oct onh radial scan , and sd - oct macular cube scan ) , and vf testing . in this report , we included three groups of participants . the first group was composed of 35 eyes of 35 advanced - glaucoma patients ( vf md 21 db ) followed for an average of 3.5 0.9 years . all eyes were followed at approximately 6-month intervals with vf and oct testing and were required to have a minimum of three vfs and three octs during follow - up for inclusion in this study ( number of tests ranged from three to eight ) . the stable glaucoma group of consisted of 50 eyes from 27 early- , moderate- , and advanced - glaucoma patients with five serial oct exams imaged every week for 5 weeks . we assume that in this short follow - up period , glaucoma changes are not likely to occur . these stable glaucoma eyes were used to train the bkds onh volume change method to reduce the likelihood that changes due to measurement variability would be classified as progression due to glaucoma . a third group of 46 eyes from 30 healthy subjects followed for an average of 2.8 0.4 years was used to estimate the aging effects . all eyes were followed at approximately 6-month intervals with vf and oct testing and had an average of six tests acquired during follow - up ( test range was 410 ) . healthy participants were recruited from the general population through advertisement , from referring practices , and from the staff and employees from the shiley eye institute , university of california , san diego . each subject was required to have at least one good - quality spectralis sd - oct ( heidelberg engineering inc . , heidelberg , germany ) circular scan , sd - oct onh cube scan , sd - oct onh radial scan , and sd - oct macular cube scan acquired on the same day . spectralis oct uses a dual - beam sd oct , a confocal laser - scanning ophthalmoscope with a wavelength of 870 nm , and an infrared reference image to obtain images of ocular microstructures . spectralis oct incorporates a real - time eye - tracking system that couples confocal laser - scanning ophthalmoscope and sd oct scanners to adjust for eye movements and to ensure that the same location of the retina is scanned over time . the image acquisition protocols included ( 1 ) high - resolution rnfl circle scan , which consists of 1536 a - scan points from a 3.45-mm circle centered on the optic disc , ( 2 ) high - resolution cube scan centered on the optic disc ( 73 b - scans with 768 a - scans each ) , ( 3 ) enhanced depth imaging ( edi ) scan centered on the optic disc ( 48 b - scans with 1024 a - scans each ) , and ( 4 ) high - resolution cubic scan centered on the fovea ( 73 b - scans with 768 a - scans each ) . quality assessment of oct scans was evaluated by imaging data evaluation and assessment ( idea ) center experienced examiners masked to the subject 's results of the other tests . raw 3d sd - oct images were exported to a numerical computing language ( matlab ; mathworks , natick , ma , usa ) . the san diego automated layer segmentation algorithm ( salsa ) was used to automatically segment ( 1 ) the bruch 's membrane opening ( bmo ) and the internal limiting membrane ( ilm ) on each onh radial scan to calculate the mrw defined as the shortest distance from bmo to ilm , and ( 2 ) the macular gcipl from the macular cube scan . the spectralis built - in segmentation algorithm was used to segment the cprnfl from the circular scan . briefly , we assumed that each b - scan consists of several interretinal layers ( e.g. , the bruch 's membrane [ bm ] layer , the retinal nerve fiber layer [ rnfl ] ) . because the interretinal layers have different thicknesses , each layer can be defined by a curve modeling its skeleton and a filter or set of filters modeling its thickness . to segment the different layers , it is sufficient to estimate their skeletons and the hyperparameters of the filters . in this study , we are interested only in the segmentation of the bm , ilm layers in the onh scans , and rnfl and ipl layers in the macular scans . in order to build connected skeletons , we considered an object - oriented approach rather than the voxel - oriented approach . therefore , short segments ( 20 voxels in our case ) are added to or deleted from the current configuration depending on their state ( connected or not ) . note that shorter segments have been considered in the termination of the skeletons for better estimation accuracy . the estimation of the model parameters and hyperparameters are addressed using a monte carlo markov chain . for the bmo identification , once we estimated the bm layer separately in each of the 48 b - scans , we utilized the whole 3d volume to estimate the bmo points to take into account the planarity of the bmo points . our aim is to properly integrate the elliptical shape of the bmo curve and to rely only on the reliable bmo points in the estimation scheme . note that our aim is not to fit an ellipse to the data but to use the elliptical shape as a bayesian an elegant way to address this task is to use the inverse artificial neural network ann - pca to model the elliptical shape of the bmo curve . the bayesian - kernel detection scheme is a bayesian - based approach that uses change in the 3d onh volume scans to classify an eye as nonprogressing or progressing . raw 3d sd - oct onh cube scans were exported to a numerical computing language ( matlab , mathworks ) . the bayesian - kernel detection scheme was used to estimate glaucoma progression from onh cube scans . we formulated the detection of glaucomatous change between baseline image and follow - up images as a missing data problem . markov random field model ( mrf ) prior was used to model the spatial dependency of changed voxels . once the change detection map was estimated , we used a kernel - based classifier for glaucoma progression detection . as prior knowledge or an agreed - upon gold standard for detection of progression in advanced glaucoma is not available , we used a one - class classifier trained only on the nonprogressing 50 stable glaucoma eyes and 36 healthy eyes . the svdd enables us to distinguish between targets ( nonprogressing eyes ) and outliers ( progressing eyes ) by defining a closed boundary around the target data . as done previously , we included two features as input for the svdd based on a glaucoma - related region of interest of the baseline image that generally consists of the rnfl , neuoretinal rim , and prelamina tissue of the onh identified using salsa . follow - up images were automatically registered by the instrument to the baseline image to detect change over time at specific locations in the region of interest . please note that retinal layer segmentation of the follow - up images is not required . the two features identified in the region of interest are the number of voxels detected as changed , and the ratio image intensity between the baseline and follow - up scans of changed voxels . an eye was defined as progressing if ( 1 ) macular gcipl , mrw , or crnfl loss was significantly ( p < 0.05 ) different from zero and faster than the 5th percentile of the healthy group or ( 2 ) 3d onh volume bkds change was classified as progressing utilizing both the healthy group and stable glaucoma group in the one - class svdd classification method . descriptive statistics were used to compare demographic characteristics by group ( healthy and glaucoma subjects ) . tests were used to compare categorical variables , and t - tests were used to compare continuous variables . mixed effects models were used to calculate the mean rates of change ( slopes ) for global cprnfl thickness , global mrw thickness , and global macular gcipl thickness loss from baseline . models included group ( healthy versus glaucoma ) , time , and the interaction term group time . the model was adjusted for age , and the correlation between eyes was accounted for in the model . statistical analysis was performed using sas , version 9.2 ( sas institute , cary , nc , usa ) . patients with primary oag and healthy subjects included in this study were recruited from the diagnostic innovations in glaucoma study ( digs ) and african descent and glaucoma evaluation study ( adages ) . the multicenter adages includes participants from the hamilton glaucoma center at the department of ophthalmology , university of california - san diego ; the new york eye and ear infirmary ; and the department of ophthalmology , university of alabama at birmingham . the diagnostic innovations in glaucoma study includes participants recruited at the university of california - san diego . all the methods adhered to the tenets of the declaration of helsinki and to the health insurance portability and accountability act . the institutional review boards at the university of california - san diego , new york eye and ear infirmary , and university of alabama at birmingham approved the methods . the african descent and glaucoma evaluation study and digs are registered as cohort clinical trials ( http://www.clinicaltrials.gov , identifiers nct00221923 and nct00221897 , september 14 , 2005 ) . methodological details of digs and adages have been described previously . in brief , for glaucoma subjects , inclusion criteria were 20/40 or better best - corrected visual acuity at baseline , spherical refraction within 5.0 diopters ( d ) , cylinder correction within 3.0 d , open angles on gonioscopy , and at least two consecutive and reliable standard automated perimetry vf examinations with either a pattern standard deviation ( psd ) or a glaucoma hemifield test ( ght ) result outside the 99% normal limits . exclusion criteria were eyes with coexisting retinal disease and eyes with nonglaucomatous optic neuropathy . for healthy subjects , inclusion criteria were 20/40 or better best - corrected visual acuity , spherical refraction within 5.0 d , cylinder correction within 3.0 d , iop < 22 mm hg with no history of elevated iop , and at least two reliable normal vfs , defined as a psd within 95% confidence limits and a ght result within normal limits . all subjects underwent an annual comprehensive ophthalmologic examination including review of medical history , best - corrected visual acuity , slit - lamp biomicroscopy , dilated funduscopic examination , and stereoscopic optic disc photography . semiannual examination included intraocular pressure ( iop ) , sd - oct imaging ( sd - oct circular scan , sd - oct onh cube scan , sd - oct onh radial scan , and sd - oct macular cube scan ) , and vf testing . in this report , we included three groups of participants . the first group was composed of 35 eyes of 35 advanced - glaucoma patients ( vf md 21 db ) followed for an average of 3.5 0.9 years . all eyes were followed at approximately 6-month intervals with vf and oct testing and were required to have a minimum of three vfs and three octs during follow - up for inclusion in this study ( number of tests ranged from three to eight ) . the stable glaucoma group of consisted of 50 eyes from 27 early- , moderate- , and advanced - glaucoma patients with five serial oct exams imaged every week for 5 weeks . we assume that in this short follow - up period , glaucoma changes are not likely to occur . these stable glaucoma eyes were used to train the bkds onh volume change method to reduce the likelihood that changes due to measurement variability would be classified as progression due to glaucoma . a third group of 46 eyes from 30 healthy subjects followed for an average of 2.8 0.4 years was used to estimate the aging effects . all eyes were followed at approximately 6-month intervals with vf and oct testing and had an average of six tests acquired during follow - up ( test range was 410 ) . healthy participants were recruited from the general population through advertisement , from referring practices , and from the staff and employees from the shiley eye institute , university of california , san diego . each subject was required to have at least one good - quality spectralis sd - oct ( heidelberg engineering inc . , heidelberg , germany ) circular scan , sd - oct onh cube scan , sd - oct onh radial scan , and sd - oct macular cube scan acquired on the same day . spectralis oct uses a dual - beam sd oct , a confocal laser - scanning ophthalmoscope with a wavelength of 870 nm , and an infrared reference image to obtain images of ocular microstructures . spectralis oct incorporates a real - time eye - tracking system that couples confocal laser - scanning ophthalmoscope and sd oct scanners to adjust for eye movements and to ensure that the same location of the retina is scanned over time . the image acquisition protocols included ( 1 ) high - resolution rnfl circle scan , which consists of 1536 a - scan points from a 3.45-mm circle centered on the optic disc , ( 2 ) high - resolution cube scan centered on the optic disc ( 73 b - scans with 768 a - scans each ) , ( 3 ) enhanced depth imaging ( edi ) scan centered on the optic disc ( 48 b - scans with 1024 a - scans each ) , and ( 4 ) high - resolution cubic scan centered on the fovea ( 73 b - scans with 768 a - scans each ) . quality assessment of oct scans was evaluated by imaging data evaluation and assessment ( idea ) center experienced examiners masked to the subject 's results of the other tests . raw 3d sd - oct images were exported to a numerical computing language ( matlab ; mathworks , natick , ma , usa ) . the san diego automated layer segmentation algorithm ( salsa ) was used to automatically segment ( 1 ) the bruch 's membrane opening ( bmo ) and the internal limiting membrane ( ilm ) on each onh radial scan to calculate the mrw defined as the shortest distance from bmo to ilm , and ( 2 ) the macular gcipl from the macular cube scan . the spectralis built - in segmentation algorithm was used to segment the cprnfl from the circular scan . briefly , we assumed that each b - scan consists of several interretinal layers ( e.g. , the bruch 's membrane [ bm ] layer , the retinal nerve fiber layer [ rnfl ] ) . because the interretinal layers have different thicknesses , each layer can be defined by a curve modeling its skeleton and a filter or set of filters modeling its thickness . to segment the different layers , it is sufficient to estimate their skeletons and the hyperparameters of the filters . in this study , we are interested only in the segmentation of the bm , ilm layers in the onh scans , and rnfl and ipl layers in the macular scans . in order to build connected skeletons , we considered an object - oriented approach rather than the voxel - oriented approach . therefore , short segments ( 20 voxels in our case ) are added to or deleted from the current configuration depending on their state ( connected or not ) . note that shorter segments have been considered in the termination of the skeletons for better estimation accuracy . the estimation of the model parameters and hyperparameters are addressed using a monte carlo markov chain . for the bmo identification , once we estimated the bm layer separately in each of the 48 b - scans , we utilized the whole 3d volume to estimate the bmo points to take into account the planarity of the bmo points . our aim is to properly integrate the elliptical shape of the bmo curve and to rely only on the reliable bmo points in the estimation scheme . note that our aim is not to fit an ellipse to the data but to use the elliptical shape as a bayesian an elegant way to address this task is to use the inverse artificial neural network ann - pca to model the elliptical shape of the bmo curve . the bayesian - kernel detection scheme is a bayesian - based approach that uses change in the 3d onh volume scans to classify an eye as nonprogressing or progressing . raw 3d sd - oct onh cube scans were exported to a numerical computing language ( matlab , mathworks ) . the bayesian - kernel detection scheme was used to estimate glaucoma progression from onh cube scans . we formulated the detection of glaucomatous change between baseline image and follow - up images as a missing data problem . markov random field model ( mrf ) prior was used to model the spatial dependency of changed voxels . once the change detection map was estimated , we used a kernel - based classifier for glaucoma progression detection . as prior knowledge or an agreed - upon gold standard for detection of progression in advanced glaucoma is not available , we used a one - class classifier trained only on the nonprogressing 50 stable glaucoma eyes and 36 healthy eyes . the svdd enables us to distinguish between targets ( nonprogressing eyes ) and outliers ( progressing eyes ) by defining a closed boundary around the target data . as done previously , we included two features as input for the svdd based on a glaucoma - related region of interest of the baseline image that generally consists of the rnfl , neuoretinal rim , and prelamina tissue of the onh identified using salsa . follow - up images were automatically registered by the instrument to the baseline image to detect change over time at specific locations in the region of interest . please note that retinal layer segmentation of the follow - up images is not required . the two features identified in the region of interest are the number of voxels detected as changed , and the ratio image intensity between the baseline and follow - up scans of changed voxels . an eye was defined as progressing if ( 1 ) macular gcipl , mrw , or crnfl loss was significantly ( p < 0.05 ) different from zero and faster than the 5th percentile of the healthy group or ( 2 ) 3d onh volume bkds change was classified as progressing utilizing both the healthy group and stable glaucoma group in the one - class svdd classification method . descriptive statistics were used to compare demographic characteristics by group ( healthy and glaucoma subjects ) . tests were used to compare categorical variables , and t - tests were used to compare continuous variables . mixed effects models were used to calculate the mean rates of change ( slopes ) for global cprnfl thickness , global mrw thickness , and global macular gcipl thickness loss from baseline . models included group ( healthy versus glaucoma ) , time , and the interaction term group time . the model was adjusted for age , and the correlation between eyes was accounted for in the model . statistical analysis was performed using sas , version 9.2 ( sas institute , cary , nc , usa ) . the study included 35 eyes of 35 glaucoma patients with very advanced glaucoma , 50 eyes from 27 stable glaucoma , and 46 eyes from 30 healthy subjects . a summary of the demographic variables and measurements at baseline of each group is shown in table 1 . glaucoma patients were significantly older ( p < 0.001 ) and had worse vf md ( p < 0.001 ) and longer follow - up ( p < 0.001 ) than healthy subjects . the advanced - glaucoma and the healthy eye groups were similar with respect to sex ( p = 0.74 ) , axial length ( p = 0.65 ) , and disc area ( p = 0.57 ) . baseline characteristics of study subjects baseline global cprnfl thickness derived from the circular scan , global mrw thickness derived from the onh radial scan , and macular gcipl thickness derived from the entire macular cube sd - oct scan measurements in healthy and advanced - glaucoma eyes are presented in table 2 . healthy eyes had thicker baseline cprnfl , mrw , and macular gcipl when compared to glaucoma eyes . baseline global cprnfl , mrw , and macular gcipl measurements by group rates of global cprnfl , mrw , and macular gcipl thickness loss in healthy and advanced - glaucoma eyes are presented in figure 1 and table 3 . in healthy eyes , mean rates ( p value ) of cprnfl , mrw , and macular gcipl were 0.32 m / y ( p < 0.001 ) , 1.41 m / y ( p < 0.001 ) , and 0.11 m / y ( p < 0.001 ) , respectively . in advanced - glaucoma eyes , the mean rates of cprnfl , mrw , and macular gcipl change were 0.08 m / y ( p = 0.39 ) , 0.29 m / y ( p = 0.43 ) , and 0.18 m / y ( p < 0.02 ) , respectively . there was no statistically significant difference between the mean rate of loss of cprnfl and mrw in advanced - glaucoma eyes and healthy eyes ( p = 0.44 and p = 0.58 ) . the mean rate of macular gcipl loss tended to be larger in advanced - glaucoma eyes than in healthy eyes , but the difference in mean rates between the two groups was not statistically significant ( p = 0.12 ) . distribution of the mean rates of change of circumpapillary retinal nerve fiber layer ( cprnfl ) , minimum rim width ( mrw ) , and macular ganglion cell inner plexiform layer ( gcipl ) thickness in healthy and advanced - glaucoma eyes . rates of global cprnfl , mrw , macular gcipl , and vf md loss by group by the structural measurement rate of change criterion , the number of identified progressing glaucoma eyes was 2 ( 5% ) using cprnfl , 4 ( 11% ) using the mrw , and 11 ( 31% ) using the macular gcipl . table 4 presents baseline global cprnfl , mrw , macular gcipl , and vf md measurements by study group ; table 5 presents the mean rates of global cprnfl , mrw , macular gcipl , and vf md loss by study group . no significant difference was found between the vf md baseline measurement and md mean rate of change of the 11 eyes that progressed based on the macular gcipl criteria and 24 eyes that did not progress ( p = 0.34 , p = 0.2 , respectively ) . however , mean macular gcipl thickness tended to be larger in the 11 eyes that progressed compared to the eyes that did not ( p = 0.09 ) . there was no significant difference in cprnfl and mrw thickness and mean rate of change between the 11 progressing eyes based on gcipl and the eyes that did not progress ( p = 0.39 , p = 0.43 and p = 0.37 , p = 0.58 , respectively ) . figure 2 shows the cprnfl , mrw , macular gcipl thickness , and vf of an eye identified as progressing by cprnfl , mrw , and macular gcipl . figure 3 shows the cprnfl , mrw , macular gcipl thickness , and vf of an eye with progression detected only by macular gcipl . baseline global cprnfl , mrw , macular gcipl , and vf md measurements by group rate of global cprnfl , mrw , macular gcipl , and vf md loss by group top row : circumpapillary retinal nerve fiber layer ( cprnfl ) , minimum rim width ( mrw ) , bottom row : macular ganglion cell inner plexiform layer ( gcipl ) thickness , and 24 - 2 visual field in one eye detected as glaucoma progressing by cprnfl , mrw , and macular gcipl ( md vf = 22.3 ) . top row : circumpapillary retinal nerve fiber layer ( cprnfl ) , minimum rim width ( mrw ) , bottom row : macular ganglion cell inner plexiform layer ( gcipl ) thickness , and 24 - 2 visual field in one eye detected as glaucoma progressing by only macular gcipl ( md vf = 28.46 ) . using the 3d whole - volume bkds method , the number of progressing glaucoma eyes identified was 13 ( 31% ) . among the 13 progressing eyes detected by the 3d whole - volume bkds , 7 eyes were also identified by macular gcipl , 3 eyes by mrw , and 1 eye by cprnfl . using the 3d whole - volume bkds method , no significant difference was found between the vf md baseline measurement and md rate of change of the 13 eyes that progressed based on the bkds and 22 eyes that did not progress ( p = 0.11 , p = 0.3 , respectively ) . the gcipl mean rate of change was significantly faster in bkds progressing eyes than nonprogressing eyes ( p = 0.03 ) . there was no significant difference in baseline cprnfl and mrw thickness and mean rate of change between the 13 bkds progressing eyes and 22 nonprogressing eyes ( p = 0.72 , p = 0.84 and p = 0.48 and p = 71 , respectively ) . baseline global cprnfl thickness derived from the circular scan , global mrw thickness derived from the onh radial scan , and macular gcipl thickness derived from the entire macular cube sd - oct scan measurements in healthy and advanced - glaucoma eyes are presented in table 2 . healthy eyes had thicker baseline cprnfl , mrw , and macular gcipl when compared to glaucoma eyes . baseline global cprnfl , mrw , and macular gcipl measurements by group rates of global cprnfl , mrw , and macular gcipl thickness loss in healthy and advanced - glaucoma eyes are presented in figure 1 and table 3 . in healthy eyes , mean rates ( p value ) of cprnfl , mrw , and macular gcipl were 0.32 m / y ( p < 0.001 ) , 1.41 m / y ( p < 0.001 ) , and 0.11 m / y ( p < 0.001 ) , respectively . in advanced - glaucoma eyes , the mean rates of cprnfl , mrw , and macular gcipl change were 0.08 m / y ( p = 0.39 ) , 0.29 m / y ( p = 0.43 ) , and 0.18 m / y ( p < 0.02 ) , respectively . there was no statistically significant difference between the mean rate of loss of cprnfl and mrw in advanced - glaucoma eyes and healthy eyes ( p = 0.44 and p = 0.58 ) . the mean rate of macular gcipl loss tended to be larger in advanced - glaucoma eyes than in healthy eyes , but the difference in mean rates between the two groups was not statistically significant ( p = 0.12 ) . distribution of the mean rates of change of circumpapillary retinal nerve fiber layer ( cprnfl ) , minimum rim width ( mrw ) , and macular ganglion cell inner plexiform layer ( gcipl ) thickness in healthy and advanced - glaucoma eyes . rates of global cprnfl , mrw , macular gcipl , and vf md loss by group by the structural measurement rate of change criterion , the number of identified progressing glaucoma eyes was 2 ( 5% ) using cprnfl , 4 ( 11% ) using the mrw , and 11 ( 31% ) using the macular gcipl . table 4 presents baseline global cprnfl , mrw , macular gcipl , and vf md measurements by study group ; table 5 presents the mean rates of global cprnfl , mrw , macular gcipl , and vf md loss by study group . no significant difference was found between the vf md baseline measurement and md mean rate of change of the 11 eyes that progressed based on the macular gcipl criteria and 24 eyes that did not progress ( p = 0.34 , p = 0.2 , respectively ) . however , mean macular gcipl thickness tended to be larger in the 11 eyes that progressed compared to the eyes that did not ( p = 0.09 ) . there was no significant difference in cprnfl and mrw thickness and mean rate of change between the 11 progressing eyes based on gcipl and the eyes that did not progress ( p = 0.39 , p = 0.43 and p = 0.37 , p = 0.58 , respectively ) . figure 2 shows the cprnfl , mrw , macular gcipl thickness , and vf of an eye identified as progressing by cprnfl , mrw , and macular gcipl . figure 3 shows the cprnfl , mrw , macular gcipl thickness , and vf of an eye with progression detected only by macular gcipl . baseline global cprnfl , mrw , macular gcipl , and vf md measurements by group rate of global cprnfl , mrw , macular gcipl , and vf md loss by group top row : circumpapillary retinal nerve fiber layer ( cprnfl ) , minimum rim width ( mrw ) , bottom row : macular ganglion cell inner plexiform layer ( gcipl ) thickness , and 24 - 2 visual field in one eye detected as glaucoma progressing by cprnfl , mrw , and macular gcipl ( md vf = 22.3 ) . top row : circumpapillary retinal nerve fiber layer ( cprnfl ) , minimum rim width ( mrw ) , bottom row : macular ganglion cell inner plexiform layer ( gcipl ) thickness , and 24 - 2 visual field in one eye detected as glaucoma progressing by only macular gcipl ( md vf = 28.46 ) . using the 3d whole - volume bkds method , the number of progressing glaucoma eyes identified was 13 ( 31% ) . among the 13 progressing eyes detected by the 3d whole - volume bkds , 7 eyes were also identified by macular gcipl , 3 eyes by mrw , and 1 eye by cprnfl . using the 3d whole - volume bkds method , no significant difference was found between the vf md baseline measurement and md rate of change of the 13 eyes that progressed based on the bkds and 22 eyes that did not progress ( p = 0.11 , p = 0.3 , respectively ) . the gcipl mean rate of change was significantly faster in bkds progressing eyes than nonprogressing eyes ( p = 0.03 ) . there was no significant difference in baseline cprnfl and mrw thickness and mean rate of change between the 13 bkds progressing eyes and 22 nonprogressing eyes ( p = 0.72 , p = 0.84 and p = 0.48 and p = 71 , respectively ) . our results suggest that even in very advanced glaucoma , structural loss can be detected in some eyes using standard global structural measures . specifically , macular gcipl had the highest proportion of eyes with detectable change ( 31% ) , followed by mrw ( 11% ) and cprnfl ( 4% ) . in addition , the 3d whole - volume bkds change method , which does not require extensive retinal layer segmentation , detected change in 37% of eyes . moreover , only the mean rate of macular gcipl change reached statistical significance , suggesting that gcipl changes can be detected even in this group of severely advanced - oag eyes . in contrast , the mean rates of change in cprnfl and mrw were not significantly different from zero . the mean rates of change in the healthy eyes were all significantly different from zero , suggesting that aging effects can be detected even in a relatively short follow - up time . these results are important as detecting change in advanced - oag eyes is challenging , as the amount of remaining measurable neural tissue is limited . moreover , determining whether the glaucoma patient is stable or progressing is critical for determining whether the treatment initiated needs to be changed or intensified , as patients with advanced glaucoma are at a high risk of losing remaining vision and becoming functionally impaired or blind from the disease . this study is unique in that it focused on identification of change in very advanced disease where the usefulness of conventional structural and functional tests that usually guide treatment decisions is reduced ( i.e. , vf and cprnfl thickness ) . while the vf md is commonly used to classify the glaucoma subject into early , moderate , and advanced disease , it may not accurately reflect the extent of structural loss . for example , one eye with only a focal cprnfl defect may have the same vf md value as another eye with a diffuse cprnfl defect . in the first case it is possible that the rnfl thickness in sectors without the focal defect can still be used to monitor further change , which is less likely the case for the second eye . therefore , this report can be considered as a proof of concept ; if structural change was detectable in this very advanced stage of the disease using existing and novel methods for change detection , then structural change is also likely to be detectable in eyes with less advanced disease . recent advances in sd - oct technology have improved image resolution , made it possible to measure retina layer thickness and onh parameters , and allowed the imaging of deeper retinal structures such as the choroid and the lamina cribrosa . for these reasons , sd - oct has been widely adopted clinically to objectively assess small changes in the retina . in the current study , we evaluated the rate of change of cprnfl thickness , mrw , and macular gcipl in advanced - glaucoma eyes and compared the number of progressing glaucoma eyes using these measurements to a new 3d optic onh whole - volume change detection method , bkds , that does not require retinal layer segmentation . retinal layer segmentation is particularly challenging in eyes with advanced disease as the layers are thin , and results are more variable than in eyes with less severe glaucoma . this issue is of particular importance for cprnfl segmentation , as the presence of large blood vessels can adversely affect segmentation of the thin cprnfl in advanced disease . similarly , differentiating between the ganglion cell layer ( gcl ) and ipl is particularly challenging when the layers are thin , as in advanced oag . for this reason , we analyzed the macular gcipl and not the gcl as a separate layer . the salsa and spectralis instrument based retinal layer segmentation of eyes included in this study were manually checked and did not have segmentation failures of the macular gcipl , mrw , or cprnfl . there are several possible reasons why we found faster cprnfl and mrw change in healthy subjects than in advanced - glaucoma patients . in addition , cprnfl and mrw atrophy is already extensive in patients with advanced glaucoma . therefore , identification of cprnfl and mrw changes is more challenging at later stages of glaucoma when the layers have already thinned . the 3d whole - volume bkds offers several advantages compared to retinal structural- and thickness - based methods . first , the 3d whole - volume bkds does not require accurate segmentation of each retinal layer on each scan . rather , a glaucoma - based region of interest , generally consisting of rnfl , neuroretinal rim , and prelamina tissue , is delineated on the baseline scan only . therefore , measurement variability inherent in retinal layer segmentation algorithms will not adversely affect the ability to detect progression . the bkds also facilitates the analysis of the entire onh 3d volume for potential glaucomatous changes that may occur in the region of interest that also includes deeper layers such as prelaminar tissue . moreover , the use of spatial dependency favors the generation of homogeneous areas , reducing the likelihood of false - positive detection of change . third , the proposed kernel - based support vector data description ( svdd ) method allows the classifier to be trained using only nonprogressing eyes . gold standard for detection of progression in advanced glaucoma that can be used to identify progressing eyes for use in training the classifier . it is important to note that we have previously demonstrated the utility of the bkds for detection of onh progression in early- to moderate - glaucoma eyes using vf progression as the gold standard . for example , in our previous publications , bkds was implemented without any retinal layer segmentation . in the current study , a region of interest was defined by segmentation of the baseline images and progression detection limited to the designated region of interest . there are several limitations to the 3d whole - volume bkds method that should be noted . one limitation of the proposed method is the inclusion of retinal blood vessels in the change detection map . although the variation of retinal blood vessels may be compensated for by the classifier , this compensation by the classification algorithm could mask small changes in the rnfl or rim area . another limitation is the lack of a parameter to assess and quantify the rate of progression , which may be helpful for clinicians to estimate the likelihood that a patient will become functionally impaired in his or her lifetime . further , it is difficult to assess whether the detected changes are due to glaucomatous changes or other tissue remodeling that is not related to progression of the disease . nevertheless , while it is unclear if the detected changes are related or not to the glaucoma , it is important for the clinician to evaluate change that is outside normal limits . limitations of the current study include the small sample size , relative short follow - up time for both healthy subjects and glaucoma patients , and the limited age range of the healthy subjects . for these reasons , the proportion of progressing eyes and rates of change measured for gcipl , mrw , and cprnfl in this specific severely advanced - glaucoma group may not be generalizable to other groups of advanced - glaucoma patients . in addition , without a gold standard definition of progression in advanced disease , it is difficult to determine whether the detected changes are glaucomatous changes or age - related changes , or , as previously mentioned , to tissue remodeling not necessarily related to oag . we utilized nonprogressing healthy and stable glaucoma eyes in the analysis to reduce the possibility that the changes identified were due to aging or variability of the measurements . nevertheless , while it is unclear if the detected changes are related or not to the glaucoma , characterizing these changes may improve our understanding of the pathophysiology of glaucoma or age - related changes in these eyes with advanced disease . another limitation is the use of the global measurements to assess the rate of change of cprnfl , mrw , and gcipl . while using global measurements results in reduced variability compared to sectoral measurements , in advanced oag , they may underestimate the changes at locations where there is a region in which neural tissue remains intact . the development of automated methods to detect the remaining neural tissue may be helpful to assess more accurately regions of interest for monitoring the structural rate of change . in conclusion , our results suggest that even in very advanced disease , structural change can be detected , and that monitoring macular gcipl and 3d whole - volume bkds change shows promise for identifying progression in advanced glaucoma . however , a larger sample of advanced - glaucoma patients with longer follow - up is needed to validate these findings . | purposeto compare spectral - domain optical coherence tomography ( sd - oct ) standard structural measures and a new three - dimensional ( 3d ) volume optic nerve head ( onh ) change detection method for detecting change over time in severely advanced - glaucoma ( open - angle glaucoma [ oag ] ) patients.methodsthirty-five eyes of 35 patients with very advanced glaucoma ( defined as a visual field mean deviation < 21 db ) and 46 eyes of 30 healthy subjects to estimate aging changes were included .
circumpapillary retinal fiber layer thickness ( cprnfl ) , minimum rim width ( mrw ) , and macular retinal ganglion cell inner plexiform layer ( gcipl ) thicknesses were measured using the san diego automated layer segmentation algorithm ( salsa ) .
progression was defined as structural loss faster than 95th percentile of healthy eyes .
three - dimensional volume onh change was estimated using the bayesian - kernel detection scheme ( bkds ) , which does not require extensive retinal layer segmentation.resultsthe number of progressing glaucoma eyes identified was highest for 3d volume bkds ( 13 , 37% ) , followed by gcpil ( 11 , 31% ) , cprnfl ( 4 , 11% ) , and mrw ( 2 , 6% ) . in advanced - oag eyes ,
only the mean rate of gcipl change reached statistical significance , 0.18 m / y ( p = 0.02 ) ; the mean rates of cprnfl and mrw change were not statistically different from zero . in healthy eyes ,
the mean rates of cprnfl , mrw , and gcipl change were significantly different from zero .
( all p < 0.001).conclusionsganglion cell inner plexiform layer and 3d volume bkds show promise for identifying change in severely advanced glaucoma .
these results suggest that structural change can be detected in very advanced disease .
longer follow - up is needed to determine whether changes identified are false positives or true progression . |
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breast cancer is the second most common cancer in humans and is the most common malignant tumor in females . breast cancer accounts for approximate 23% of new cancer cases and 14% of total cancer mortality . triple - negative breast cancer ( tnbc ) , which is characterized by a lack of estrogen receptor ( er ) , progesterone receptor ( pr ) , and human epidermal growth factor receptor 2 ( her2 ) according to immunohistochemical ( ihc ) and/or fluorescent in situ hybridization , results is a special type of breast cancer and accounts for 1220% of all breast cancer occurrences . compared with the other 3 subtypes of breast cancers , tnbc presents an aggressive biological behavior and poor prognosis . given that tnbc is insensitive to mammography screening , patients may be diagnosed at a higher grade . compared with the other 3 subtypes , tnbc are prone to distant recurrence and death within 5 years of diagnosis . wnt protein is a large secreted protein family that participates in numerous aspects of cellular processes . wnt signaling can be divided into 2 parts : the canonical n - catenin - dependent signaling pathway and the non - canonical catenin - independent signaling pathway . wnta5a is often dysfunctional , and its protein is recognized as either tumor - suppressive or tumor - promoting . wnt5a expression is elevated in melanoma , and its high expression is associated with poor prognosis in melanoma . moreover , wnt5a expression is found to be positive in gastric cancer , and its high expression is associated with bad outcomes . however , in certain types of cancer , the loss or decreased expression of wnt5a is associated with unfavorable prognosis . . found that wnt5a is up - regulated in primary breast cancers compared with normal breast tissues . furthermore , the up - regulation of wnt5a is crucial for the macrophage - induced invasion of invasive breast cancer . found that , compared with mcf7 cell line , wnt5a has higher expression in mda - mb-231 cell line . moreover , in breast cancer patients with brain metastasis , wnt5a is highly expressed in brain tissues . however , the loss or low expression of wnt5a also has poor prognostic value in breast cancer [ 1518 ] . in mcf7 and 4t1cell lines , however , the clinical significance of wnt5a protein expression in tnbcs remains unclear . in the present study , we aimed to detect the clinical significance of wnt5a expression in tnbcs . a total of 425 paraffin - embedded samples of breast - invasive ductal carcinomas were obtained from the department of pathology of the affiliated tumor hospital of harbin medical university . informed consents were obtained from the subjects , and the study was performed with the approval of the ethics committee of harbin medical university . the median age of the tnbc patients was 49 years old ( range from 34 years to 69 years ) . each sample was routinely tested for er , pr , her2 , ki67 , and p53 by immunohistochemistry . ihc markers were assayed in paraffin - embedded formalin - fixed tissue stained with hematoxylin and eosin by using antibodies to the proteins er , pr , and her-2 ( zhongshan bio co. , china ) . samples that showed nuclear staining for er or pr in more than 1% of the cells were considered er - positive or pr - positive , respectively . positive staining for her2 was defined on the basis of the percentage of tumor cells and the intensity of membrane staining . her2 was scored from 0 to 3 + on the basis of the method recommended for the dako hercep test . tumors were recognized as positive for her2 if immunostaining was scored as 3 + or when the her-2 fluorescence in situ hybridization amplification ratio was greater than 2.2 ( 21 ) . patients were followed up for at least 3 months and up to 82 months ( median 75 months ) . all patients received a definite pathological diagnosis , and none received any therapy before surgery . recurrence - free survival ( rfs ) was used as the assessment for prognostic analyses . rfs was defined as the time interval from the date of surgery to the date of local , regional , and distant recurrences and breast cancer death . according to the scarff - bloom - richardson system , the invasiveness of cancers can be determined on the basis of the extent of cell mitosis frequency , tubule formation , and nuclear pleomorphism . invasiveness in patients was classified as low grade ( grade i ) , moderate grade ( grade ii ) , and high grade ( grade iii ) . the occurrence of lymph node metastases ( lnm ) and tumor size ( negative : < 2 cm ; positive : 2 cm ) were also recorded for each patient . the molecular subtypes of breast cancer were classified according to the criteria established in the st . gallen international breast cancer conference 2011 ( 24 ) : luminal a type : er and/or pr - positive and her2-negative and ki67 low ( < 14% ) ; luminal b type : er- and/or pr - positive , her2-negative , and ki67 high ( 14% ) ; ( her2-positive ) er- and/or pr - positive , her2-overexpressed or amplified , and any ki67 ; her2-positive type : er- and pr - negative , and her2-overexpressed or -amplified ; tnbc type : er - negative , pr - negative , and her2-negative . by using a punching machine , the selected area of the paraffin block was punched out , and a 3-mm tissue core was placed onto a recipient block . invasive ductal carcinomas and corresponding normal breast sample blocks were cut with a microtome to 44m and arrayed as triplicates . each tissue core was assigned a unique tma location number linked to a database containing other clinicopathological data . tissue sections were dried at 70c for 3 h. after deparaffinization and hydration , tissue sections were washed in phosphate - buffered saline ( pbs ; 33 min ) . the washed sections were treated with 3% h2o2 in the dark for 5 min to 20 min . after washing with distilled water , each section was then treated with 300 ml to 500 ml of wnt5a mouse monoclonal antibodies ( abcam , usa ; a dilution of 1:200 ) solution at 4c overnight . after washing with pbs ( 35 min ) , each section was incubated with 300 ml to 500 ml of secondary antibody at room temperature for 30 min . after washing with pbs ( 35 min ) , each section was treated with 300 ml to 500 ml of diaminobenzidine ( dab ) working solution at room temperature for 3 min to 10 min , and then washed with distilled water . wnt5a were all cytoplasmically stained . in our study , wnt5a was expressed in most tumor cells ( 75% ) ; therefore , only staining intensity was calculated in the statistical analyses . according to staining intensity ( negative=0 , weak=1 , moderate=2 , and strong=3 ) , we recognized 0,1 as wnt5a - negative and 2,3 as wnt5a - positive . the staining on each sample chicago , usa ) . the chi - square test was used to examine any differences in categorical variables . the influences of different variables on tnbc were assessed by cox regression analyses including univariate and multivariate analysis . hazard ratios ( hrs ) and their 95% confidence intervals ( cis ) were recorded to evaluate the relative risk of tnbc . a total of 425 paraffin - embedded samples of breast - invasive ductal carcinomas were obtained from the department of pathology of the affiliated tumor hospital of harbin medical university . informed consents were obtained from the subjects , and the study was performed with the approval of the ethics committee of harbin medical university . the median age of the tnbc patients was 49 years old ( range from 34 years to 69 years ) . each sample was routinely tested for er , pr , her2 , ki67 , and p53 by immunohistochemistry . ihc markers were assayed in paraffin - embedded formalin - fixed tissue stained with hematoxylin and eosin by using antibodies to the proteins er , pr , and her-2 ( zhongshan bio co. , china ) . samples that showed nuclear staining for er or pr in more than 1% of the cells were considered er - positive or pr - positive , respectively . positive staining for her2 was defined on the basis of the percentage of tumor cells and the intensity of membrane staining . her2 was scored from 0 to 3 + on the basis of the method recommended for the dako hercep test . tumors were recognized as positive for her2 if immunostaining was scored as 3 + or when the her-2 fluorescence in situ hybridization amplification ratio was greater than 2.2 ( 21 ) . patients were followed up for at least 3 months and up to 82 months ( median 75 months ) . all patients received a definite pathological diagnosis , and none received any therapy before surgery . recurrence - free survival ( rfs ) was used as the assessment for prognostic analyses . rfs was defined as the time interval from the date of surgery to the date of local , regional , and distant recurrences and breast cancer death . according to the scarff - bloom - richardson system , the invasiveness of cancers can be determined on the basis of the extent of cell mitosis frequency , tubule formation , and nuclear pleomorphism . invasiveness in patients was classified as low grade ( grade i ) , moderate grade ( grade ii ) , and high grade ( grade iii ) . the occurrence of lymph node metastases ( lnm ) and tumor size ( negative : < 2 cm ; positive : 2 cm ) were also recorded for each patient . the molecular subtypes of breast cancer were classified according to the criteria established in the st . gallen international breast cancer conference 2011 ( 24 ) : luminal a type : er and/or pr - positive and her2-negative and ki67 low ( < 14% ) ; luminal b type : er- and/or pr - positive , her2-negative , and ki67 high ( 14% ) ; ( her2-positive ) er- and/or pr - positive , her2-overexpressed or amplified , and any ki67 ; her2-positive type : er- and pr - negative , and her2-overexpressed or -amplified ; tnbc type : er - negative , pr - negative , and her2-negative . by using a punching machine , the selected area of the paraffin block was punched out , and a 3-mm tissue core was placed onto a recipient block . all breast invasive ductal carcinomas and corresponding normal breast sample blocks were cut with a microtome to 44m and arrayed as triplicates . each tissue core was assigned a unique tma location number linked to a database containing other clinicopathological data . tissue sections were dried at 70c for 3 h. after deparaffinization and hydration , tissue sections were washed in phosphate - buffered saline ( pbs ; 33 min ) . the washed sections were treated with 3% h2o2 in the dark for 5 min to 20 min . after washing with distilled water was then treated with 300 ml to 500 ml of wnt5a mouse monoclonal antibodies ( abcam , usa ; a dilution of 1:200 ) solution at 4c overnight . after washing with pbs ( 35 min ) , each section was incubated with 300 ml to 500 ml of secondary antibody at room temperature for 30 min . after washing with pbs ( 35 min ) , each section was treated with 300 ml to 500 ml of diaminobenzidine ( dab ) working solution at room temperature for 3 min to 10 min , and then washed with distilled water . wnt5a were all cytoplasmically stained . in our study , wnt5a was expressed in most tumor cells ( 75% ) ; therefore , only staining intensity was calculated in the statistical analyses . according to staining intensity ( negative=0 , weak=1 , moderate=2 , and strong=3 ) , we recognized 0,1 as wnt5a - negative and 2,3 as wnt5a - positive . the staining on each sample chicago , usa ) . the chi - square test was used to examine any differences in categorical variables . the influences of different variables on tnbc were assessed by cox regression analyses including univariate and multivariate analysis . hazard ratios ( hrs ) and their 95% confidence intervals ( cis ) were recorded to evaluate the relative risk of tnbc . breast tma was used to evaluate wnt5a expression in tnbc tissues and was compared with normal breast tissues . the rate of decreased wnt5a- expression accounts for 61.1% of breast cancer tissues in this study ( figure 1 ) . we also analyzed the association between wnt5a expression level and various clinicopathological factors ( table 1 ) . we found that negative wnt5a expression was significantly associated with positive lnm ( p=0.07 ) and positive ki67 status ( p=0.02 ) . however , no difference was found between wnt5a expression and other clinicopathological features . to study whether wnt5a expression is associated with rfs , we utilized kaplan meier survival analysis and log - rank test . the kaplan meier survival curves are shown in figure 2 . among the 90 patients , patients with negative -wnt5a- expression showed significantly poorer outcome ( 68.5433.121 months ) in terms of rfs than patients with positive - wnt5a - expression ( 75.1432.869 ) were used to evaluate the relationship between wnt5a expression and clinicopathological characteristics on prognosis ( table 2 ) . by using cox regression analysis , the univariate analyses of rfs showed negative wnt5a expression ( hr=3.779 , 95% ci=1.30210.967 ) and lnm ( or=3.581 , 95% ci=1.23310.401 ) as a significant poor prognosis factor . no other features were discovered to be associated with prognosis . by using a cox regression model , multivariate analysis was conducted on the same set of patients for wnt5a expression and for pathological factors of survival time . by using multivariate analysis , we discovered that the negative expression of wnt5a ( hr=3.779 , 95% ci=1.30210.967 ) was an independent prognostic predictor ( table 2 ) . breast tma was used to evaluate wnt5a expression in tnbc tissues and was compared with normal breast tissues . the rate of decreased wnt5a- expression accounts for 61.1% of breast cancer tissues in this study ( figure 1 ) . we also analyzed the association between wnt5a expression level and various clinicopathological factors ( table 1 ) . we found that negative wnt5a expression was significantly associated with positive lnm ( p=0.07 ) and positive ki67 status ( p=0.02 ) . to study whether wnt5a expression is associated with rfs , we utilized kaplan meier survival analysis and log - rank test . the kaplan meier survival curves are shown in figure 2 . among the 90 patients , patients with negative -wnt5a- expression showed significantly poorer outcome ( 68.5433.121 months ) in terms of rfs than patients with positive - wnt5a - expression ( 75.1432.869 ) ( p=0.008 , log - rank test ) . both univariate and multivariate survival analyses were used to evaluate the relationship between wnt5a expression and clinicopathological characteristics on prognosis ( table 2 ) . by using cox regression analysis , the univariate analyses of rfs showed negative wnt5a expression ( hr=3.779 , 95% ci=1.30210.967 ) and lnm ( or=3.581 , 95% ci=1.23310.401 ) as a significant poor prognosis factor . no other features were discovered to be associated with prognosis . by using a cox regression model , multivariate analysis was conducted on the same set of patients for wnt5a expression and for pathological factors of survival time . by using multivariate analysis , we discovered that the negative expression of wnt5a ( hr=3.779 , 95% ci=1.30210.967 ) was an independent prognostic predictor ( table 2 ) . the loss of wnt5a has been reported to act as a negative prognostic factor in breast cancer . moreover , janna et al . found that the loss of wnt5a expression is associated with tnbc , but they did not clearly state the function of this expression in tnbc . the purpose of our study was to determine whether the loss of wnt5a presents a pivotal prognostic value in tnbc . in our study , 55 of the 90 tnbc patients were wnt5a - negative ( accounting for approximately 61.1% ) . in tnbc , the loss rate of wnt5a is higher than that ( 44% and 40.4% ) in unclassified breast cancer . in the present study , we found that negative wnt5a expression in the cytoplasm of cancer cells is associated with lymph metastasis ( p=0.07 ) . a previous study indicated that in the mda - mb-231 cell line , which is a tnbc cell line , the re - expression of wnt5a could inhibit cell migration . moreover , in the 4t1 cell line , which expresses very low wnt5a , breast cancer cell metastasis to the lung can be inhibited when the wnt5a protein is re - expressed . this finding is similar to that in the current study . in mmtv - pyvmt and mmtv - pyvmt : wnt5a/ tumors , the absence of wnt5a enhances the tumor growth . moreover , in the absence of wnt5a , ki67 staining illustrates a significant elevation in proliferation , which is consistent with our results . in the present study , although statistically insignificant , the finding showed a trend wherein negative wnt5a was associated with large tumor size . we speculate that the loss of wnt5a might result in tumor growth via ki67 proliferation . both univariate and multivariate analyses found that patients with low level of wnt5a expression presented significantly poorer rfs rates than patients with higher wnt5a expression . thus , the loss of wnt5a expression is an independent unfavorable survival factor for patients with tnbc . that finding is different from the research conducted by sfholm et al . , which found that reconstitution of wnta5a signaling in 4t1 mouse breast cancer cells ( which lack er , pr , and her2 ) could impair migration and invasion , and that recombinant wnt5a- and wnt5a - derived foxy-5 peptide may be a potential therapeutic strategy for triple - negative breast cancer . additionally , lnm is an independent poor survival factor in univariate analysis , but under multivariate analysis , it had no significant association with the prognosis of tnbc . therefore , we speculated that tnbc risk was influenced by confounding factors , but the genetic factor was the decisive factor . the results of this study suggest that down - regulated wnt5a predicted a poor prognosis of tnbc . firstly , this was a case - only study , and there was no control group . secondly , gene - gene and gene - environmental interactions were not considered in our study . tnbc onset is influenced by genetic and environmental factors , and a single gene can not determine the development of it . based on the above , the result of our study was insufficient to provide an effective therapy method for tnbc . our study shows that the loss of wnt5a is associated with lymph node metastasis and ki67 proliferation in tnbc . low level of wnt5a protein was correlated with poorer survival rate in patients with tnbc . our results suggest that wnt5a might serve as a tumor suppressor gene in tnbc and that wnt5a might be a potential new drug target in breast cancer treatment . | backgroundwingless - type mmtv integration site family member 5a ( wnt5a ) has been documented to either overexpress or be lost in several malignancies .
our study aimed to investigate the expression and clinical significance of wnt5a protein in triple - negative breast cancer ( tnbc).material / methodsby using immunohistochemistry , wnt5a expression was evaluated in 90 tnbc specimens .
the association between wnt5a expression and clinic - pathological factors was assessed by using the chi - square test .
the survival analysis of patients was conducted by using the kaplan - meier and log - rank tests .
cox regression was utilized for the univariate and multivariate analyses of prognostic factors.resultsresults showed that negative wnt5a expression correlated with positive lymph node metastasis ( lnm ) ( p=0.007 ) and ki67 proliferation ( p=0.002 ) .
patients with negative wnt5a expression had significantly poorer recurrence - free survival ( rfs ) than patients with positive wnt5a expression ( p=0.008 ) .
multivariate cox regression analysis revealed that decreased wnt5a expression was an independent prognostic factor for rfs ( p=0.014).conclusionsnegative wnt5a protein expression might contribute to the tumor progression and poor prognosis of tnbc and might be a new therapy target in tnbc . |
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they divide
the inner part of the cell from the outer environment as well as dictate
the intercellular transport and cell fusion / division . membranes also
provide the matrix in which important proteins ( transmembrane pumps ,
channels , receptors etc . ) reside . in nature , biological membranes
are very complex systems , consisting of a mixture of different lipids ,
sterols , and proteins . these constituting parts are involved in a
number of processes making the cell membrane a very dynamic structure . it has been proposed that protein lipid interactions regulate
the functionality of proteins , making the lipid bilayer more than
a passive hydrophobic slab . most studies are , however , performed on pure phospholipid bilayers
since phospholipids are a major part of biological membranes . before
one can study biological membranes in their true essence experimental techniques
used to study lipid bilayers span from x - ray and neutron scattering
to ir / raman and nmr . the biologically most relevant phase for membranes is the
liquid - crystalline phase ( l-phase ) , which means
that the aliphatic chains of the lipid have a certain degree of freedom
which result in a disordered structure . although a large number of
experimental studies have been performed on membrane systems and already
shed some light on the dynamical and structural properties , a more
detailed understanding of these systems is desirable . due to the difficulties
of studying these systems in the correct phase , computer simulation
methods are an ideal complement to experiments . molecular dynamics
( md ) computer simulations have become an essential tool for studying
membrane on an atomistic level during the past two decades . if a sufficiently large system is chosen to be simulated ,
the quality
of the simulations is limited by two factors : ( 1 ) sampling or time
scale and ( 2 ) the empirical potential energy function or force field
( ff ) employed . the sampling issue is always present and it has been
found that fluctuations in simulations of pure lipid bilayers happen
on a multinanosecond time scale . as a
consequence of this , long simulations ( > 100 ns ) are needed in order
to study equilibrium properties of lipid bilayers . for systems with
proteins embedded , the other issue , i.e. , the ability of the potential energy function
to provide realistic dynamics and thermodynamic properties of the
system , is extremely crucial . two major types of ffs are available
for lipid bilayers : united atom ( ua ) and all - atomistic ( aa ) . in ua
ffs all nonpolar hydrogens have been included in the heavier atoms ,
creating pseudoatoms . the popular berger lipids , the g53a6l ff , the
43a1-s3 ff , and the potential developed
by ulmschneider and ulmschneider and
kukol are examples of these ffs . the
aa ffs have explicit hydrogens included and the most common ones for
lipid simulations are charmm and the general amber ff ( gaff ) . until now , these have been the most widely used
aa ffs , although several problems have been reported regarding older
versions of charmm and gaff ffs . other ffs are nonadditive ( polarizable ) or coarse - grained . in some cases , an aa description is preferred and
recent studies have shown that there exists a unique designated hydrogen
bond network around the choline head group . in order to describe and study this in full detail , an accurate
aa model is crucial . aa description may also be crucial for describing
order parameters of the glycerol group and in the description of double
bonds in unsaturated lipids . an inaccurate ff often results in a lipid
bilayer in a too ordered phase , a gel phase ( l ) , at a higher temperature than observed in experiments and this
leads to a contracted surface area which results in a too low area
per lipid ( al ) . most often , this has been a problem for
the models including explicit hydrogens . in order to solve this problem , a positive surface tension can be applied , so the simulations are
performed in a npt ensemble . another solution is to fix the total simulation box area leading to
a npat ensemble . neither of these two solutions is
appealing since they restrict the membrane from stretching laterally
and suppress membrane undulations . therefore , the appropriate ensemble
for studying lipid bilayers as well as insertions to the bilayer is
the isothermal isobaric ensemble ( npt ) which
has been explained in more depth earlier . it has proven to be very difficult
to obtain accurate ffs for lipid
bilayers and often specialized ffs have to be used ; e.g. , charmm uses
specialized parameters for lipids that differ from the parameters
used for proteins to describe the same type of atoms . lipid
bilayers are very peculiar systems with a polar head group in contact
with the aqueous environment and a strong attraction of the tails
driven by the hydrophobic effect and van der waals ( vdw ) forces . a
complexity like this poses a challenge for the modeling community .
especially , the fact that in order to study complexes including both
lipids and proteins we need ffs that work well together implies further
complications . previous work has shown that the interactions between
amino acids and lipid molecules need to be carefully evaluated before
embarking on larger simulations . in the present paper , we present an aa ff for fully saturated phosphocholine
lipids based primary on high - level ab initio calculations together
with some limited empirical input . partial atomic charges for the
whole lipid , lennard - jones ( lj ) parameters , and torsion potentials
for the aliphatic chains are recalculated . the introduction of the
scaling factor of 0.8333 for 14 coulombic interactions and
0.5 for 14 vdw interactions makes the parameters set presented
here compatible with the amber ffs for proteins and biomolecules . computing of partial atomic charges from the electrostatic potential
( which is a quantum mechanical observable where s the charges are not )
makes these very essential ff parameters physically grounded . the
fact that all charges are computed at the same level of theory and
with the same procedure together with the lj parameters and torsional
potential results in a ff with balanced terms that have been developed
in a consistent manner . this is important since it makes it easy to
improve the ff in the future by using even more precise ab initio
methods and this also creates a good ground for transferability of
the computed parameters . with a ff well balanced and capable of describing
the physics and chemistry of a lipid bilayer , computer simulations
can be extended to do more than just reproduce experimental findings . different properties and phenomena can be predicted by accurate models
and the fundamentals of molecular biology can be better understood
with the help of modeling . naturally , verification against experimental
findings plays a key role in development of these models , so a large
number of properties for the lipid bilayers have been computed and
compared with experimental data . however , due to experimental inconsistencies
it has been proposed that this should not be the only property investigated . therefore , different thicknesses ,
scattering form factors , volumes , and order parameters have been calculated
and compared to experimental data . due to the distinct thermal dependency
of the aliphatic chains of the lipids , simulations at different temperatures were performed in order to verify that the set of parameters proposed
here captures this effect . the final comparisons show that the ffs
presented here reproduce the experimental findings very well and therefore
is highly suited for simulations of lipid bilayers in the correct
phase . the main focus of the parametrization
procedure has been on the hydrophobic tails of the lipids , since these
parameters were previously shown to have strong influence on simulated
properties of bilayers , and the partial atomic charges for the whole
lipid . as model compounds for the hydrophobic tails , the assumption
that the hydrophobic core behaves similar to a bulk alkane liquid
has proven to be valid a number of times . if the fitting is done
simultaneously for a series of alkanes , the results are generally
better . furthermore , studies on nmr relaxation
rates have shown that the hydrophobic part of the lipid bilayer behaves
similar to bulk alkane solutions . the following potential energy function was chosen for this work1which is a standard form for ffs like amber ,
charmm , and gromos . as a starting point for our parametrization , we
took parameters of the charmm36 ( c36 ) ff . parameters for all covalent bonds and angles as well as lj and torsional
parameters for the lipid head group were taken from the ff described
by klauda et al . we then derived new
parameters ( as described in detail below ) : partial atomic charges
for the whole lipid and lj and torsion parameters describing the lipid
tails . we started our parametrization by computing partial atomic
charges
for typical configurations of alkanes and averaging over these configurations .
because of the known difficulties of reproducing the vdw dispersion
interaction by ab initio methods , experimental heats of vaporization
and densities were used during the fitting of the lj parameters . first ,
the new charges were used with the original parameters from c36 ( lj
and torsional ) and the lj parameters were then altered until satisfactory
agreement between simulations and experiments was obtained . after
this , the torsional potentials were fitted from ab initio computations
for the model compound . after one round in the parametrization scheme ,
it was necessary to refit the lj parameters again and the torsional
potentials until self - consistency was obtained . the introduction of scaling factors for the 14
interactions with the charmm ffs for lipids together with a new set
of charges have earlier been proven to be successful . below , we present more detailed descriptions of each parametrization
step . for the lipid tails hexadecane it is well - known
that partial atomic charges are conformation dependent but previous ffs have been parametrized from optimized
geometries . in order to address this issue , we performed a 10 ns long
md simulation with pure hexadecane with ff parameters earlier derived
by our group . after the simulation , 54
random conformations were extracted and used for computing the charges
which were then averaged over all conformations in order to obtain
a final set . in this way , we obtained boltzmann - averaged charges over
an ensemble of conformations in a procedure equivalent to the one
used by sonne et al . we hope that by
averaging over an ensemble of conformations the effects of the conformational
dependence of partial charges are minimized . in computation of atomic
charges , a dielectric constant of 2.04 was used to mimic the dielectric
environment of the membrane s hydrophobic part . atomic
charges for the lipid head group were obtained in a similar fashion
where 26 random conformations were chosen from a 20 ns long simulation
of an equilibrated bilayer ( dmpc ) with the same ff parameters used
in the initial simulation of hexadecane . a large part of the hydrophobic
parts of the lipids were then cut off in order to save cpu time and
the cropped lipids were placed in dielectric continuum with
= 78.4 in order to mimic the aqueous environment . inclusion of solvent
effects results in a ff with implicitly polarized charges optimized
for condensed phase simulations . this has been proven to give reliable
results without any performance loss . for each molecular conformation , the charges were computed
using
the restricted electrostatic potential approach ( resp ) with the dft method using the b3lyp exchange - correlation
functional and the cc - pvtz basis set . kollman scheme by single - point calculations and fitted during
the two - stage procedure developed by cornell et al . all solvent effects were modeled by placing the molecule
in a polarizable continuum with different dielectric constant ( see
above ) with the iefpcm continuum solvent model . the quantum mechanical calculations were performed with the gaussian09
software package , and the resp calculations
were performed with the red software . in subsequent molecular dynamics simulations , the way the
atomic charges have been calculated and used in md
simulations makes them compatible with the amber03 ff and since the charges in all amber ffs are derived from
the resp the lipid ff presented here is compatible with most members
of the amber ff family . this is of importance since there is a growing
interest in simulating membrane proteins in their native environment and also peptide partitioning in biological membranes . ongoing work aims to clarify which amber biomolecular ffs that work
sufficiently well together with the current parameters . a preliminary
test of the compatibility of the lipid parameters and the amber03
ff is presented further down . boltzmann averaging over charges
introduces temperature dependency
on the charges and in order to see the impact of temperature , simulations
with different temperatures ( 298 , 303 , 310 , 318 , and 325 k ) were performed
with hexadecane using the methodology described above . no explicit
temperature dependence could be found over this range of temperatures ,
making the charges reliable and robust with respect to temperature ,
at least within the interval tested here ( data not shown ) . by reproducing experimental
densities correct -values for the lj potential heats of vaporization are important since they describe the interactions
between the molecules which is primarily determined by the well - depth
( ) of the lj potential . the average density is straightforward
to obtain from md simulations and the heat of vaporization was computed
according to2where egas was obtained as an average of eight separate 50 ns long ,
in vacuo simulations with different starting conformations taken from
a condensed - phase simulation . test simulations in condensed phase lasted
for 5 ns and production simulations for 100 ns at 298.15 k with the
partial atomic charges obtained in the previous step . the simulation
length of the condensed phase is longer than usually considered but
it is necessary since a variation of parameters during the fitting
procedure can freeze the system and this transition can occur on a
relatively long time scale . the lj 1 - 4
interactions between the hydrogens of the lipid carbon chains were
scaled by a factor of 0.5 and for the remaining interactions special
1 - 4 interactions were specified . in order to make sure that the alkanes
intermolecular interactions are of the right magnitude and that the
molecules have the correct size , the lj parameters and
were fitted simultaneously . the potential energy surface ( pes )
for octane was scanned in order to obtain an accurate torsion potential
for the hydrophobic tails of the lipids . the relative energies between different conformations
were obtained by employing the hybrid method for interaction energies
( hm - ie ) . applying this method makes it
possible to estimate intermolecular and intramolecular interactions
with the precision of very high level ab initio methods , namely ccsd(t ) with a large basis set ( lbs ) . assuming that
the effect of using a larger basis set is additive , the following
expression can be used to estimate the intermolecular energies3where sbs is the abbreviation for the small
basis set . this energy was estimated at a ccsd(t)/cc - pvqz level of
theory ; i.e. , lbs and sbs are equivalent to cc - pvqz and cc - pvdz , respectively . the geometry of the molecules was optimized with the second order
of mller plesset perturbation theory ( mp2 ) with the cc - pvdz basis set and then single - point
calculations were used to estimate the relative energies with the
hm - ie scheme . it has been proven that mp2/cc - pvdz gives reasonable
geometries whereas it is necessary to use more exact methods for the
relative energies . since longer
carbon chains give lower rotational barriers and due to the fact that the number of medium - range 1,3-interactions
increases the stability of the compounds , a slightly larger model compound ( octane ) was used than that used
in parametrization of charmm . different
conformations were used during the scanning of the pes and used in
the fitting procedure , resulting in a total number of roughly 200
conformations estimated at a ccsd(t)/cc - pvqz level of theory . more
detailed information regarding the fitting procedure can be found
in the supporting information along with
all ff parameters derived in this work . a number or properties
were computed for the bulk alkane systems and several lipid bilayers
( dlpc , dmpc , and dppc ) in order to test the quality of the newly derived
parameters . for bulk alkane liquids , uncorrected ( dpbc ) and corrected ( d ) self - diffusion
coefficients were determined as well
as c longitudinal relaxation times ( t1 ) and the number of gauche bonds per molecule . the ability
of the newly derived ff to reproduce experimental data for lipid bilayers
was verified by computing a series of structural and dynamic properties : al , volume per lipid ( vl ) , isothermal area compressibility modulus ( ka ) , thermal area expansivity ( at ) , and lateral diffusion coefficient . three types of bilayer thicknesses were computed and compared to experiments :
head - to - head distance ( dhh ) , the luzzati
thickness ( db ) , the hydrophobic core thickness ( 2dc ) ,
and the two corresponding thermal contractivities , dbt and dct . electron density
profiles along the bilayer normal were obtained from simulations with
the contributions from different groups ( see figure 1 ) . from the total electron density , the scattering form factors , f(q ) , were obtained via fourier transformation . deuterium order parameters ( |scd| ) were
also computed and compared to experimental findings . more information
regarding the computed properties for both alkane liquids and lipid
bilayers can be found in the supporting information . all simulations were performed
in the isothermal isobaric ensemble , npt ,
at a pressure of 1 atm . the pressure was held constant by using the
parrinello rahman barostat with
a coupling constant of 10.0 ps with an isothermal compressibility
of 4.5 10 bar . for
the bulk liquids an isotropic pressure coupling was used and for the
bilayer simulations a semi - isotropic pressure coupling scheme was
used . the temperature was kept constant by the nos hoover
thermostat with a coupling constant of 0.5 ps . long - range electrostatic interactions were treated by a particle - mesh
ewald scheme with a real - space cutoff at 1.4
nm with a fourier spacing of 0.10 nm and a fourth - order interpolation
to the ewald mesh . van der waals
interactions were truncated at 1.5 nm and treated with a switch function
from 1.4 nm . the inclusion of long - range
corrections should eliminate the lj cutoff dependency in the simulations .
due to the fact that lipid bilayers are inhomogeneous systems the
method introduced by lage et al . to add long - range corrections a time step of
2 fs was used with a leap - frog integrator . the lincs algorithm was used to freeze all covalent bonds in the
lipid , and the analytical settle method
was used to hold the bonds and angle in water constant . the choice of water model can be explained by the fact that tip3p
is the default water model in major ffs such as amber and charmm and
since one of the aims of the work presented here was to create a lipid
ff compatible with amber this was a natural choice . further , earlier
work of hgberg et al . has shown
that there is flexibility in the choice of water model for aa simulations
of lipid bilayers . atomic coordinates were saved every 1 ps and the
neighbor list was updated every 10th step . bulk liquids were
simulated with a simulation box consisting of 128 molecules for the
larger alkanes and 256 for the smaller alkanes ( hexane and heptane )
at a temperature of 298.15 k. the lipid bilayer systems were prepared
using the charmm - gui with 128 lipids in total , 64
in each leaflet . in order to achieve proper hydration , 30 tip3p water
molecules three different lipid types were simulated ,
dlpc ( 12:0/12:0 ) , dmpc ( 14:0/14:0 ) , and dppc ( 16:0/16:0 ) . these system
were investigated under a range of temperatures ; see table 1 for an overview of all simulations performed . all
lipid bilayer systems were equilibrated for 40 ns before production
runs were initiated which lasted for 300500 ns . all md simulations
were performed with the gromacs software
package ( versions 4.5.3 and 4.5.4 ) . all analysis were made with the
analysis tools that come with the mdynamix software package . the calculations of free energies of solvation in
water and cyclohexane
were performed by using thermodynamic integration over 35
values in the range between 0 and 1 . a soft core potential ( scp ) was
used to avoid singularities when the solute is almost decoupled from
the solvent . the -parameters used for the scp and the simulation
workflow were set following the methodology described by sapay and
tieleman . the amino acid analogues were
solvated with 512 and 1536 molecules of cyclohexane and water , respectively . our initial charge calculations
implied that the major part of the alkanes have almost zero ( less
than 0.005 ) charge of carbons and hydrogens with small but noticeable
dipole moments at the end of the chains . therefore , charges were set
to zero for the inner methylene groups ( both for carbons and hydrogens ) .
as a result of this the total charge on the outermost methylene
groups was set to + 0.033 which is balanced by the charge of 0.033
on the methyl groups . the partial atomic charges used during the simulations
of bulk alkane solutions were the same as presented for the lipid
tail in figure 1 . the major parts of the charges in the aliphatic tails are set to
zero which is similar to most ua ffs but differs from all
version of charmm ffs having a charge of + 0.09 on all aliphatic hydrogens . the present set of charges differ from a majority of other ffs due
to the presence of the small dipole moments at the end of the lipid
tails . charges within parentheses are the charges on the hydrogen(s ) attached
to the heavier atom . 5 , 4 , and 3 are torsional angles that are discussed
in the results section . the final lj parameters for methylene and methyl
groups are shown
in table 2 and the final torsional parameters
in table 3 . the same number of terms in the
fourier sum with the same multiplicity and phase shift as used in
c36 gave the best result . the results of computations with the final optimized
parameter
set for heat of vaporization , density , and diffusion are summarized
in table 4 . the linear trend in hvap is reproduced perfectly by the simulations
with only a slight overestimation for the longest alkanes resulting
in a rms error of only 0.9% . further , the rms error for the density
is 0.8% which means that the correct balance between size and interactions
in the ff is obtained . during the parametrization procedure the alkane
system can easily freeze due to incorrect parameters in a multinanosecond
simulation . a too high value of
combined with too small atomic sizes ( ) gave rise to a stacked
lamellae phase which was also observed in the present investigation . this behavior will affect the ffs ability to describe a lipid bilayer
in the l-phase at the correct temperature in a tensionless
ensemble . the freezing sometimes occurred after only a couple of nanoseconds ,
making it straightforward to avoid this problem . in some cases , longer
simulations were needed just as it was for ulmschneider and ulmschneider . due to the possibilities of these phase transitions ,
all simulations were run for 100 ns . as the thermodynamic properties for the alkanes converged
and resulted
in good agreement with experimental data , the ffs ability to reproduce
dynamic properties such as diffusion and nmr relaxation times was
investigated . since the ff only has been parametrized against thermodynamic
data , it is of utmost interest to test the dynamics of the alkanes .
the uncorrected diffusion coefficients , dpbc , in table 4 are all underestimated due to
the finite box size in the simulations . once corrections for the hydrodynamics
and long - range effects were added , the
rms error decreases from 20% to 8.6% . similar trends have been reported
by klauda et al . and davis et al . figure 2 shows the c nmr t1 relaxation times obtained
from simulations compared to experiments ( in the supporting information more details
on how to obtain c nmr t1 from simulations are discussed ) . the dynamics of the chain is important
since it will affect how the inner part of the bilayer behaves when
solutes and proteins are inserted . for the shortest alkane chain ( heptane )
the relaxation time is overestimated which indicates that the local
chain dynamics is too rapid . the agreement between simulations and
experiments becomes better when the chain size is increased . the trend
seen here follows the general trends found in the recent literature . albeit the fact that the longer
chains have relaxation times closer to the experimental ones , the
dynamics around the torsional angles are still slightly too slow .
as can be seen further on , this does not affect the lipid bilayer
properties but purposes a future improvement of the ff . schemes where
high - level ab initio data are used together with available experimental
data in order to fine - tune parameters have been suggested before . this method could be used to eventually reach
the limit of accuracy of fixed point charge ffs . the statistics
of different conformations of alkane chains were
also investigated by computing the fraction of gauche conformations
in condensed phase . in table 5 the average
number of gauche and trans bonds per alkane molecule are presented . the experimental estimation of 35% gauche conformations per molecule is reproduced ; however , this comparison should
be done with some care since the interpretation of the experimental
data is not trivial . still , it is reassuring that a high enough number
of gauche conformations occur since in some previous ffs the trans
conformations have been too pronounced , resulting in lipid bilayers
that are too contracted when simulated under zero surface tension . computed c nmr t1 relaxation
times for alkanes for selected carbons compared to experimental data . for tridecane , estimated by ftir . transfer free energies from a polar environment to
an apolar environment
have been used earlier to investigate the compatibility of lipid protein
ff combinations . it has been shown that this
scale , which is referred to as the radzicka scale , correlates with studies where the same amino acid analogues
have been transferred from aqueous solution to an explicit membrane
environment . the computed free energy of transfer , g , together with free energy of solvation in water and cyclohexane
are presented in figure 3 . the free energies
of hydration indicate that the amber03 ff ( used to describe amino
acids ) is slightly too hydrophobic with the md simulations performed
here when compared to experimental data . the rms in ghyd is 4.86 kj mol which does not differ much from
the rms values obtained by shirts et al . and sapay and tieleman . the largest
errors are for hie ( 7.77 kj mol ) , asn ( 6.61 kj
mol ) , and met ( 6.93 kj mol ) which also correlates with earlier work . computed free energies of solvation of the
amino acid analogues in cyclohexane are in better agreement with experimental
data and have a rms error of 2.76 kj
mol . largest errors in gcyc are obtained for trp ( 5.68 kj mol ) , cys ( 4.86 kj mol ) , and hie ( 4.34 kj mol ) . the transfer free energies also are in good agreement
with experimental findings with a rms error of 3.40 kj mol . the residues that contributes mostly to the error are gln ( 6.75 kj
mol ) , asn ( 6.26 kj mol ) , and
phe ( 5.14 kj mol ) . overall , the free energies
agree well with experiments and the partitioning of amino acid analogues
between the polar and apolar solvents suggests that the lj parameters
presented here are compatible with the amber03 ff . since there are
no intermolecular interactions of coulombic nature present in the
cyclohexane simulations , it is likely that other amber ffs will work
together with the ff presented here . the data presented here shows
that despite the fact that a different cutoff scheme was applied than
the one used in the derivation of amber03 , the two parameter sets
are very compatible . for future work , we will test explore this compatibility
further by performing simulations of membrane - bound proteins in their
native environment . however , one should bear in mind that this hydrophobicity
scale is missing some crucial points as protein backbone contributions
etc . and therefore is only a crude measurement . free energy of solvation
for aminoacid analogues in water , ghyd ( left ) , cyclohexane , gcyc ( center ) ,
and free energy of transfer from water to
cyclohexane , g ( right ) . the light gray
area corresponds to an offset of kbt and the dark gray to an offset of 2kbt . partial atomic
charges for the head group of the lipid were boltzmann - averaged over
a number of conformations generated from a 20 ns md simulation and
are presented in figure 1 . the sum of the individual
atoms in the groups agree very well with the work of hgberg
et al . and sonne et al . which both describe aa ffs the charge distribution in
the head group , due to the long - range character of electrostatic interactions ,
has a major impact on the fluidity of the lipid bilayer . compared to most ua ffs and the all - atomistic
c36 for the former , certain charge groups are usually constrained
to a certain charge , as + 1 for the choline group and 1 for
the phosphate group . in the present paper , no constraints were imposed ,
resulting in a slightly different charge distribution in the head
group , + 0.75 for the choline group and 0.86 for the phosphate
group . n dipole moment of the head group ,
making it a weaker dipole in the case of our charges and a stronger
dipole when the charge groups have predetermined values . a decrease
in dipole moment reduces the attraction of the lipid head group with
its neighbors in the membrane plane resulting in a more upright position . in figure 4 , the probability distribution
of the angle between the bilayer normal and the p the most probable angle is around
71 for all lipids compared to the experimental estimate of 72. this means that the dipole lies almost completely
in the lipid bilayer plane . due the larger dipole moment in certain
ffs , an angle of around 90 has been reported for lipid bilayer
simulations , where s other reported values
have been in within the range of 6086. the torsional angles shown in figure 1 ( 5 , 4 , and 3 ) are all in
gauche conformations which agrees with the experimental findings of
akutsu and nagamori . this means that
the intramolecular interactions in the lipid head group are described
well and that its conformation is not fully extended . this might be
important when studying membrane - bound proteins and the interactions
between small solutes and lipid bilayers since the conformations of
the head group alter the dipole moment . when using the popular berger
lipids , the head group is in a more extended conformation which is
in disagreement with experiments other
parts of the head group that are important are the charges describing
the electron distribution of the glycerol / carbonyl region . previous
studies have shown that the dipole moments of the ester groups correlate
with the area per lipid , where a stronger dipole results in a higher
area per lipid . a more polar ester group
results in a higher water density in this region which in turn increases
the area per lipid . in the c36 ff the polarity of this group was increased
and it has been proposed that this polarity is one of the reasons
for the success of the popular berger ff . the charges presented in figure 1 are similar
to previously suggested charges . interestingly ,
the ester oxygen bears almost the same charge in the present ff as
the sn-1 ester oxygen in the charge set proposed
by sonne et al . area per lipid for dlpc , dmpc , and dppc at various
temperatures is presented in table 6 . the simulations
using the ff described here give values that are in excellent agreement
with experiments for these lipids . it should be mentioned that experimental
data have been reported in a wide range , see , e.g. , table 1 in ref ( 47 ) . in table 6 generally ,
area per lipid is one of most computed properties from simulated lipid
bilayers but the difficulties in obtaining accurate experimental data make these comparisons not conclusive . it has
been proposed that the area per lipid alone can not be an indicator
of the quality of a ff and the summary
of published experimental and simulation values needs to be used for
ff validation . therefore , other properties than
just area per lipid must be computed and verified against experiments
and only a comparison with an ensemble of different experimental values
can really verify the ff and the methodology used . below , such a comparison
for a range of temperatures for the simulated lipid bilayers is presented .
despite the above arguments , area per lipid can still be used to indicate
whether or not the lipid bilayer ends up in the correct phase . incorrect
parametrization may lead to underestimated areas indicating that the
lipid bilayer is in the l-phase . this is not the case in the present
investigation , which can be seen from table 6 showing area per lipid corresponding to l-phase
for the range of temperatures , in good agreement with experimental
results . volume per lipid is a property which can be more rigorously
defined ,
and the interpretation of these properties from experiments is more
straightforward compared to area per lipid . in table 8 it is likely that this lack of consensus is due to the
parameters for the head group since the agreement between simulations
and experiments for bulk alkanes in table 4 is very good . the isothermal area compressibility is a property that can be
difficult to obtain accurate values for in md simulations . often the
variance in area per lipid is underestimated resulting in a too high
value of ka . as can be
seen from table 8 , the agreement between simulations
and experiments is very good . since the fluctuations in area per lipid
occur on a long time scale , relatively long simulations
are needed and therefore the simulations were run for 400500
ns . as a consequence of this , however , in a small system thermal fluctuations
could be suppressed resulting in a size dependency since in a larger system undulations make the bilayer more
compressible . poger and mark studied
the impact of system size by using lipid bilayers consisting of 128
and 722 lipids . it was found that ka for both systems converged to the same value ,
but roughly double the sampling time was required for the smaller
system to reach the value of the larger . possibly , longer simulations
could have made the difference between the computed ka and the experimental data even smaller . based on these
conclusions , the system - size dependencies of the systems studied here
were not explored . computed thermal area expansivities are shown
in table 7 and compared to experimental findings . the values
obtained from simulations are systematically underestimating this
property , which means that the lipid bilayer does not have the same
tendency to expand as the temperature is increased . reported values are expressed
in the unit 10 k. three types of membrane thicknesses
were analyzed : the distance between the phosphate peaks in the electron
density , the luzzati thickness ( db ) , and the hydrophobic thickness ( 2dc ) . db is calculated
according to4where dz is the repeat z - spacing along the the bilayer
normal of the simulation box , i.e. , the z - dimension
of the simulation box . w(z ) is
the probability distribution of water in the z - direction
and is calculated from the time - averaged histograms of the water distribution
along the z - axis with a thickness of dz5where nw is the
time average of water molecules per slice and dv is
the time - averaged volume of each slice . 2dc is computed in a similar way to db6where ch(z ) = ch2 + ch3 . the probability distributions are calculated
according to7as can been seen from table 8 , the agreement between
simulations and experiments is excellent for the phosphate consistent with previous work , the
luzzati thickness is slightly underestimated . since the db is a measure of how deep water is allowed to penetrate the lipid
bilayer , it is important to have good agreement between the simulation
and experiments in order to reassure that the models used are describing
the physics of the system in a correct manner . the polarity of the
ester group will determine how deep the water is willing enter the
lipid bilayer in order to form hydrogen bonds with the carbonyl dipole . the hydrophobic thickness is an important property of a
lipid bilayer since this property has been proposed to dictate a number
of cellular processes via , e.g. , hydrophobic mismatch . the differences between simulations and experiments
for 2dc are small for all temperatures ,
which is consistent with the computed values for db . the decrease in bilayer thickness with increasing
temperature can
be explained by the increase in chain distortion . gauche
isomerization , this phenomenon gives rise to the major part of the
differences observed . as the chain disorder increases , the lipid bilayer
becomes more compact in the direction of the membrane normal ( the z - direction ) . in an expansion of the lipid bilayer in the xy - plane , the area is increased and the thickness has to
be decreased . in table 9 the number of gauche
bonds for the dppc lipid simulated in a bilayer at various temperatures
are presented . the small temperature dependency
is captured by the simulations and for dmpc and dppc the hydrophobic
thickness thermal contractivity is slightly overestimated whereas
for dlpc it is underestimated to a small degree . the agreement for
the bilayer thickness contractivity between simulations and experiments
for the former two lipids is better . taking all these properties
into consideration , we can conclude
that the current ff balance the hydrophilic interactions of the lipids
well with the hydrophobic effect . water is not allowed to penetrate
the structure too deeply , and the hydrophobic core of the membrane
is kept intact , which is important . this might be shown important
in future simulations of realistic models of biological membranes
in the presence of proteins and membrane - associated molecules . the deuterium
order parameter is a property that is determined from experiments
without model assumptions , and the methods used are robust which results
in measured vales that are reproducible . as a result of this , |scd| is one of the most vital properties of a
set of ff parameters for which to get accurate values . computed deuterium
order parameters ( |scd| ) together with
experimental data for the aliphatic tails of dlpc , dmpc , and dppc
are presented in figure 5 . the splitting of order parameter
for the second carbon in the sn-2 tail observed in
experiments is also present in the simulations for each lipid type ,
however , not to the same extent . experimental studies have shown that
the upper part of the two lipid tails are aligned differently with
the sn-1 tail in a more perpendicular position relative
the bilayer normal than the sn-2 tail . as can be seen in figure 5 , this difference
is also present in the simulations for all lipids . the agreement in
order parameters indicates that the simulated lipid bilayers have
the correct internal ( disordered ) structure and that the bilayers
are all in the l-phase . a large discrepancy between
experimental findings and simulations where |scd| would have been overestimated by the latter would indicate
a lipid bilayer in a too ordered phase , i.e. , the l-phase . deuterium order parameter , |scd| , from
simulations and experiments for the lipid tails of dlpc ( top ) , dmpc ( middle ) , and dppc ( bottom )
at 303 , 303 , and 323 k , respectively . the number of gauche bonds in the aliphatic chains
of the lipids
can be computed in order to further test if the lipid bilayer is in
the correct phase during the simulations . in table 9 the number of gauche bonds from simulations of a dppc lipid
bilayer is compared to experiments . simulations
performed at 323 k agree well with the experimental estimates , and
this together with the calculated deuterium order parameter and area
per lipid indicates that the lipid bilayer is in the correct phase . a higher percentage of trans conformations ( and low number of gauche
bonds ) induces order in the system resulting in a gel phase . in figure 6 three simulation
snapshots are shown at different temperatures where the increase in
gauche bonds per lipid molecule and general disorder with increasing
temperature are well illustrated . snapshots from simulations of a dppc lipid bilayer at different
temperatures : 293 k ( left ) , 323 k ( center ) , and 353 k ( right ) . the
choline groups are rendered in blue , the phosphorus groups in cyan ,
the glycerol / carbonyl groups in red , and the hydrophobic tails in
white . in md simulations it is straightforward to obtain electron density
profiles and it is possible to divide the total electron density into
the constituting groups of the lipid molecule as defined in figure 1 . the overall electron densities for the studied
lipids are presented in figure 7 together with the contributions from each group .
from these plots the head - to - head distance ( dhh ) can be obtained ( see previous sections ) . other valuable
information such as the fact that water molecules penetrate the lipid
bilayer down to the carbonyls of the aliphatic tails can also be extracted ,
which is in agreement with experiments . furthermore , it is evident that water does not penetrate the hydrophobic
core of the lipid bilayer , in agreement with experimental findings . for all lipids , the leaflets are interdigitated and the peaks corresponding to the head groups
are broad , indicating that they are all in the l-phase since in gel phase the leaflets are less interdigitated and
the head group s electron density peaks are divided into two
sharp peaks instead of one broad peak . one
inherent problem with experimental electron density curves is that
they are all based on certain assumptions and models which makes direct
comparison between simulations and experiments difficult . electron density profiles are
obtained from an inverse fourier transformation of the scattering
form factors . a better way of comparing simulations and experiments
is to compute the form factors from the total electron density profiles
obtained from the simulations . this comparison together with
the order parameters should serve as the major part of the testing
procedure when the quality of a ff is established . f(q ) and |scd| ( at least
for the lipid tail ) can be obtained from experiments in a unambiguous
manner and are reproducible and therefore should receive the most
focus . in figure 8 form factors obtained from md simulations are compared
to experimental form factors at various temperatures . the most common temperatures to perform
experiments and simulations for dlpc , dmpc , and dppc are 303 and 323
k. for these temperatures , the agreement between simulations and experiments
is very good , providing a clear proof that the ff presented here gives
the correct structure for a fully hydrated lipid bilayer . the position
where the form factor goes to zero is reproduced in an excellent manner ,
and the relative peak heights agree very well with the available experimental
data . a direct indication of this is that all the lipid bilayers have
the correct thickness and structure . a small offset can be seen for
the third peak of dmpc , but the difference is very small . when the
temperature is increased , the agreement is slightly decreased . still ,
the simulations give more than reasonable agreement with experiments .
the largest discrepancy with experimental data is found for dlpc ,
for the third peak , when the temperature is increased from 303 to
323 and 333 k. the insets in figure 8 show
the neutron scattering ffs obtained from simulations compared to experiments . the sdp model introduced by kuerka
et al . , the agreement is good and the points where the form
factors have their minima are reproduced by the simulations . these
findings extend the evidence that the ff presented here is highly
suitable for simulating lipid bilayers . electron density profiles
from simulations of dlpc ( top ) , dmpc
( middle ) , and dppc ( bottom ) lipid bilayers . contributions from individual
components are shown with colors : n , choline groups , dark blue ; p ,
phosphate groups , cyan ; co , ester groups , red ; ch2 : , methylene
groups , light gray ; ch3 , methyl groups , dark gray . form factors for the simulated lipid bilayers ( lines )
compared
to available experimental data ( points ) at various temperatures . in the insets , experimental f(0 ) values
are shown as black squares . in membranes the lipids are free
to diffuse in the plane that is perpendicular to the bilayer normal .
to test the parameter set proposed here , the einstein relation
was used to calculate dl via the mean - squared
displacement ( msd ) . in order to correct for the artificial center
of mass motion of each monolayer , the diffusion
of lipids in a bilayer occurs on two different time scales : on a short
time scale ( < 5 ns ) where the center of mass of the lipid is in
motion due to the conformational freedom of the acyl chains and the
head group is relatively fixed in space , and on the longer time scale the whole lipids move in the monolayer
more freely . in the present investigation , only the long time diffusion
was investigated . for dlpc , dmpc , and dppc dl is underestimated
with respect to the experimental data . the values obtained for dmpc at 303 k are close to other values
obtained from md simulations . the drastic decrease in dl observed
for dppc when the temperature is lowered is expected due to the phase
transition . as in the latter case the lipids are in a gel phase , the
configurational freedom for the acyl tails is very limited , resulting
in a very low diffusion coefficient . a large drop in lateral diffusion
( about 2 orders of magnitude ) when going from the l-phase to the l-phase has been reported earlier
by marrink and co - workers in coarse - grained simulations . the increase in the lateral mobility of the
lipids with an increase in temperature can be observed which is related
to the area per lipid . as the temperature is increased , the area per
lipid is increased , making it easier for the lipid to move in the
two - dimensional liquid . for dmpc , the lateral diffusion coefficients
obtained from simulations when the temperature is increased are about
half of the experimental values as can be seen in the fifth and sixth
columns of table 10 . in order to enhance dl accelerated md simulations could be applied where the sampling of the conformational
space is improved . one possible explanation
for the systematic underestimation of the lateral diffusion could
be the system size . in a lipid bilayer patch of 128 lipids , no efficient
collective movement can occur that could result in a decrease in dl . this is similar to the case of bulk alkanes
when the diffusion coefficients computed in a box of limited size
were underestimated , but after correction on collective motion they occur in very good agreement with experimental
results ( table 4 ) . the agreement between simulations
and experiments for the normalized values ( with respect to the highest
temperature ) in table 10 supports this , indication
that there could be a systematic underestimation which could originate
from this effect . to further test the ffs ability
to describe the nature of a lipid bilayer under various thermal conditions ,
simulations of a dppc bilayer in gel phase were conducted . the phase
transition from the l-phase to the l-phase occurs at 314 k for dppc , so
the simulations were performed at a temperature of 293 k in order
to ensure that the lipid bilayer entered the correct phase . in order
to prepare the gel phase , the size of the carbon atoms for the special
1 - 4 lj interactions was increased so that the bilayers entered the
gel phase without any major problems of metastable configurations .
for the production run , the 1 - 4 lj interactions were set to the original
values with a equilibration time of 40 ns and a production simulation
of 300 ns with the same procedure as for the bilayers in the l-phase . this approach to force a lipid bilayer into
the l-phase is similar to the methodology
described by schubert et al . simulations
where the temperature was slowly decreased to 293 k without the biased
1 - 4 interactions resulted in a lipid bilayer in the rippled phase
( p ) . as this is a metastable state at 293
k the system would eventually reach the more stable l-phase . since the dynamics of the system is slowed down by roughly
2 orders of magnitude , biased intramolecular interactions were introduced
merely to force the lipid bilayer into the correct phase on a shorter
time scale . thus , we can conclude that the correct phase behavior
is to be expected . we believe that if the parameter set would have
given rise to a more fluid phase than l at
293 k this would have been observed during the 300 ns production simulation
since melting is a process that generally occurs on a smaller time
scale than freezing . in figure 6 the obtained
gel phase , l , it is evident that
the bilayer entered the gel phase and that the hydration number ( 30
water molecules per lipid ) did not play a significant role in accordance
with the findings of marrink et al . the area per lipid is overestimated
by roughly 0.02 nm , which is the only discrepancy
in the comparison between simulations and experiment . the tilt of
the lipids with respect to the normal of the bilayer is characteristic
of the l-phase and the tilt angle obtained
from simulations is in very good agreement with experimental data . the head - to - head distance also agrees well with experimental findings . the significant decrease in number of gauche
defect per chain ( table 5 ) illustrates that
the bilayer has entered the gel phase and that the conformations of
the chains is of importance in controlling the bilayers fluidity . these findings strengthen the present ffs ability
to model lipid
bilayers under a range of temperatures and shows that the correct
phase behavior can be expected . there is a growing interest in simulating
membranes in other phases than the liquid crystalline one and the
data presented here makes this ff a good candidate for these simulations . it is critical for computer simulations
of lipid bilayers to have
sets of parameters obtained in a clearly defined and physically grounded
procedure , in order to produce reliable and meaningful results . here , the development of new aa lipid ff has resulted in a consistent ff
where the hydrophilic and hydrophobic forces balance each other , which
is crucial . this balance is what drives the formation of the different
structures that can be formed by lipids . as model compounds for the
aliphatic chains of saturated lipids , a series of alkanes was chosen
and many experimentally accessible properties for these bulk liquids
were reproduced in the simulations . furthermore , a large number of
important properties for lipid bilayer are in very good agreement :
area and volume per lipid , membrane thicknesses , isothermal area compressibility ,
and trans / gauche ratio . but , most importantly , the key properties ,
nmr order parameters and scattering form factors , are excellently
reproduced which is a clear proof of the abilities of the current
ff . simulations performed with dppc in the gel phase show that the
ff gives the correct phase behavior as well . to our knowledge , this
is the first reported set of parameters that reproduces all these
properties consistently under a range of temperatures . the ability
of the ff to reproduce a lipid bilayer in a liquid - crystalline phase
in a tensionless ensemble and describe the physics of lipid in a membrane
accurately from more than just the point of view of one or two properties
makes it an excellent choice for more advanced simulations , including
several lipid types and important membrane - inserted molecules . most
importantly , |scd| and f(q ) , which are properties that can be determined
without any empirical input from an experimental point view , are in
very good agreement with available experimental data . as protein
simulations in membranes become more popular and since
the ff presented here is compatible with the amber , ffs for biomolecule
simulations can easily be extended to include transmembrane proteins . ongoing work in our group aims to expand the ff to unsaturated and
polyunsaturated lipids , lipids with different head groups , and cholesterol . a ff that has been developed in a consistent manner for a large number
of lipids will be important in future modeling of biological membranes . | an all - atomistic force field ( ff ) has been developed
for fully
saturated phospholipids .
the parametrization has been largely based
on high - level ab initio calculations in order to keep the empirical
input to a minimum .
parameters for the lipid chains have been developed
based on knowledge about bulk alkane liquids , for which thermodynamic
and dynamic data are excellently reproduced .
the ffs ability to simulate
lipid bilayers in the liquid crystalline phase in a tensionless ensemble
was tested in simulations of three lipids : 1,2-diauroyl - sn - glycero-3-phospocholine ( dlpc ) , 1,2-dimyristoyl - sn - glycero-3-phosphocholine ( dmpc ) , and 1,2-dipalmitoyl - sn - glycero-3-phospcholine ( dppc ) . computed areas and volumes per lipid ,
and three different kinds of bilayer thicknesses ,
have been investigated .
most importantly nmr order parameters and scattering form factors
agree in an excellent manner with experimental data under a range
of temperatures .
further , the compatibility with the amber ff for
biomolecules as well as the ability to simulate bilayers in gel phase
was demonstrated .
overall , the ff presented here provides the important
balance between the hydrophilic and hydrophobic forces present in
lipid bilayers and therefore can be used for more complicated studies
of realistic biological membranes with protein insertions . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
adenosquamous carcinoma ( asc ) is defined as a tumor in which both glandular and squamous elements are histologically malignant . incidence rate of carcinoma of amv is 3.8 per 10,00,000 in men and 2.7 per 10,00,000 in women . asc of the amv has rarely been discussed in the literature . here , we report a case of a 58-year - old male with primary asc of the amv . a 58-year - old male presented with abdominal pain , jaundice and vomiting for one month . he also had associated history of weight loss and anorexia with no h / o pruritus and clay colored stool . the laboratory examination revealed a serum total bilirubin level of 6.7 mg / dl , an aspartate aminotransferase ( sgot ) level of 102 . demonstrated large deformed and bulky papilla with ulcerated lesion with infiltration in to duodenum causing 1st and 2nd part of duodenal ( d1d2 ) stenosis . contrast enhanced computed tomography scan ( cect ) of the abdomen revealed a soft - tissue mass in the periampullary region with dilated distal portion of the common bile duct ( cbd ) and main pancreatic duct . there was a growth at the ampullary region with involvement of cbd leading to grossly dilated cbd and main pancreatic duct . the gross specimen revealed a growth of 4 3 cm in the amv ( fig . adenosquamous carcinoma ( asc ) is defined as a tumor in which both glandular and squamous elements are histologically malignant . four hypotheses regarding its histogenesis are summarized as follows : ( 1 ) pluripotent epithelial stem cells capable of inducing the malignant transformation of both cell types ; ( 2 ) squamous metaplasia in the intestinal mucosa ; ( 3 ) adenocarcinoma transforming into squamous cell carcinoma ; and ( 4 ) collision of both malignant tumors . although we do not understand the histogenesis of asc , it exhibits a more aggressive biologic behavior and is associated with a worse prognosis than conventional adenocarcinoma according to previous reports . preoperative diagnosis is difficult because of the lack of defining characteristics in imaging studies and the difficulty in acquiring both malignant components by limited biopsy . all of the patients have a dismal prognosis , and most of them have early distal metastasis after surgery . a review of the literature revealed only five reported cases of asc of the amv . we have summarized these reports , including the clinical course and final prognosis of asc of the amv , in table 1 . asc of the amv is a virulent disease with a worse prognosis than that of adenocarcinoma . a review of the data revealed that the most common procedure performed was pancreaticoduodenectomy followed by ampullectomy . most patients with asc of the amv experienced early distal metastasis and short survival after surgery . our limited experience makes it difficult to determine the clinical course of this disease and the efficacy of surgical intervention . it is hard to draw a conclusion on whether performing major surgery in these patients is beneficial , because of the potentially high morbidity . one report presented two patients with prolonged survival ( 19 and 46 months after surgery ) . as noted by lee et al . complete surgical resection may prolong patient survival ; however , this finding was not supported by other reports . because of findings of early recurrence and metastasis after surgery , micrometastasis is suspected , however we could not detect it prior to surgery . currently , there is no established diagnostic tool for detecting micrometastasis . positron emission tomography may be another diagnostic tool for detecting micrometastasis , but the efficacy of this tool requires more evidence for confirmation . some authors have suggested conservative management of patients with asc of the ampulla of vater diagnosed before surgery , to reduce postoperative morbidity and achieve comparable survival as observed in patients undergoing surgery . although there was no published report evaluating the efficacy of chemoradiation against this disease . in past , the diagnostic term adenoacanthoma world health organization tumor classification system makes a clear distinction between an asc , in which both glandular and squamous elements are histologically malignant and adenoacanthoma which represents adenocarcinoma that contains foci of benign squamous metaplasia . pancreaticoduodenectomy is treatment of choice though early recurrence and distal metastasis may be encountered after surgery . follow - up should be more frequent to detect possible early recurrence and distal metastasis in patients with asc of the amv . 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 on request . nitin r. nangare : introduction ; dr . mayank a. vekariya : presentation of case and discussion ; dr . | introductionprimary adenosquamous carcinoma ( asc ) of the ampulla of vater ( amv ) is extremely rare .
carcinoma of the ampulla of vater tends to manifest early due to biliary outflow obstruction , as opposed to pancreatic neoplasms that often are advanced at the time of diagnosis .
periampullary carcinomas are treated by pancreaticoduodenectomy ( pd ) .
adenosquamous carcinoma carries very dismal prognosis.presentation of casehere we present a case of 58-year - old male who was presented with abdominal pain , jaundice and anorexia with no history of ( h / o ) pruritus and clay colored stool .
all blood investigations were normal except liver function tests ( lfts ) .
ultrasonography ( usg ) of abdomen suggestive of periampullary mass with dilated pancreatico - biliary tree .
endoscopic retrograde cholangiopancreatography ( e.r.c.p . ) demonstrated large deformed and bulky papilla with ulcerated lesion with infiltration in to duodenum .
exploratory laprotomy proceeds whipple 's pancreaticoduodenectomy done .
histopathology revealed adenocarcinoma of the ampulla of vater .
immunohistochemistry was confirmatory of adenosquamous carcinoma.discussionadenosquamous carcinoma ( asc ) is defined as a tumor in which both glandular and squamous elements are histologically malignant . compared to adenocarcinoma , asc of the amv
is a rare malignancy .
preoperative diagnosis is difficult because of the lack of defining characteristics in imaging studies and the difficulty in acquiring both malignant components by limited biopsy .
periampullary carcinomas are treated by pancreaticoduodenectomy.conclusionadenosquamous carcinoma is a very rare form of cancer of the amv .
pancreaticoduodenectomy is the treatment of choice though early recurrence and distal metastasis may be encountered after surgery .
follow - up should be more frequent to detect possible early recurrence and distal metastasis . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the european union ( eu ) is committed to improving the health of its ageing population , with growing initiatives to promote active and healthy ageing ( supplementary figure s1 ) . its preferred measure is healthy life years ( hly ) , the average remaining years spent free of activity limitation . health expectancies , of which hly is one , were developed to assess whether increases in life expectancy ( le ) are due to improvements in medical technology , which keep the old and frail alive ( resulting in an expansion of morbidity ) , or because the onset and progression of disabling diseases are being postponed ( compression of morbidity ) . as well as assessing the relationship between le and hly historically , the compression of morbidity concept can be used prospectively to set hly targets , e.g. the european innovation partnership on active and healthy ageing ( eipon - aha ) target , which seeks to increase hly at birth by 2 years during the next 10 years , averaging 2.4 months per year . if achieved , disability would be compressed at the eu level , as projected le increases , at 1.9 years ( men ) and 1.5 years ( women ) ( supplementary table s1 ) , are less than the eipon - aha target of 2 years . although the eipon - aha target is generally supported , especially among those fearing that broader welfare may be sacrificed for economic growth , there are concerns that an eu - wide target ignores disparities between member states . these are large in 2005 , the gaps in hly at age 50 years were a remarkable 14.5 years for men and 13.7 years for women , exceeding gaps in le at age 50 years ( men : 9.1 years ; women : 6.1 years ) . eurostat projections assume a convergence hypothesis that envisages the le gap across the eu27 reducing between 2010 and 2020 , by 1.6 years for men and 1 year for women ( supplementary table s1 ) , though this is a stronger assumption than the 0.9 years ( men ) and 0.2 years ( women ) projected during the same period by the united nations . nevertheless , though the eu , as a whole , is currently projected to reach the target , male le is projected to exceed 2 years for nine countries and female le for four countries ( supplementary table s1 ) . we assess whether the eipon - aha target is attainable for the eu27 as a whole and what effect this will have on individual countries and the health gaps between them . first , we examine how le and hly has changed during the past 5 years in the eu27 countries . then we project the changes in hly forward from 201020 under various scenarios to investigate ( i ) the scope to achieve the target for the eu27 as a whole and in its constituent countries and ( ii ) the impact on disparities in hly between countries . to document previous trends in le and hly , we obtained values of hly and le at birth from the eurohex website ( www.eurohex.eu ) , these being identical to values from eurostat . hly are calculated by sullivan s method in which the age- and sex - specific prevalence of the health state is applied to a standard life table . for hly , the underlying health state question is the global activity limitation indicator ( gali ) , included in the eu statistics of income and living conditions survey ( see supplementary material for the exact form of the gali ) . although some countries began data collection in 2004 , hly were only available for all 25 pre-2007 eu countries ( eu25 ) since 2005 . overall hly trends are complicated because some countries made substantial changes to the gali question used to calculate hly , particularly between 2007 and 2008 . consequently , to explore preliminary trends further , we concentrated on the six countries ( belgium , france , greece , ireland , slovenia and uk ) where the gali question was essentially unchanged between 2005 and 2009 , but also included romania , which had retained the same question since eu accession in 2007 . values were also included for countries involved in the pilot phase in 2004 ( belgium , france , greece and ireland ) to provide longer time series . to assess trends , we estimated annual change in hly , le and the proportion of remaining life spent healthy ( hly / le ) and 95% confidence intervals from the country and sex - specific linear regression models . further details of methods , including sample size and non - response rates for the eu statistics of income and living conditions 2009 , are given in supplementary material . we then estimated hly in 2010 in each country by multiplying eurostat projections of le in 2010 by the ratio hly / le obtained either from country- and sex - specific linear regression models of hly / le on year ( seven countries retaining same hly question ) or extrapolating the average of hly / le in 2008 and 2009 to 2010 ( 20 countries and eu27 ) . full details of methods are in the online supplementary material . projecting hly / le rather than hly ensured that hly could not exceed le . to assess the validity of these assumptions , we compared projected 2010 hly with preliminary true values for 20 countries and found close agreement . the mean difference ( men and women ) between the predicted and actual 2010 hly was 0.2 years , and 84% of the values were within 1.5 years , although the limits of agreement ( 3.4 to 3.7 years ) were somewhat wider . hly values for eu27 were the average of hly values for the individual countries weighted according to population size . reducing inequalities is a key concern for the eu ; therefore , the other three scenarios explored relevant implications , first alone ( scenario 2 ) , then in combination with the eipon - aha target for each country ( scenario 3 ) and finally in combination with the requirement that each country s gain in hly was at least at the level of le gains during the period , thus avoiding expansion of disability ( scenario 4 ) . in all three inequality scenarios , we considered two reductions : a 50% gap reduction between countries with the highest and lowest values for 10 years , equating to eliminating the hly gap in 20 years ( a ) , and a 30% reduction for the 10 years , thus eliminating the hly gap in around 33 years ( b ) . in 2005 , male le in the eu25 was 75.9 years , and female le was 81.9 years ( supplementary figure s2 ) . between 200509 , le for the eu25 increased steadily , by just over 1 year or 3.0 months per year for men , and by 1.0 years or 2.4 months per year for women . le increased least in denmark ( 0.6 years for women ) and ireland ( 0.1 years for men ) and most in cyprus ( 2.7 years for women ) and latvia ( 2.8 years for men ) . differential gains in le narrowed the gap in le from 13.2 years in 2005 to 11.9 years in 2009 for men but a small increase for women from 7.3 years in 2005 to 7.6 years in 2009 . in 2005 , male hly at birth for the pre-2007 eu25 was 61.0 years , 80% of remaining life . female hly was greater , at 62.3 years , though women could expect to live less of their remaining life healthy ( 76% ) . similar levels of hly for men and women contrast with women s higher overall le and therefore co - occur with a greater number of years with disability [ unhealthy life years ( uhly ) ] ( supplementary figure s2 ) . in contrast to the increase in le for men and women between 2005 and 2009 , hly showed little change : for men an increase of 0.3 years or 0.8 months per year and for women a decrease of 0.4 years or 1.0 months per year . as the eipon - aha target equates to 2.4 months increase per year , it was not reached for the eu25 overall during the period 200509 . turning to the restricted set of countries with consistent wording of the gali question , even here , real increases in le at birth from 200409 were not universal ( supplementary table s2 ) . during this period , romania ( both sexes ) and ireland ( men ) had no significant increase in le , and romanian men had a significant decrease in hly . significant increases in hly were observed in belgium ( both sexes ) , and for men in ireland , slovenia and the uk , all were equating to increases of at least 2 hly for 10 years . in slovenia , the hly increase was also accompanied by a significant increase in the proportion of remaining life spent healthy . projections of hly at birth are presented in table 1 ( men ) and table 2 ( women ) . in 2010 , the gap between countries in male hly was estimated to be 17.5 years ( from slovakia 52.6 years to sweden 70.1 years ) and in female hly 18.9 years ( from slovakia 52.7 years to malta 71.5 years ) . hly projections between 2010 and 2020 were explored under four main scenarios ( tables 1 and 2 ) . scenario 1 assumed that recent annual changes in hly ( previously calculated ) would continue for the whole period . eu27 hly at birth then increases by 1.4 years ( men ) and 0.5 years ( women ) , failing to reach the 2-year eipon - aha target , although it would be reached in nine countries for male hly but only one country ( belgium ) for female hly . crucially , the hly gap between countries would increase further to 18.3 years ( men ) and 19.5 years ( women ) by 2020 . table 1hly at birth in 2010 , current annual increase in hly and projected increase in hly between 2010 and 2020 under various scenarios for eu27 countries , malescountryprojected increase in hly at birth between 2010 and 2020hly 2010scenario1 : current trends prevailscenario 2 : reduction of hly gaps byscenario 3 : hly increase 2 years for country and reduction in hly gaps byscenario 4 : no increase in uhly for country and reduction in the hly gaps bya:50%b:30%a:50%b:30%a:50%b:30%slovakia52.61.88.85.310.87.310.26.7latvia52.92.28.65.210.67.210.06.6estonia54.52.17.84.79.86.79.26.1hungary55.52.17.34.49.36.48.75.8germany56.71.26.74.08.76.08.15.4lithuania56.72.56.74.08.76.08.15.4finland58.41.45.93.57.95.57.34.9poland58.72.15.73.47.75.47.14.8portugal58.81.45.73.47.75.47.14.8austria58.91.25.63.47.65.47.04.8romania59.52.25.33.27.35.26.74.6slovenia61.18.14.52.76.54.75.94.1czechrepublic61.31.74.42.66.44.65.84.0denmark61.91.34.12.56.14.55.53.9netherlands62.21.14.02.46.04.45.43.8bulgaria62.42.33.82.35.84.35.23.7france62.61.23.82.35.84.35.23.7italy62.91.23.62.25.64.25.03.6ireland63.41.93.42.05.44.04.83.4spain63.61.33.32.05.34.04.73.4cyprus64.61.32.71.64.73.64.13.0belgium64.78.02.71.64.73.64.13.0luxembourg64.91.32.61.64.63.64.03.0uk65.01.02.61.54.63.54.02.9greece66.12.22.01.24.03.23.42.6malta69.41.50.30.22.32.21.71.6sweden70.11.20.00.02.02.01.41.4eu2761.31.44.42.66.44.65.84.0a : hly2010 values based on 200809 values.b : hly2010 values based on 2004/200509 values . table 2hly at birth in 2010 , current annual increase in hly and projected increase in hly between 2010 and 2020 under various scenarios for eu27 countries , femalescountryprojected increase in hly at birth between 2010 and 2020hly 2010scenario1 : current trends prevailscenario 2 : reduction of hly gaps byscenario 3 : hly increase 2 years for country and reduction in hly gaps byscenario 4 : no increase in uhly for country and reduction in the hly gaps bya:50%b:30%a:50%b:30%a:50%b:30%slovakia52.71.39.45.711.47.710.97.2latvia55.41.58.14.810.16.89.66.3portugal56.91.07.34.49.36.48.85.9germany57.81.06.94.18.96.18.45.6hungary58.31.66.64.08.66.08.15.5estonia58.51.36.53.98.55.98.05.4finland58.81.06.43.88.45.87.95.3austria60.01.05.83.57.85.57.35.0netherlands60.11.05.73.47.75.47.24.9denmark60.71.35.43.37.45.36.94.8lithuania61.01.55.33.27.35.26.84.7slovenia61.00.35.33.27.35.26.84.7romania61.15.25.23.17.25.16.74.6italy61.90.94.82.96.84.96.34.4france62.62.74.52.76.54.76.04.2poland62.81.44.42.66.44.65.94.1czechrepublic63.01.34.32.66.34.65.84.1spain63.00.84.32.66.34.65.84.1belgium64.39.13.62.25.64.25.13.7ireland64.81.73.42.05.44.04.93.5luxembourg65.01.23.32.05.34.04.83.5cyprus65.51.13.01.85.03.84.53.3unitedkingdom66.01.62.81.74.83.74.33.2bulgaria66.01.82.81.74.83.74.33.2greece66.70.52.41.54.43.53.93.0sweden69.31.21.10.73.12.72.62.2malta71.51.30.00.02.02.01.51.5eu2761.90.54.82.96.84.96.34.4a : hly2010 values based on 200809 values.b : hly2010 values based on 2004/200509 values . hly at birth in 2010 , current annual increase in hly and projected increase in hly between 2010 and 2020 under various scenarios for eu27 countries , males a : hly2010 values based on 200809 values . hly at birth in 2010 , current annual increase in hly and projected increase in hly between 2010 and 2020 under various scenarios for eu27 countries , females a : hly2010 values based on 200809 values . b : hly2010 values based on 2004/200509 values . under scenario 2a , eliminating the hly gap in 20 years would result in gains for the next decade of 4.4 hly for eu27 men ( table 1 ) and 4.8 hly for eu27 women ( table 2 ) , more than double the eipon - aha target . for slovakia , which currently has the lowest hly at birth for men and women , this means an increase of 9 hly ( male hly : 8.8 , female hly : 9.4 ) during the decade , substantially more than the increase of under 2 hly ( male hly : 1.8 , female hly : 1.3 ) expected if recent annual changes continue ( scenario 1 ) . if the goal of eliminating the hly gap in a 20-year period was relaxed , then the eipon - aha target could be achieved by a 2123% reduction in the gap over the next 10 years , in women and men , respectively , with more realistic increases for slovakia ( male hly : 4.0 , female hly : 3.3 ; data not shown ) , although now the gap would only be closed in 45 years . moreover , with this reduction , the eipon - aha target would be achieved by under half of the countries ( male : 13 countries , female : 5 countries ) . scenario 3 therefore added the constraint that each country also had to achieve the eipon - aha target of an increase in 2 hly during the decade resulting in slovakia needing to achieve even greater increases of 11 hly ( male hly : 10.8 , female hly : 11.4 ) or more than one hly per year . for scenario 4 , in addition to closing the gap for the same periods as scenarios 2 and 3 , projected increases in le were included by requiring that the number of remaining years spent unhealthy ( le - hly ) did not increase . in 2005 , male le in the eu25 was 75.9 years , and female le was 81.9 years ( supplementary figure s2 ) . between 200509 , le for the eu25 increased steadily , by just over 1 year or 3.0 months per year for men , and by 1.0 years or 2.4 months per year for women . le increased least in denmark ( 0.6 years for women ) and ireland ( 0.1 years for men ) and most in cyprus ( 2.7 years for women ) and latvia ( 2.8 years for men ) . differential gains in le narrowed the gap in le from 13.2 years in 2005 to 11.9 years in 2009 for men but a small increase for women from 7.3 years in 2005 to 7.6 years in 2009 . in 2005 , male hly at birth for the pre-2007 eu25 was 61.0 years , 80% of remaining life . female hly was greater , at 62.3 years , though women could expect to live less of their remaining life healthy ( 76% ) . similar levels of hly for men and women contrast with women s higher overall le and therefore co - occur with a greater number of years with disability [ unhealthy life years ( uhly ) ] ( supplementary figure s2 ) . in contrast to the increase in le for men and women between 2005 and 2009 , hly showed little change : for men an increase of 0.3 years or 0.8 months per year and for women a decrease of 0.4 years or 1.0 months per year . as the eipon - aha target equates to 2.4 months increase per year , it was not reached for the eu25 overall during the period 200509 . turning to the restricted set of countries with consistent wording of the gali question , even here , real increases in le at birth from 200409 were not universal ( supplementary table s2 ) . during this period , romania ( both sexes ) and ireland ( men ) had no significant increase in le , and romanian men had a significant decrease in hly . significant increases in hly were observed in belgium ( both sexes ) , and for men in ireland , slovenia and the uk , all were equating to increases of at least 2 hly for 10 years . in slovenia , the hly increase was also accompanied by a significant increase in the proportion of remaining life spent healthy . projections of hly at birth are presented in table 1 ( men ) and table 2 ( women ) . in 2010 , the gap between countries in male hly was estimated to be 17.5 years ( from slovakia 52.6 years to sweden 70.1 years ) and in female hly 18.9 years ( from slovakia 52.7 years to malta 71.5 years ) . hly projections between 2010 and 2020 were explored under four main scenarios ( tables 1 and 2 ) . scenario 1 assumed that recent annual changes in hly ( previously calculated ) would continue for the whole period . eu27 hly at birth then increases by 1.4 years ( men ) and 0.5 years ( women ) , failing to reach the 2-year eipon - aha target , although it would be reached in nine countries for male hly but only one country ( belgium ) for female hly . crucially , the hly gap between countries would increase further to 18.3 years ( men ) and 19.5 years ( women ) by 2020 . table 1hly at birth in 2010 , current annual increase in hly and projected increase in hly between 2010 and 2020 under various scenarios for eu27 countries , malescountryprojected increase in hly at birth between 2010 and 2020hly 2010scenario1 : current trends prevailscenario 2 : reduction of hly gaps byscenario 3 : hly increase 2 years for country and reduction in hly gaps byscenario 4 : no increase in uhly for country and reduction in the hly gaps bya:50%b:30%a:50%b:30%a:50%b:30%slovakia52.61.88.85.310.87.310.26.7latvia52.92.28.65.210.67.210.06.6estonia54.52.17.84.79.86.79.26.1hungary55.52.17.34.49.36.48.75.8germany56.71.26.74.08.76.08.15.4lithuania56.72.56.74.08.76.08.15.4finland58.41.45.93.57.95.57.34.9poland58.72.15.73.47.75.47.14.8portugal58.81.45.73.47.75.47.14.8austria58.91.25.63.47.65.47.04.8romania59.52.25.33.27.35.26.74.6slovenia61.18.14.52.76.54.75.94.1czechrepublic61.31.74.42.66.44.65.84.0denmark61.91.34.12.56.14.55.53.9netherlands62.21.14.02.46.04.45.43.8bulgaria62.42.33.82.35.84.35.23.7france62.61.23.82.35.84.35.23.7italy62.91.23.62.25.64.25.03.6ireland63.41.93.42.05.44.04.83.4spain63.61.33.32.05.34.04.73.4cyprus64.61.32.71.64.73.64.13.0belgium64.78.02.71.64.73.64.13.0luxembourg64.91.32.61.64.63.64.03.0uk65.01.02.61.54.63.54.02.9greece66.12.22.01.24.03.23.42.6malta69.41.50.30.22.32.21.71.6sweden70.11.20.00.02.02.01.41.4eu2761.31.44.42.66.44.65.84.0a : hly2010 values based on 200809 values.b : hly2010 values based on 2004/200509 values . table 2hly at birth in 2010 , current annual increase in hly and projected increase in hly between 2010 and 2020 under various scenarios for eu27 countries , femalescountryprojected increase in hly at birth between 2010 and 2020hly 2010scenario1 : current trends prevailscenario 2 : reduction of hly gaps byscenario 3 : hly increase 2 years for country and reduction in hly gaps byscenario 4 : no increase in uhly for country and reduction in the hly gaps bya:50%b:30%a:50%b:30%a:50%b:30%slovakia52.71.39.45.711.47.710.97.2latvia55.41.58.14.810.16.89.66.3portugal56.91.07.34.49.36.48.85.9germany57.81.06.94.18.96.18.45.6hungary58.31.66.64.08.66.08.15.5estonia58.51.36.53.98.55.98.05.4finland58.81.06.43.88.45.87.95.3austria60.01.05.83.57.85.57.35.0netherlands60.11.05.73.47.75.47.24.9denmark60.71.35.43.37.45.36.94.8lithuania61.01.55.33.27.35.26.84.7slovenia61.00.35.33.27.35.26.84.7romania61.15.25.23.17.25.16.74.6italy61.90.94.82.96.84.96.34.4france62.62.74.52.76.54.76.04.2poland62.81.44.42.66.44.65.94.1czechrepublic63.01.34.32.66.34.65.84.1spain63.00.84.32.66.34.65.84.1belgium64.39.13.62.25.64.25.13.7ireland64.81.73.42.05.44.04.93.5luxembourg65.01.23.32.05.34.04.83.5cyprus65.51.13.01.85.03.84.53.3unitedkingdom66.01.62.81.74.83.74.33.2bulgaria66.01.82.81.74.83.74.33.2greece66.70.52.41.54.43.53.93.0sweden69.31.21.10.73.12.72.62.2malta71.51.30.00.02.02.01.51.5eu2761.90.54.82.96.84.96.34.4a : hly2010 values based on 200809 values.b : hly2010 values based on 2004/200509 values . hly at birth in 2010 , current annual increase in hly and projected increase in hly between 2010 and 2020 under various scenarios for eu27 countries , males a : hly2010 values based on 200809 values . hly at birth in 2010 , current annual increase in hly and projected increase in hly between 2010 and 2020 under various scenarios for eu27 countries , females a : hly2010 values based on 200809 values . b : hly2010 values based on 2004/200509 values . under scenario 2a , eliminating the hly gap in 20 years would result in gains for the next decade of 4.4 hly for eu27 men ( table 1 ) and 4.8 hly for eu27 women ( table 2 ) , more than double the eipon - aha target . for slovakia , which currently has the lowest hly at birth for men and women , this means an increase of 9 hly ( male hly : 8.8 , female hly : 9.4 ) during the decade , substantially more than the increase of under 2 hly ( male hly : 1.8 , female hly : 1.3 ) expected if recent annual changes continue ( scenario 1 ) . if the goal of eliminating the hly gap in a 20-year period was relaxed , then the eipon - aha target could be achieved by a 2123% reduction in the gap over the next 10 years , in women and men , respectively , with more realistic increases for slovakia ( male hly : 4.0 , female hly : 3.3 ; data not shown ) , although now the gap would only be closed in 45 years . moreover , with this reduction , the eipon - aha target would be achieved by under half of the countries ( male : 13 countries , female : 5 countries ) . scenario 3 therefore added the constraint that each country also had to achieve the eipon - aha target of an increase in 2 hly during the decade resulting in slovakia needing to achieve even greater increases of 11 hly ( male hly : 10.8 , female hly : 11.4 ) or more than one hly per year . for scenario 4 , in addition to closing the gap for the same periods as scenarios 2 and 3 , projected increases in le were included by requiring that the number of remaining years spent unhealthy ( le - hly ) did not increase . we have previously reported large inequalities in health across the eu in 2005 when the hly indicator was first introduced . we have examined trends between 2005 and 2009 both within countries and overall , and we are the first to show that , far from diminishing , inequalities have persisted . indeed , although le at birth for the eu increased by 1 year for men and women between 2005 and 2009 , hly increased by less than half a year for men and decreased by almost half a year for women . by applying projections of current trends in different scenarios , we explored the extent to which individual countries and the eu27 as a whole might reach the eipon - aha target of increasing hly at birth by 2 years by 2020 . we found that if recent trends continue , the eipon - aha target could be met by several countries , predominantly eastern european ones including estonia , latvia , lithuania and hungary , despite le in these countries being projected to increase more than in western european countries . on the other hand , france ( women ) and romania ( both sexes ) are unlikely to meet the target , as , extrapolating recent trends , they will lose rather than gain hly in the next decade . overall , the eipon - aha target would not be reached by the eu27 as a whole , and more critically , hly inequalities would increase further . as le at birth is expected to increase by 22.5 years every decade in low mortality countries , increasing hly at birth by 2 years is a minimum to avoid expansion of uhly at the european level . nevertheless , this strategy will not automatically reduce the existing hly gaps between countries ; therefore , this average target should be accompanied by a second one , focussed on reducing the hly disparities . our other scenarios looked at the impact of reducing the gaps alone , alongside an increase of 2 hly for each country and finally together with no increase in uhly . the large gains in hly , of between 0.5 and one hly per year during the decade , required by the baltic and eastern european countries for ultimate elimination of hly gaps in 20 or 30 years would only be reached by one country , i.e. slovenia . to obtain hly in 2010 , we assumed that the proportion of remaining life spent healthy in most countries would remain at the average of 2008 and 2009 values . this is an obvious limitation but a standard approach , not unrealistic when compared with preliminary 2010 hly values for 20 countries . when time series are available for these countries , we can replace this assumption by the empirical trends , as we have already done for the seven countries with available data . a further limitation is that , for these seven countries , the time series were too short to adequately estimate anything other than a linear trend , but again longer time series will enable the linear trend assumption to be tested . we concentrated on the effect of reducing the overall gap , which will depend strongly on outliers . nevertheless , our findings appear relatively robust , as , at least with the scenario of continued current trends , the interquartile range for predicted hly in 2010 for men is 16.2 years , 1.3 years less than the overall gap , and would still increase , to 16.8 years , by 2020 . an inevitable limitation of analyses such as these is that they must predict the future on the basis of past knowledge ; although the future will be different , it is difficult to know in what way . consequently , this article looks at the implications of achieving certain objectives while recognizing that there are many factors that can not be taken into account to determine whether they can be achieved . though short - term effects on mortality have been shown ( increased suicides but reduced traffic deaths ) , it is much more difficult to identify long - term effects on morbidity or mortality . one possibility that must be considered is whether the substantial gains in survival at older ages seen in recent decades , and thought to be driven largely by improvements in the management of chronic disorders , such as hypertension and its complications , particularly in older patients , will continue given policies of austerity reducing access to health care in some countries . finally , particular caution must be exercised in relation to projections for two of the baltic states , latvia and lithuania , which experienced transient declines in le during the period 200407 , followed by rapid increases , causing difficulties when calculating our baseline scenario . increasing hly by 2 years and reducing gaps will require action on many fronts as well as measures designed to address both the level and distribution of hlys . there is growing recognition of the imperative to address socio - economic inequalities in health at both national and international levels , which include housing quality , access to health care , unemployment , work conditions as well as education , perhaps the most often cited determinant of health and one we have shown to account for some of the variation in hly across the eu25 . socio - economic inequalities in health across the eu25 have been estimated to account for 20% of the total cost of health care and 15% of the total costs of social security benefits . policies must also address factors that predominantly cut lives short prematurely , such as deaths from injuries and violence or smoking and those that leave people alive but disabled , such as poor diet , obesity and inadequate physical activity . a particular priority will be to reduce exposure to risk factors in middle age , and especially smoking , coupled with policies that help people to remain confident and engaged with life , factors shown to correlate with successful ageing . these considerations highlight the importance of comprehensive health improvement strategies , reaching from the underlying determinants of health to responses to established disease . conditions such as cognitive impairment and cardiovascular disease impact on both disability and mortality , others ( arthritis and visual impairment ) affect disability only , whereas cancer predominantly affects mortality . countries need to consider the balance between investment in life - saving medical interventions , rehabilitation for disabling conditions or prevention ( of both disabling and fatal conditions ) , as the former could result in increased le but with a likelihood of increased disability prevalence . an important barrier to reducing the health gaps across the eu is the lack of evidence of what works in narrowing inequalities . a recent systematic review found little evidence , apart from some , albeit not strong , in the areas of housing and work conditions . population laboratory for international comparative research , which should be fully exploited alongside the more systematic diffusion of successful national interventions to other countries as recently highlighted by futurage . the prime concern for both the lisbon strategy and its successor europe 2020 is to create sustainable economic growth in europe . global target , like the eipon - aha target , moves attention from inter - country differences and , alongside the current economic crisis with its winners and losers , is likely to increase health inequalities . the eu , in accordance with the goal of health 2020 of reducing health inequalities , should urgently invest in research to monitor , understand and close the unacceptable level of health inequalities across european countries . the joint action ehleis is co - funded by the european commission , ( grant number dg sanco 20102301 ) , the french ministry of health and the french national solidarity fund for autonomy ( cnsa ) . the funding source had no role in the study design ; in the collection , analysis and interpretation of data ; in the writing of the report ; or in the decision to submit the paper for publication , although eurostat is responsible for data collection . key points
the eu is committed to active and healthy ageing , but its target for hly applies to the eu as a whole and assumes convergence across countries.on the basis of recent trends , the eu target will not be achieved , and inter - country disparities will widen further.achievement of the eu target will require action on many fronts but especially to reduce health inequalities.to be effective , policies must be based on evidence of what works , although that evidence is currently limited .
the eu is committed to active and healthy ageing , but its target for hly applies to the eu as a whole and assumes convergence across countries.on the basis of recent trends , the eu target will not be achieved , and inter - country disparities will widen further.achievement of the eu target will require action on many fronts but especially to reduce health inequalities.to be effective , policies must be based on evidence of what works , although that evidence is currently limited . the eu is committed to active and healthy ageing , but its target for hly applies to the eu as a whole and assumes convergence across countries . on the basis of recent trends , the eu target will not be achieved , and inter - country disparities will widen further . achievement of the eu target will require action on many fronts but especially to reduce health inequalities . to be effective , policies must be based on evidence of what works , although that evidence is currently limited . | background : the european innovation partnership on active and healthy ageing seeks an increase of two healthy life years ( hly ) at birth in the eu27 for the next 10 years .
we assess the feasibility of doing so between 2010 and 2020 and the differential impact among countries by applying different scenarios to current trends in hly .
methods : data comprised hly and life expectancy ( le ) at birth 200409 from eurostat .
we estimated hly in 2010 in each country by multiplying the eurostat projections of le in 2010 by the ratio hly / le obtained either from country and sex - specific linear regression models of hly / le on year ( seven countries retaining same hly question ) or extrapolating the average of hly / le in 2008 and 2009 to 2010 ( 20 countries and eu27 ) . the first scenario continued these trends with three other scenarios exploring different hly gap reductions between 2010 and 2020 .
results : the estimated gap in hly in 2010 was 17.5 years ( men ) and 18.9 years ( women ) . assuming current trends continue , eu27 hly increased by 1.4 years ( men ) and 0.9 years ( women ) , below the european innovation partnership on active and healthy ageing target , with the hly gap between countries increasing to 18.3 years ( men ) and 19.5 years ( women ) . to eliminate the hly gap in 20 years
, the eu27 must gain 4.4 hly ( men ) and 4.8 hly ( women ) in the next decade , which , for some countries , is substantially more than what the current trends suggest .
conclusion : global targets for hly move attention from inter - country differences and , alongside the current economic crisis , may contribute to increase health inequalities . |
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characterized by a triad of rapidly progressive glomerulonephritis , hemoptysis and the presence of anti - glomerular basement membrane ( anti - gbm ) antibodies , goodpasture syndrome is a rare , life - threatening autoimmune disease . the autoantibodies are targeted against the non - collagen domain 1 of the 3 chain of type iv collagen . the 3 chain of type iv collagen is restricted to the basement membranes of the kidney , cochlear tissue , lung , bruch 's membrane of the retina and the testis . the autoantibodies initiate destruction of the basement membranes of the kidney glomeruli , resulting in a focal necrotizing glomerulonephritis , which can be rapidly progressive and cause renal failure . antoantibody - mediated damage of alveolar basement membranes leads to diffuse pulmonary hemorrhage , which if severe enough , can lead to respiratory failure . pulmonary symptoms often precede renal symptoms , and may occur weeks to months earlier . the trigger for this autoantibody formation is unknown ; however , environmental factors are believed to unmask the usually hidden epitopes , eliciting autoantibody formation . postulated environmental factors include viral infection , hydrocarbon solvent exposure and smoking [ 3 , 4 ] . diffuse pulmonary hemorrhage in patients with anti - gbm autoantibodies is seen almost exclusively in active smokers . additionally , this autoimmune disease is associated with certain human leukocyte antigen ( hla ) subtypes , such as hladrb1 * 1501 and * 1502 and hla - dr4 , has a slight male preponderance and is known to occur in two age peaks : the third decade and the fifth and sixth decades [ 5 , 6 ] . here , we report an unusual case of a 40-year - old man with clinical evidence of goodpasture 's syndrome , a negative anti - gbm antibody serum result , eosinophilia and delta granule pool storage deficiency . a 40-year - old caucasian man was admitted to the hospital complaining of a 3-week history of slight cough and increased shortness of breath on exertion and a 2-week history of occasional hemoptysis . the patient 's medical history was scarce as he had not seen a doctor in over 20 years . upon questioning , he reported a 33 pack - year history of smoking , a 27 -kg weight gain over the previous 5 years due to inactivity , no sexual contact or activity , no contact with sick people and no intravenous drug use in the past or present . besides being morbidly obese , he had no other known medical illnesses . chest x - ray imaging revealed bilateral haziness , with extensive involvement of the right lung and the left lower lobe . on physical examination , the patient was afebrile , and he had coarse breath sounds bilaterally with rare rhonci . a complete blood count demonstrated a white blood count of 9.6 10 cells / l ( 4.411.3 10 cells / l ) with 0.96 10 eosinophils / l ( 00.45 10 eosinophils / l ) . he was put on nasal oxygen to improve oxygenation ; however , despite this intervention , blood gases worsened . bronchoscopy was performed , which revealed diffuse airway bleeding ; however , an active bleeding site was not identified . a significant amount of blood was seen in the patient 's mouth on intubation , and suctioning through a nasogastric tube yielded 200 ml of coffee ground contents. an extensive workup was done in an attempt to discern the etiology of his hemorrhagic lung disease . tests included those for autoimmune diseases , acquired hemolytic anemias , microbial infection and coagulation disorders . a cold agglutinin titer was reported positive ; however , a coomb 's test was negative . rheumatoid factor and c - reactive protein levels were elevated at 23 u / l ( < 20 u / l ) and 44.67 mg / dl ( 03 mg / dl ) , respectively . he tested negative for anti - neutrophil cytoplasmic antibodies ( ancas ) and anti - gbm antibodies . one of two blood cultures revealed gram - positive cocci in clusters , later identified as oxacillin - resistant staphylococcus hominis . the patient 's international normalized ratio remained slightly elevated at around 1.4 during his stay , with an unknown etiology . blood was consequently sent for platelet electron microscopy study , which revealed platelets with an average of 2.79 delta granules per platelet ( 46 delta granules / platelet ) consistent with delta granule storage pool deficiency . the patient 's condition continued to deteriorate during his 14-day hospital stay , with worsening anemia requiring receipt of multiple blood products , worsening oxygenation refractory to increased fraction of inspired oxygen ( fio2 ) , hypertension , hypernatremia , hypocalcemia , hypermagnesemia , blood urea nitrogen and creatinine reaching as high as 35 mmol / l ( 2.98.2 mmol / l ) and 230 mol / l ( 831 mol / l ) , respectively , indicating kidney failure , and alanine amino transferase ( alt or serum glutamic pyruvic transaminase ) and aspartate amino transferase ( ast or serum glutamic oxaloacetic transaminase ) of 371 u / l ( 436 u / l at 37c ) and 173 u / l ( 833 u / l at 37c ) , respectively , indicating liver failure . still , the patient developed progressive lung consolidation bilaterally , persistent hypoxia and recurrence of a significant output of coffee ground material through his nasogastric tube . eventually , chest x - rays revealed bilateral white out of the lungs and an accumulation of right pleural fluid . a lung biopsy was considered at various points during his decline ; however , the patient was never well enough to undergo such a procedure . by the 14th day in the hospital , the patient was oozing blood from multiple mucosal sites and areas of needle sticks , and the patient was deemed critically ill with a poor prognosis due to worsening kidney failure and anemia . sections of the lungs revealed extensive intra - alveolar hemorrhage , edema , hemosiderin - laden macrophages , focal neutrophilic infiltration , wide - spread chronic inflammation and total disruption of the normal lung architecture ( figures 1 and 2 ) . microscopic examination of the kidneys revealed extensive tubular cell nuclei dropout , consistent with acute tubular necrosis , which was caused by his persistent hypoxia secondary to his respiratory failure , and mesangial cell proliferation in the glomeruli , fibrin in the bowman spaces and occasional early crescent formation secondary to inflammatory destruction of the basement membranes in the renal glomeruli . the most significant study involved immunofluorescence , which revealed focal faint linear immunoglobulin g ( igg ) deposits along the alveolar septa and strong extensive peripheral linear igg deposits in the capillary loops of the renal glomeruli ( figures 3 and 4 ) . 3.(a ) low - power photomicrograph of linear igg deposits in alveolar septa on immunofluorescence in a lung tissue section . ( b ) high - power photomicrograph of linear igg deposits in alveolar septa on immunofluorescence in a lung tissue section . 4.photomicrograph of linear igg deposits along the gbm of a kidney tissue section shown via immunofluorescence . ( a ) low - power photomicrograph of linear igg deposits in alveolar septa on immunofluorescence in a lung tissue section . ( b ) high - power photomicrograph of linear igg deposits in alveolar septa on immunofluorescence in a lung tissue section . photomicrograph of linear igg deposits along the gbm of a kidney tissue section shown via immunofluorescence . goodpasture syndrome is a rare autoimmune disorder characterized by glomerulonephritis , hemoptysis and autoantibodies directed against a non - collagenous domain of the 3 chain of collagen type iv present in a few tissue basement membranes . if not diagnosed and treated early , patients with goodpasture syndrome may develop respiratory and renal failure and die , or become dependent on dialysis if they survive . many clinicians diagnose this syndrome with a variety of assays used to detect serum anti - gbm antibodies . renal and lung tissue biopsies are also viewed as valuable in making the diagnosis , as the anti - gbm antibodies may be detected in these specimens by immunohistochemistry or immunofluorescence techniques . a linear pattern of immunoglobulin along the basement membrane of renal glomerular capillaries and alveolar septa seen by immunofluorescence or immunohistochemistry is consistent with goodpasture syndrome . the latter diagnostic tests are important in suspected clinical cases in which the serum anti - gbm antibody test is negative , as up to 15% of patients with goodpasture syndrome may have no detectable circulating antibodies . this was the case in our patient , in which his repeated anti - gbm antibody tests came back negative ; however , the presence of these antibodies was confirmed by immunofluorescence in lung and renal tissue sections . further interesting findings in our patient , which to our knowledge have not been reported with any other case of goodpasture syndrome , were those of delta granule pool storage deficiency and eosinophilia . there are at least a few cases of goodpasture syndrome to our knowledge that have been reported to occur in the context of negative anti - gbm serum results . reported a case of a 55-year - old woman who presented with dyspnea and hemoptysis , bronchoscopy revealing diffuse alveolar hemorrhage , negative anti - gbm antibody by enzyme - linked immunosorbant assay , positive perinuclear anca at a dilution of 1:160 and glomerular crescents in renal biopsies under light microscopy . immunofluorescence also demonstrated weak linear staining of the gbms with anti - complement 3 ( anti - c3 ) sera and strong linear staining with anti - igg sera . another case of anti - gbm serum antibody - negative goodpasture syndrome reportedly occurred in a 44-year - old female with a 3 year history of recurrent hemoptysis and shortness of breath , yet clinically normal renal function . initially , she had been treated for klebsiella pneumoniae after a positive sputum culture . on repeat presentation with hemoptysis during this period , her illness had been labeled as recurrent acute respiratory distress syndrome . on her fourth presentation with hemoptysis , other test results included a positive rheumatoid factor , autoantibodies , weakly positive anti - double stranded deoxyribonucleic acid negative cytoplasmic - anca and extensive ground glass appearance on a chest computed tomography scan . lung biopsies showed linear deposition of igg in the alveolar capillary basement membranes using immunoperoxidase staining , and linear igg and c3 were seen along the renal capillary walls with immunofluorescence . finally , a case of a 27-year - old male with goodpasture syndrome and undetectable anti - gbm circulating antibodies was reported by hellmann et al . this patient , similar to our patient , developed dyspnea and hemoptysis , which eventually led to respiratory failure requiring intubation and mechanical ventilation . also like our patient , he was a heavy smoker and tested negative for ancas , anti - nuclear antibodies and a multitude of other autoantibodies . his disease was confirmed on histological lung section by the presence of a faint but linear deposition of igg antibodies along the alveolar basement membranes . several proposed mechanisms for why serum anti - gbm tests may be negative in some patients with goodpasture syndrome have been offered . one proposed explanation is that the antibodies are of high affinity and are removed from the patient 's plasma by being bound to the kidney and alveolar basement membranes . on the contrary , the circulating serum antibodies may be of lower affinity than those that are tissue bound and thus are more easily washed away during the wash - out step of the assay . finally , the assay 's renal tissue may have a different epitope than that present in the native kidney [ 1 , 7 ] . for patients who present promptly with hemoptysis and subsequently receive steroid therapy early in the disease process , negative anti - gbm serum results along with only focal linear deposits in tissues may be due to steroid - induced immune suppression . two unusual aspects of our patient 's case , unknown to us to have occurred concomitantly in other patients with goodpasture syndrome , include delta granule pool storage deficiency and eosinophilia . delta granule pool storage deficiency is a heterogeneous disorder presenting with abnormalities in the second wave of platelet aggregation , a bleeding tendency and a decreased number of delta granules in platelets . most commonly , patients experience mucocutaneous hemorrhage , excessive bleeding postoperatively and postpartum , epistaxis and easy bruising . no evidence of parasites was found during the autopsy or in microscopic examination of tissues , nor ova or parasites detected in stool samples . also , no vasculitis was observed in tissue samples on histologic examination . in an article published in the cleveland clinic journal of medicine on diffuse alveolar hemorrhage , eosinophilia is said to be rare or mild in goodpasture syndrome ; however , the parameters of mild are not defined . still , it is possible that his eosinophilia was the result of an allergy developed from his reported unsanitary living conditions , which included an abundance of trash , dead mice and black mold . the diffuse pulmonary hemorrhage associated with goodpasture syndrome occurs almost exclusively in active smokers , and is a postulated cause of unmasking the epitope which elicits the formation of the anti - gbm autoantibodies . thus , his 33 pack - year smoking history put him at risk of developing this autoimmune disease , as well as increased his risk of diffuse pulmonary alveolar hemorrhage . additionally , his platelet disorder likely worsened his hemorrhagic lung condition by increasing his likelihood to bleed excessively . finally , his negative anti - gbm antibody serum result and rapidly progressive condition prevented the ability to attain a lung or renal biopsy to establish the etiology of his disease , and thus , the correct treatment could not be determined . this case demonstrates the difficulty in diagnosing goodpasture syndrome in an unusual patient in whom the anti - gbm antibody serum results are negative and a biopsy is unattainable , and the grave consequences in missing such a diagnosis . | goodpasture syndrome is a rare , life - threatening autoimmune disease characterized by a triad of rapidly progressive glomerulonephritis , a hemorrhagic pulmonary condition and the presence of anti - glomerular basement membrane ( anti - gbm ) antibodies .
the antibodies initiate destruction of the kidney glomeruli , resulting in a focal necrotizing glomerulitis , which may progress rapidly to renal failure .
autoantibody - mediated damage of alveolar basement membranes leads to diffuse pulmonary hemorrhage , which in some cases may be severe enough to cause respiratory failure .
many clinicians use a variety of assays to detect serum anti - gbm antibodies ; however , these tests may be falsely negative in up to 15% of patients with goodpasture syndrome . here
, we report an unusual case of a 40-year - old man with clinical evidence of goodpasture syndrome , a negative anti - gbm antibody serum result , eosinophilia and delta granule pool storage deficiency .
after a 14-day hospital stay and extensive workup , as well as treatment with antibiotics , steroids and ventilator support for respiratory failure , the patient continued to deteriorate and entered multisystem organ failure .
the family decided to withdraw ventilator support , and the patient expired .
immunofluorescence testing for anti - gbm autoantibodies on lung and kidney tissues during an autopsy confirmed the diagnosis of goodpasture syndrome . |
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a 33-year - old man with ebstein anomaly and klinefelter syndrome was referred to severance cardiovascular hospital for the surgical treatment of severe tricuspid regurgitation ( tr ) . the patient underwent surgical repair of carpentier type b ebstein anomaly when he was nine years old using the danielson technique , which involves transversal plication of the atrialized right ventricle ( rv ) and anterior tricuspid annuloplasty . echocardiography showed the typical features of ebstein anomaly , but the patient also had a completely obliterated atrialized portion of the rv and reduced true tricuspid annulus size . the septal and posterior leaflets were displaced into the rv and were attached to the ventricular wall . although the leading edge of the anterior leaflet was mobile , its basal portion was tethered to the right ventricular wall . although the patient was relatively asymptomatic , surgical repair was indicated for severe tr and right atrial dilatation . the operation was performed through a redo median sternotomy . under cardiopulmonary bypass and cardioplegic myocardial protection we noted that the atrialized rv had been horizontally obliterated in the previous operation , and that the stitches for the circumferential plication annuloplasty had been placed along the anterior annulus of the tricuspid valve . the posterior and septal leaflets were displaced to the ventricular apex and adhered to the underlying myocardium . the anterior leaflet was large , but partially tethered to the ventricular wall . we performed a cone reconstruction , including complete surgical delamination and recruitment of all undelaminated leaflet tissues . the antero - inferior portion of the anterior leaflet and the posterior leaflet were detached from the annulus and their anomalous attachment to the rv as a single piece . the abnormal muscular bands and tissues between the leaflets and the ventricular wall were then divided in order to mobilize each leaflet . the septal leaflet was carefully delaminated and mobilized , and a leaflet cone was constructed using the mobilized tissue and subsequently attached to the true annulus . post - repair echocardiography revealed edge - to - edge apposition of the newly constructed tricuspid valve at the true annulus level , and the tr was measured as trivial with no stenosis . the postoperative course was uneventful , and the patient was discharged on postoperative day seven . an echocardiogram performed 15 months after the operation revealed a competent tricuspid valve without any deterioration of its function ( fig . she had previously been diagnosed with ebstein anomaly and underwent surgical repair using the carpentier method at the age of 27 years at another hospital . holter monitoring showed a normal sinus rhythm with symptomatic paroxysmal atrial flutter and frequent premature ventricular complexes . although the anterior leaflet was large and mobile , severe tr took place due to failed coaptation of the leaflets . the right atrium and we noted that the atrialized portion of the rv had been plicated vertically in her previous operation . a suture line for the previous anterior leaflet reattachment was noted on the antero - inferior portion of the tricuspid annulus . the anterior leaflet was large and mobile , while the posterior leaflet was incompletely mobilized from the ventricle . after the performance of the maze procedure with cryoablation and tricuspid valve repair , the anterior leaflet was detached from the annulus , and the posterior leaflet was surgically delaminated from the myocardium . although the septal edge of the anterior leaflet was able to be mobilized , the septal leaflet was too small to be delaminated . a leaflet cone was constructed using the available leaflet tissue and reattached to the true tricuspid annulus . although the inflow velocity through the tricuspid valve mildly increased , a bidirectional cavopulmonary shunt was not placed because the severity of the tricuspid stenosis was mild , with a mean pressure gradient of 3 mmhg . she was extubated 17 hours after the operation and discharged in good condition on postoperative day 16 . she was followed up 14 months later , and was found to be in nyha functional class i at that time . follow - up holter monitoring revealed a normal sinus rhythm with asymptomatic premature atrial and ventricular contractions , and no evidence of atrial tachyarrhythmia was observed . a 33-year - old man with ebstein anomaly and klinefelter syndrome was referred to severance cardiovascular hospital for the surgical treatment of severe tricuspid regurgitation ( tr ) . the patient underwent surgical repair of carpentier type b ebstein anomaly when he was nine years old using the danielson technique , which involves transversal plication of the atrialized right ventricle ( rv ) and anterior tricuspid annuloplasty . echocardiography showed the typical features of ebstein anomaly , but the patient also had a completely obliterated atrialized portion of the rv and reduced true tricuspid annulus size . the septal and posterior leaflets were displaced into the rv and were attached to the ventricular wall . although the leading edge of the anterior leaflet was mobile , its basal portion was tethered to the right ventricular wall . although the patient was relatively asymptomatic , surgical repair was indicated for severe tr and right atrial dilatation . the operation was performed through a redo median sternotomy . under cardiopulmonary bypass and cardioplegic myocardial protection we noted that the atrialized rv had been horizontally obliterated in the previous operation , and that the stitches for the circumferential plication annuloplasty had been placed along the anterior annulus of the tricuspid valve . the posterior and septal leaflets were displaced to the ventricular apex and adhered to the underlying myocardium . the anterior leaflet was large , but partially tethered to the ventricular wall . we performed a cone reconstruction , including complete surgical delamination and recruitment of all undelaminated leaflet tissues . the antero - inferior portion of the anterior leaflet and the posterior leaflet were detached from the annulus and their anomalous attachment to the rv as a single piece . the abnormal muscular bands and tissues between the leaflets and the ventricular wall were then divided in order to mobilize each leaflet . the septal leaflet was carefully delaminated and mobilized , and a leaflet cone was constructed using the mobilized tissue and subsequently attached to the true annulus . post - repair echocardiography revealed edge - to - edge apposition of the newly constructed tricuspid valve at the true annulus level , and the tr was measured as trivial with no stenosis . the postoperative course was uneventful , and the patient was discharged on postoperative day seven . an echocardiogram performed 15 months after the operation revealed a competent tricuspid valve without any deterioration of its function ( fig . she had previously been diagnosed with ebstein anomaly and underwent surgical repair using the carpentier method at the age of 27 years at another hospital . holter monitoring showed a normal sinus rhythm with symptomatic paroxysmal atrial flutter and frequent premature ventricular complexes . although the anterior leaflet was large and mobile , severe tr took place due to failed coaptation of the leaflets . the right atrium and we noted that the atrialized portion of the rv had been plicated vertically in her previous operation . a suture line for the previous anterior leaflet reattachment was noted on the antero - inferior portion of the tricuspid annulus . the anterior leaflet was large and mobile , while the posterior leaflet was incompletely mobilized from the ventricle . after the performance of the maze procedure with cryoablation and tricuspid valve repair , the anterior leaflet was detached from the annulus , and the posterior leaflet was surgically delaminated from the myocardium . although the septal edge of the anterior leaflet was able to be mobilized , the septal leaflet was too small to be delaminated . a leaflet cone was constructed using the available leaflet tissue and reattached to the true tricuspid annulus . although the inflow velocity through the tricuspid valve mildly increased , a bidirectional cavopulmonary shunt was not placed because the severity of the tricuspid stenosis was mild , with a mean pressure gradient of 3 mmhg . she was extubated 17 hours after the operation and discharged in good condition on postoperative day 16 . she was followed up 14 months later , and was found to be in nyha functional class i at that time . follow - up holter monitoring revealed a normal sinus rhythm with asymptomatic premature atrial and ventricular contractions , and no evidence of atrial tachyarrhythmia was observed . the anatomic and functional features of the anomaly cause tr , which ultimately results in the dilation of the right atrium and ventricle and provides a substrate for the development of atrial and ventricular arrhythmias . various surgical techniques have been developed to repair ebstein anomaly , and most techniques use a large anterior leaflet as a functional monocusp valve to achieve tricuspid valve competence [ 24 ] . in contrast , the cone repair technique creates a cone - like valve structure that allows leaflet - to - leaflet coaptation , resembling the normal tricuspid valve . this procedure has been clinically shown to lead to lower morbidity and mortality , with a low incidence of tricuspid valve replacement or reoperation . the most important part of the procedure involves the surgical delamination of the available leaflet tissue . in case 1 , the posterior and septal leaflets were successfully delaminated and incorporated into the leaflet cone to build a competent and non - stenotic valve . in case 2 , the anterior leaflet was large and mobile , while the septal leaflet was too small to be incorporated into the cone structure . it may be assumed that the lack of adequate leaflet tissue was the reason for the mild post - repair tricuspid stenosis . although no surgical treatment was necessary for tricuspid stenosis in this patient , a bidirectional cavopulmonary shunt can alleviate significant stenosis by reducing systemic venous return into the right atrium . another major aspect of the cone repair procedure is the vertical plication of the atrial portion of the rv to obliterate the atrialized rv space and to reduce the tricuspid annulus size . in our cases , this procedure was not performed , because the atrialized portion of the rv had been completely obliterated by the transverse and vertical plication method used in the previous operations . moreover , no further annular plication was required due to the aggressive annular size reduction performed in the previous operations . the scar tissue at the tricuspid annulus from the previous repair provided a firm and safe base to reattach the newly - created leaflet cone . the feasibility of the cone technique for tricuspid valve re - repair has already been shown by dearani et al . . in the present cases , the cone technique for tricuspid valve repair was successfully performed , and postoperative echocardiography confirmed the presence of competent tricuspid valves at the true annulus level . therefore , both patients were able to avoid valve replacement , and their nyha functional classes improved . furthermore , no deterioration of tricuspid valve function was observed in either case over a year of follow - up . in conclusion , the cone repair technique is useful for reconstructing a competent tricuspid valve in patients with ebstein anomaly , and is feasible even in patients who have undergone previous tricuspid valve repair . | the management of recurrent tricuspid regurgitation after tricuspid valve repair in patients with ebstein anomaly is difficult , and tricuspid valve replacement is most commonly performed in such patients .
we report two cases of recurrent tricuspid regurgitation in patients with ebstein anomaly that were successfully re - repaired using the cone technique .
the cone repair technique is a useful surgical method for reconstructing a competent tricuspid valve , and can be applied in patients who have undergone previous tricuspid valve repair . |
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it has been reported that thiopurines are effective at maintaining remission in steroid - dependent or steroid - refractory cd and uc and have steroid - sparing effect.123 they have also shown efficacy in preventing postoperative recurrence of cd.45 although their clinical implications are limited , thiopurines remain a mainstay of treatment of patients with ibd in the biologic era . however , possible toxicities such as bone marrow suppression , hepatotoxicity , and increased risk of opportunistic infections and malignancies interfere with the use of these potent drugs in clinical practice . at present , there exist several questions regarding the optimization of thiopurine therapy . to balance the efficacy with safety and tolerability , thiopurine s - methyltransferase ( tpmt ) pretreatment screening , prudent dose escalation , and careful monitoring using novel biomarkers have been suggested.678 however , most of the current guidelines for thiopurine use in ibd have been established based on western data . it has been widely accepted that notable differences in the context of epidemiology , genetics , and clinical characteristics exist between western countries and east asia , including korea.910 therefore , there is a definite need to establish management strategies that are more suitable for korean patients . for this reason , the korean association for the study of intestinal diseases ( kasid ) developed a set of consensus statements on thiopurine use in ibd . the aim of these statements is to offer guidance to clinicians on the appropriate use of thiopurines in the management of ibd . the committee for the development of consensus statements comprised five members of kasid ( working group ) . the committee conducted extensive literature review regarding thiopurine use in ibd and developed initial statements . the statements consisted of four parts : ( 1 ) pre - treatment evaluation and management strategy , ( 2 ) treatment with thiopurines , ( 3 ) safety of thiopurines , and ( 4 ) monitoring of side effects or efficacy of therapy . the quality of evidence and classification of recommendation was assessed according to the grading of recommendation assessment , development and evaluation ( grade ) system.11 in this system , the quality of evidence of each statement was categorized as high , moderate , low , or very low . evidence based on randomized controlled trials was initially classified as high - quality evidence , but the rating could be downgraded for several reasons including study limitations , inconsistency of results , indirectness of evidence , imprecision , and reporting bias . although data from observational studies ( e.g. , cohort and case - control studies ) were initially classified as low - quality evidence , the rating could be upgraded if the magnitude of the treatment effect is very large , if there is evidence of a dose - response relation , or if all plausible biases were found to decrease the magnitude of an apparent treatment effect.1112 based on the grade system , the strength of recommendations was classified as strong or weak , determined by four key factors including the balance between the desirable and undesirable effects , quality of evidence , values and preferences , and costs ( resource allocation).13 the definitions of the levels of evidence and recommendations are summarized in table 1 . prior to the consensus meeting , consensus statements were circulated to academic gastroenterologists with experience in managing patients with ibd and were revised in accordance with their comments and opinions regarding each statement . the consensus meeting was held on april 18 , 2015 in seoul , korea , wherein 38 ibd experts participated . during the meeting , the working group presented statements and all participants voted on their level of agreement regarding each statement . a consensus statement was accepted if at least 75% of participants voted 1 ( strongly agree ) or 2 ( agree ) on a scale of 1 to 5 ( with 3 , 4 , and 5 indicating uncertain , disagree , and strongly disagree , respectively ) . if a statement was not accepted , the wording of the statement was discussed and revised , and then re - voting was conducted . thiopurine - induced leukopenia is more common in east asians , namely , chinese , japanese , and koreans , compared to caucasians . quality of evidence : moderate classification of recommendation : strong level of agreement : strongly agree 55% , agree 40% , uncertain 5% thiopurines have been widely used for the treatment of patients with ibd . recommended dosages for the treatment of ibd are 2.0 - 2.5 mg / kg / day for aza and 1.0 - 1.5 mg / kg / day for 6-mp.67 however , one of the major drawbacks of the use of thiopurines is the development of leukopenia ( defined as white blood cell [ wbc ] count < 3,000/mm ) , occurring in up to 5% of caucasian patients with ibd treated with these agents.141516 interestingly , the occurrence of thiopurine - induced leukopenia is considerably high in asians.171819202122 in a study by lee et al.,17 thiopurine - induced leukopenia was observed in 116 ( 31.2% ) of 372 korean patients with cd at a median aza dose of 1.34 mg / kg / day . in a study by yang et al.,18 leukopenia was noted in 346 ( 35.4% ) of 978 korean patients with cd at a median aza dose of 1.70 mg / kg / day . in another korean multicenter study,19 leukopenia developed in 110 ( 39.6% ) of 278 patients with ibd at a mean aza dose of 1.80 mg / kg / day . in two japanese ibd cohorts with wild - type tpmt,2021 leukopenia was observed in 18 ( 15.8% ) of 114 patients and 7 ( 10.0% ) of 70 patients , although the total daily dose of aza was only 50 mg in the majority of patients . in a study by fangbin et al.,22 leukopenia ( wbc < 3,500/mm ) was noted in 36 ( 18.1% ) of 199 chinese patients with ibd . since all these east - asian studies used doses lower than the recommended dosage of aza , more frequent and severe leukopenia is expected with standard doses of thiopurines in this population . assessment of the tpmt genotype or enzyme activity before initiating thiopurine therapy is of limited value in east - asian populations compared to that in caucasians . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 29% , agree 55% , uncertain 16% aza and 6-mp are metabolized by tpmt . tpmt mutations that result in decreased enzymatic activity are associated with a greater risk of thiopurine - induced leukopenia.23242526 therefore , in order to mitigate the risk of leukopenia with thiopurines , the us food and drug administration recommends an assessment of tpmt status prior to initiating therapy.7 however , only about a quarter of ibd patients with thiopurine - induced leukopenia carry a tpmt mutation , thus raising questions on the utility of tpmt pre - testing.272829 moreover , while the frequency of tpmt mutations is lower in asians ( 1 - 3%)1819202122 than that in caucasians ( ~10%),30 the occurrence of thiopurine - induced leukopenia in asians is considerably high , as described in the statement 1.171819202122 consequently , tpmt mutations are present in only 0 to 5.6% of asian patients with ibd who develop thiopurine - induced leukopenia.1819202122 in addition , homozygotes for tpmt mutant alleles are extremely rare ( ~0.01% ) in the japanese population 20 whereas their frequency is about 0.6% among caucasians.31 taken together , assessment of tpmt genotype / phenotype prior to thiopurine therapy is of limited value in east - asian populations , when compared with caucasians . gradual increase in the dosage of thiopurines over several months until the target dose is usually preferable to beginning the medication at the target dose at the onset , in east asians . quality of evidence : low classification of recommendation : weak level of agreement : strongly agree 58% , agree 42% the recommend target dose of aza and 6-mp for ibd treatment is 2.0 - 2.5 mg / kg / day and 1 - 1.5 mg / kg / day , respectively , if the patients can tolerate the drugs.67 however , the best approach to attain the target dose has not been established yet . the traditional " dose escalating approach " is to initiate the therapy at 50 mg daily , then increase the dose by 25 mg every 1 - 2 weeks to a target dose along with monitoring for leukopenia and other potential adverse events.7 the main concern regarding this approach is under - dosing of patients with a suboptimal response rate . an alternate approach is to start immediately at the full standard dose based on tpmt activity . according to this approach , patients with normal tpmt activity receive the standard dose while the patients with intermediate tpmt activity receive 50% of the standard dose . however , patients with low tpmt activity ( 0.3% of general population ) should not be treated with thiopurines.32 interestingly , a questionnaire - based survey showed that 33% of us gastroenterologists initiated therapy at 50 mg of aza , while 28% administered a full dose of aza at 2.5 mg / kg / day , in clinical practice.33 interestingly , the usual approach to prescribing thiopurines in east asia is to start at a low dose and to gradually increase the dose.34353637 according to a survey on clinical practice patterns in the treatment of ibd in korea , 80% of the responders initiated aza at 50 mg / day , 68% increased the dose by 25 mg , and 56% increased the dose every 4 weeks.34 this gradual dose increment policy could be useful for reducing aza - induced myelotoxicity.35 however , it may delay the time to clinical response . in china , a dose step - up strategy is typically carried out by gradually increasing the dose of aza to the target dose under close monitoring of laboratory results and clinical response.36 in japan , lower doses of aza ( 50 mg / day or 0.6 - 1.2 mg / kg / day ) are employed for the treatment of patients with ibd.2037 unlike western data , several studies have suggested that lower doses of aza or 6-mp may be effective and safe for the treatment of patients with ibd in east asia.17363738 however , these studies did not compare the efficacies of lower doses with those of standard doses ; moreover , they did not analyze the 6-thioguanine nucleotide ( 6-tgn ) levels . nevertheless , considering higher incidences of myelotoxicity 171819202122 and limited value of the assessment of tpmt genotypes,1920212227282939 the dose escalating approach starting with low doses can be an effective and safe strategy in east asians . determination of nudt15 genotypes before initiating thiopurine therapy may identify patients with a predisposition to the development of thiopurine - induced early leukopenia . quality of evidence : moderate classification of recommendation : strong level of agreement : strongly agree 13% , agree 76% , uncertain 11% as described in statement 2 , assessment of the tpmt genotype or enzyme activity may play a limited role in the prevention of thiopurine - induced leukopenia in east asians . recently , a non - synonymous single nucleotide polymorphism in nudt15 variant was identified as a significant risk factor for thiopurine - induced early leukopenia in korean patients with cd.18 the nudt15 allele encoding p.arg139cys was found in 89.4% of the early leukopenia cases but was found in only 6.8% of the controls , suggesting that the presence of the nudt15 allele had a sensitivity of 89.4% and specificity of 93.2% to early leucopenia . although rare , this single nucleotide polymorphism was also associated with thiopurine - induced leukopenia in caucasians.18 the frequency of nudt15 risk allele is much higher in east asians than that in caucasians ( 10.4% in koreans , 7% in japanese , 13% in chinese , and 2% in an admixed american population).18 the results of this study were reproduced by a study in children with acute lymphoblastic leukemia conducted in the united states.39 therefore , pre - treatment determination of nudt15 genotypes may help to identify patients susceptible to thiopurine - induced early leukopenia in diverse populations , especially in east asians . there is not enough evidence to recommend thiopurine monotherapy for induction of remission in active cd or uc . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 42% , agree 53% , uncertain 5% the data for thiopurines for remission induction in active uc is limited.4041 in a meta - analysis of 4 randomized controlled trials ( n=89 ) evaluating the efficacy of aza/6-mp for the induction of clinical remission of uc , the mean efficacy of aza was not superior to placebo or 5-aminosalicylate ( 75% vs. 64% , respectively ; or , 1.59 ; 95% ci , 0.59 - 4.29 ; p=0.13).41 thiopurines may be effective for inducing remission in active cd as compared to placebo ( or , 2.43 ; 95% ci , 1.62 - 3.64).42 however , a recent cochrane meta - analysis including 11 randomized trials reported that thiopurines offered no significant advantage over placebo for induction of clinical remission in active cd ( 48% vs. 37% , respectively ; rr , 1.23 ; 95% ci , 0.97 - 1.55).43 usually , thiopurines have been limited in the use of remission induction in active cd or uc because of the delay in the onset of their action.44 thiopurines may take 3 - 6 months to achieve full clinical efficacy.45 therefore , in patients with active cd or uc requiring rapid symptom relief , concomitant therapy with systemic corticosteroids and thiopurines is a more reasonable approach . however , the addition of thiopurines to corticosteroids makes no benefit in terms of remission induction.44 thiopurines have the benefit in corticosteroid sparing effect for active cd with a pooled or of 3.69 ( 95% ci , 2.12 - 6.42).42 thiopurines are effective for the maintenance of remission in patients with cd and uc . quality of evidence : high classification of recommendation : strong level of agreement : strongly agree 66% , agree 32% , uncertain 2% thiopurines are effective for maintenance of remission in both uc and cd.23 aza appears to be more effective than placebo for the maintenance of remission in uc.3 according to a meta - analysis of 4 studies including 232 patients with uc , 44% ( 51/115 ) of patients administered aza failed to maintain remission compared to 65% ( 76/117 ) of placebo - administered patients ( rr , 0.68 ; 95% ci , 0.54 - 0.86).3 aza or 6-mp may be effective as maintenance therapy in patients with uc who have failed to respond or can not tolerate 5- aminosalicylate and in patients who require repeated courses of corticosteroids . in case of patients with cd , aza or 6-mp also had a positive effect in maintaining remission.124647 a meta - analysis of 8 studies including 503 patients with cd revealed that the overall remission rate was 71% ( 147/208 ; 95% ci , 64 - 77% ) for aza treatment compared to that for placebo ( 55% [ 141/255 ] ; 95% ci , 49 - 61%).2 aza ( 1.0 - 2.5 mg / kg / day ) is effective in reducing the risk of disease recurrence over a 6-month to 2-year period . higher doses of aza ( 2.5 mg / kg / day ) are more effective than the lower doses ( 1.0 or 2.0 mg / kg / day ) in preventing disease recurrence . in addition , aza has corticosteroid - sparing effects that reduce corticosteroid - related side effects . previously , a korean study also reported that complete withdrawal of corticosteroids was achieved in 70.9% of patients with cd receiving treatment with aza.17 aza or 6-mp may also benefit patients dependent on corticosteroids or in cases where corticosteroid treatment has failed . among korean patients with steroid - dependent uc , the 3-year success rate was higher in the aza therapy group ( 71.2% ) than that in the aza intolerance group ( 25.0%).38 however , withdrawals due to adverse events were more commonly observed in patients treated with aza ( peto or , 3.74 ; 95% ci , 1.48 - 9.45 ) compared to placebo.2 optimal duration of treatment with thiopurines is uncertain . prolonged or indefinite use of thiopurines may be considered to maintain remission . the risk and benefits of continuing thiopurine therapy should be balanced and discussed with individual patients . quality of evidence : low classification of recommendation : weak level of agreement : strongly agree 68% , agree 29% , uncertain 3% conclusive evidence suggests that thiopurines are effective for maintenance of remission in both cd and uc.23 well - tolerated and effective therapy is generally continued to maintain the remission of ibd . there is limited data regarding the factors predicting responses to aza and the uncertainty regarding the optimal duration of treatment.48 in patients with cd , several studies showed relapse rates of 14 - 41% at 1 year after cessation of thiopurines with a cumulative increase with time.495051 furthermore , patients with cd who discontinued aza after more than 3 years of efficacious treatment had a higher probability of relapse than those who continued therapy.52 a recent meta - analysis showed that there is a clear benefit of continuing aza/6-mp therapy for at least 18 months to maintain remission in patients with cd.53 in patients with uc , relapse rates after cessation of aza treatment were reported to be 35 - 77% and 65 - 75% at 1 and 5 years , respectively.165455 there was no difference in the relapse rates between cd and uc . the duration of aza treatment did not affect the relapse rates after cessation of treatment ( p=0.68).16 according to the european crohn 's and colitis organisation ( ecco ) guidelines,56 for patients with cd who have been treated with thiopurines as part of maintenance therapy , discontinuation may be considered after 4 years of remission . to summarize these data , thiopurines should be used for at least 18 months after achieving remission and could be continued for over 4 years . therefore , considering the high cumulative relapse rates after cessation of thiopurine treatment , risk stratification is an important issue for patients with ibd receiving thiopurines . meanwhile overall , around 10 - 28% of patients reported side effects and of which 50 - 80% discontinued the thiopurines as a result.57 in addition to risk factors for disease relapse , adverse events with long - term use such as serious infections and malignancies must be taken into account . thus , benefits and risks of continuing thiopurines should be considered and discussed with each patient individually . the combination of anti - tumor necrosis factor ( tnf)- agents and thiopurines is more effective than that of monotherapy with anti - tnf- agents or thiopurines alone in inducing remission in patients with moderate to severe cd . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 26% , agree 58% , uncertain 16% quality of evidence : high classification of recommendation : strong level of agreement : strongly agree 70% , agree 24% , uncertain 6% several studies have reported the effectiveness of combination of anti - tnf agents with thiopurines in case of rheumatic diseases and ibd . thiopurines improved the pharmacokinetics of anti - tnf agents and decreased the formation of anti - drug antibodies.58 in the sonic ( the study of biologic and immunomodulator naive patients in crohn 's disease ) trial that included immunosuppressant - naive cd patients , the corticosteroid - free remission rates and mucosal healing rates were higher in those treated with a combination of infliximab plus aza than those treated with infliximab or aza alone.59 the corticosteroid - free remission rates at week 26 were 57% in those receiving combination therapy , 44% in those receiving infliximab alone , and 30% in patients receiving aza alone . at week 50 , the corticosteroid - free remission rates were 72% , 61% , and 55% . additionally , mucosal healing rates were evaluated at week 26 and they were 44% , 30% , and 16.5% , respectively.59 in the getaid ( groupe d'etude therap - utique des affections inflammatoires du tube digestif ) study , infliximab plus aza/6-mp was found to be more effective than aza/6-mp alone for the induction of remission in patients with corticosteroid - dependent cd.60 in case of adalimumab , a recent meta - analysis study demonstrated that adalimumab monotherapy was inferior when compared to combination therapy with adalimumab plus thiopurines ( or , 0.78 ; p=0.02 ) for induction of remission in patients with cd . however , the rate of remission at 1 year and the need for dose escalation was similar in both groups.61 therefore in patients with moderate to severe cd , especially in case of immunosuppressant - naive patients with cd , a combination therapy with anti - tnf and thiopurines seems to be preferred mode of treatment in order to allow the patients to achieve the highest rate of rapid disease control and limited tissue damage . however , the benefit of combination therapy in immunosuppressant treatment - failed patients remains unclear . the combination of anti - tnf- agents and thiopurines is more effective than that of monotherapy with anti - tnf- agents or thiopurines alone in inducing remission in patients with moderate to severe uc . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 18% , agree 66% , uncertain 16% quality of evidence : high classification of recommendation : strong level of agreement : strongly agree 55% , agree 42% , disagree 3% the main question that remains to be answered in this clinical situation is whether combination therapy with infliximab and thiopurines is clearly superior to monotherapy with infliximab or thiopurines alone , in patients with uc . recently , the uc success ( efficacy and safety of infliximab and azathioprine monotherapy or in combination in moderate to severe uc ) trial was performed to provide a suitable explanation for the above question.62 this study was a randomized , double - blind clinical trial for evaluating the efficacy and safety of 16 weeks of infliximab plus aza , infliximab or aza monotherapy in patients with moderate to severe uc.62 at week 16 , a greater proportion of patients in the infliximab plus aza achieved corticosteroid - free remission ( 39.7% ) compared to 22.1% receiving infliximab alone ( p=0.017 ) and 23.7% receiving aza alone ( p=0.032 ) . mucosal healing at week 16 occurred in 62.8% of patients receiving infliximab plus aza , compared to 54.6% receiving infliximab alone ( p=0.295 ) and 36.8% receiving aza alone ( p=0.001 ) . adverse events that required discontinuation of therapy were higher in the aza alone ( 8% ) than in the infliximab alone ( 3% ) or the infliximab plus aza group ( 4% ) . although there are no high quality reports regarding the superiority of combination therapy except for the uc success trial , infliximab - based combination therapy can be more therapeutically useful in patients with moderate to severe uc who previously do not appear to respond adequately to corticosteroid therapy . the combination of adalimumab and immunomodulator appeared mildly superior to adalimumab monotherapy for the induction of remission in cd.61 however , it is still unclear in case of uc . selection between anti - tnf- monotherapy and combination therapy with an immunomodulator may be individualized based on the risk of relapse and adverse events . quality of evidence : low classification of recommendation : no level of agreement : strongly agree 32% , agree 65% , uncertain 3% as described in statements 8 and 9 , combination therapy with infliximab and thiopurines is more effective than monotherapy with either agent alone in inducing corticosteroid - free clinical remission and mucosal healing in patients with cd and uc who are naive to either agent.5962 meanwhile , the clinical benefits of continuing immunomodulators in patients with ibd who are refractory to these drugs when starting anti - tnf therapy are debatable . however , combination therapy with anti - tnf agents and immunomodulators may increase the risk of serious infections and malignancies . in a study with longitudinal cohort of 8,581 cd patients,63 monotherapy with steroids , immunomodulators , or anti - tnf agents was associated with an increased risk of tuberculosis , candidiasis , herpes zoster , and sepsis as compared to patients not receiving these medications . in contrast , according to the treat ( crohn 's therapy , resource , evaluation and assessment tool ) registry,64 cd severity and use of prednisone or narcotic analgesics showed higher risks of serious infection , although an increased risk was also observed with infliximab . moreover , treatment with immunomodulators including thiopurines and methotrexate was not a significant predictor of serious infection , and combination therapy with infliximab and immunomodulators did not increase the risk of infection , compared with infliximab monotherapy . therefore , relationship between combination therapy with infliximab and immunomodulators and development of serious infections remains unclear . the risk of neoplasm in ibd patients receiving anti - tnf therapy is controversial.6566 the combination of anti - tnf agents and immunomodulators is associated with an increased risk of malignancies such as lymphoma and non - melanoma skin cancer.676869 the risks of these cancers are largely driven by the use of concomitant immunomodulators , particularly thiopurines.70 in order to decide between the combination therapy versus monotherapy approach , we should weigh the increased risk of complications induced by combination therapy against the increased risk of relapse observed with monotherapy . according to a decision analytic model , the benefits of combination therapy with infliximab and aza would outweigh the risks in cd patients who are naive to either agent , unless serious infections occurred in 20% or more of the population or lymphoma in 3.9% or more.71 old age ( > 65 years ) is associated with a greater risk of serious infections and lymphoproliferative disorder.7273 hepatosplenic t - cell lymphoma usually occurs in young men ( < 35 years ) receiving thiopurine therapy for more than 2 years.74 factors associated with the risk of relapse after cessation of concomitant immunomodulators include increased inflammatory markers , evidence of mucosal activity on endoscopy , short duration in remission before stopping therapy , and undetectable trough levels of anti - tnf.70 therefore , withdrawal of concomitant immunomodulators may be considered in patients in deep remission for a considerable period and with detectable anti - tnf trough levels , especially in elderly patients or young male patients . currently , there is no consensus on the duration of combination therapy with an anti - tnf agent and an immunomodulator . use of thiopurines is associated with an increased risk of lymphoma and non - melanoma skin cancers . quality of evidence : moderate classification of recommendation : strong level of agreement : strongly agree 51% , agree 49% according to a recent meta - analysis by kotlyar et al.,75 the overall standardized incidence ratio ( sir ) for lymphoma was 4.92 ( 95% ci , 3.10 - 7.78 ) in patients with ibd exposed to thiopurines : 2.80 ( 95% ci , 1.82 - 4.32 ) in 8 population studies and 9.24 ( 95% ci , 4.69 - 18.2 ) in 10 referral studies . in 8 population studies , an increased risk was noted among current users ( sir , 5.71 ; 95% ci , 3.72 - 10.1 ) but not among former users ( sir , 1.42 ; 95% ci , 0.86 - 2.34).75 young male patients ( < 30 years ) had the highest sir , and aged patients ( > 50 years ) showed the highest absolute risk.75 according to a recent meta - analysis involving 60,351 ibd patients , the pooled adjusted hazards ratio of developing non - melanoma skin cancers after exposure to thiopurines was 2.28 ( 95 % ci , 1.50 - 3.45).76 however , the results of this meta - analysis must be interpreted carefully owing to a marked heterogeneity between studies . there is not enough evidence to suggest that the modestly increased risk of lymphoma and non - melanoma skin cancers outweighs the benefit of thiopurines in ibd.6776 use of thiopurines is not associated with an increased risk of postoperative complications . quality of evidence : low classification of recommendation : weak level of agreement : strongly agree 30% , agree 67% , uncertain 3% postoperative complications in ibd include abdominal sepsis , wound problem , anastomotic leakage , early reoperation as well as complicated systemic diseases leading to increased morbidity and mortality . in case of cd , a study by colombel et al.77 reported that early postoperative complications did not increase in patients with cd treated perioperatively with immunosuppressive agents . however , in a study by myrelid et al.,78 preoperative thiopurine therapy was associated with postoperative intra - abdominal septic complications in case of abdominal surgery for patients with cd . as for uc , in a study by schaufler et al.,79 preoperative exposure to thiopurines was not associated with increased postoperative complications in a cohort undergoing colectomy for uc or ibd - u . recently , a meta - analysis involving 21 studies with 6,899 ibd patients evaluated whether the preoperative use of immunosuppressive agents was associated with increased postoperative complications in patients with ibd.80 of these 21 studies , 8 studies with 1,674 patients reported the impact of preoperative thiopurines on postoperative outcomes . the pooled risk ratio estimates for total complications and infectious postoperative complications were 0.97 ( 95% ci , 0.69 - 1.36 ) and 1.23 ( 95% ci , 0.66 - 2.29 ) , thus suggesting no association between preoperative use of thiopurines and postoperative complications.80 however , most studies were retrospectively designed and there were large variations in the patient populations and outcome definitions.80 thiopurines are considered to be safe and well tolerated during pregnancy . discontinuing thiopurine therapy during pregnancy may precipitate a flare resulting in adverse neonatal outcomes . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 16% , agree 71% , uncertain 13% the peak incidence of ibd occurs in women of childbearing age . management of patients with ibd during pregnancy requires a challenging balance between optimal disease control and drug safety considerations . fears about the potential harm of ibd medications to the developing fetus are very common . in a survey conducted in australia , 36.1% of participants believed that any form of ibd medication was harmful to the unborn fetus.81 however , discontinuation of therapy may result in disease relapse during pregnancy , which carries a greater risk of adverse fetal outcomes ( a higher rate of fetal loss , preterm birth , and low birth weight ) , according to a number of reports.828384858687 conflicting data exist regarding the association between thiopurine use for ibd treatment in pregnancy and adverse pregnancy outcomes . a few studies reported an increased risk of fetal loss , preterm delivery , low birth weight , and congenital abnormalities when thiopurines were used during pregnancy.888990 however , these outcomes might have been caused by the underlying disease rather than thiopurines . a majority of recently conducted controlled or cohort studies have not shown an increase in congenital abnormalities.9192939495 according to a recent pharmacological study conducted on thiopurines in pregnant patients with ibd , although unborn children were exposed in utero to the pharmacologically active thiopurine metabolites 6-tgn , no major teratogenicity was observed.96 in another study , thiopurine use during pregnancy did not affect long - term development or immune function of children up to 6 years of age.97 moreover , two recent meta - analyses did not demonstrate an increased risk of fetal loss , low birth weight , or congenital abnormalities despite intrauterine exposure to thiopurines.9899 however , an association with preterm birth was noted in one out of the two meta - analyses studies.98 in addition , 60% of newborns exposed to thiopurines in utero were anemic at birth.96 existing guidelines suggest that thiopurines should not be discontinued during pregnancy.100101102103 in a worldwide survey among 175 ibd experts , 155 ( 89% ) physicians replied that they would continue aza therapy throughout pregnancy.4 the potential risks and benefits of thiopurine therapy should be discussed with a patient , ideally prior to conception . breastfeeding could be advised for women on thiopurine maintenance therapy willing to nurse their infants . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 23% , agree 64% , uncertain 13% treatment with thiopurines is widely used to maintain remission in ibd . breastfeeding by patients with ibd patients on thiopurines is probably safe because the presence of only trace amounts of aza/6-mp metabolites has been noted in breast milk . in a prospective study , the concentration of 6-mp was measured in 31 breast milk samples collected from 10 mothers receiving aza.104 6-mp was detected in low concentrations ( 1.2 and 7.6 ng / ml , as compared to therapeutic immunomodulator level of 50 ng / ml in serum ) in two samples , but was not detected in any of the other 29 samples.104 additionally , aza/6-mp metabolites were undetectable in the neonatal blood.97104 the majority of thiopurine metabolites are excreted in milk in the first 4 hours after intake of the drug.105 hence , mothers should be counseled to use a breast pump 4 hours after medication intake to discard the first portion of milk produced after aza intake in order to minimize the infant 's exposure to the drug . breastfeeding by ibd patients undergoing thiopurine therapy did not affect long - term development or immune function . one retrospective study showed no differences in infection rate in children breastfed by mothers receiving thiopurines for ibd as compared to children breastfed by mothers without immunomodulator therapy.92 another recent observational study showed no differences in any of the global medical and psychosocial health status in 9 breastfed infants ( for median 7 months , range 3 - 13 months ) and the formula - fed group.106 thiopurines can be administered safely to women with ibd during lactation . therefore , breastfeeding should be recommended after considering its beneficial therapeutic effects for the nursing mother with ibd . appropriate counseling for lactation should be available for all lactating women with ibd in order to discuss the risks and benefits of thiopurine therapy . close follow - up of full blood counts is recommended in all patients taking thiopurines . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 28% , agree 67% , uncertain 5% thiopurines have been reported to increase the risk of myelotoxicity , particularly leukopenia . myelotoxicity is one of the more common dose - dependent adverse effects with potentially serious clinical consequences . the use of complete blood counts ( cbc ) to monitor bone marrow suppression is of particular importance.107 most cases of severe leukopenia occur abruptly early on in treatment.107 in a study by lewis et al . , the incidence of severe leukopenia ( wbc < 1,000/mm ) was highest in the first 8 weeks of thiopurine therapy with the median time from onset of therapy to first documentation of severe leukopenia being 24.5 days.108 there is currently no clear consensus on optimal frequency of blood monitoring.109 several monitoring guidelines have been proposed.15107110111 a western study stated that a cbc should be obtained weekly for one month , biweekly for the second month , monthly for four months and , bimonthly when the patient is stable after six months of treatment . although further studies are needed in order to better define the optimal interval of cbc monitoring , it may be scheduled biweekly for the first two months , and then every 4 - 12 weeks thereafter , in general . the dose adjustment of thiopurines through monitoring of 6-tgn and 6-methylmercaptopurine ( 6-mmp ) levels is expected to improve efficacy and reduce side effects in patients treated with thiopurines . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 18% , agree 69% , uncertain 13% the variation between clinical efficacy and side - effect profile of thiopurines is may be attributed to individual differences in drug metabolism . 6-mp and its prodrug aza are converted to 6-tgn , which is the main active metabolite responsible for therapeutic efficacy and myelotoxicity . on the other hand , metabolism of these products also yields 6-mmp , high levels of which are associated with increased hepatotoxicity . these findings have led to strategies for advantageously shifting thiopurine metabolism toward optimal 6-tgn levels while decreasing 6-mmp levels in an attempt to benefit a greater portion of patients undergoing thiopurine treatment . although the relationship between 6-tgn levels and efficacy was first described in patients with ibd in 1996,109 this correlation was confirmed in the landmark study in 2000 by dubinsky et al.,112113 which demonstrated a positive correlation of therapeutic response with 6-tgn levels in 92 pediatric patients with ibd . the frequency of therapeutic response increased at 6-tgn levels > 235 pmol/810 red blood cells ( rbc ) while hepatotoxicity correlated with elevated 6-mmp levels ( > 5700 pmol/810 rbc).113 since then , several prospective studies have reported a correlation between 6-tgn levels and clinical response.114115116117 a meta - analysis of 12 studies with 941 patients concluded that patients with 6-tgn levels above 230 - 260 pmol/810 rbc were more likely to be in remission than those below the threshold value.8 a more recent pooled analysis including 17 studies of 2,049 patients showed that the pooled or for clinical remission among patients with 6-tgn levels over a cut - off value between 230 and 260 pmol/810 rbc was 3.15.118 although the dose adjustment of thiopurines through monitoring of 6-tgn and 6-mmp levels is expected to improve efficacy and reduce side effects in patients treated with thiopurines , further studies are needed in order to confirm these findings . for decades , thiopurine agents have been a mainstay in treating ibd and will play an important role in the future . however , complex metabolism and various side effects limit their successful application in clinical practice . thiopurine - induced leukopenia is especially very common and prescreening for tpmt has a limited value in korean patients with ibd . in addition , use of novel biomarkers such as nudt15 , may help identify patients who are at a high risk for thiopurine - induced leukopenia . at present , optimal duration of thiopurine treatment in addition to long - term outcomes of combination therapy with anti - tnf agents although thiopurine treatment during pregnancy and lactation is considered safe and well tolerated , it is important to balance the risks and benefits . | background / aimsfor decades , thiopurines have been the mainstay of inflammatory bowel disease ( ibd ) treatment and will play an important role in the future . however , complex metabolism and various side effects limit the use of these potent drugs in clinical practice . the korean association for the study of intestinal diseases developed a set of consensus statements with the aim of guiding clinicians on the appropriate use of thiopurines in the management of ibd.methodssixteen statements
were initially drafted by five committee members .
the quality of evidence and classification of recommendation were assessed according to the grading of recommendations assessment , development and evaluation system .
the statements were then circulated to ibd experts in korea for review , feedback , and then finalized and accepted by voting at the consensus meeting.resultsthe consensus statements comprised four parts : ( 1 ) pre - treatment evaluation and management strategy , including value of thiopurine s - methyltransferase screening , dosing schedule , and novel biomarkers for predicting thiopurine - induced leukopenia ; ( 2 ) treatment with thiopurines with regards to optimal duration of thiopurine treatment and long - term outcomes of combination therapy with anti - tumor necrosis factors ; ( 3 ) safety of thiopurines , especially during pregnancy and lactation ; and ( 4 ) monitoring side effects or efficacy of therapy using biomarkers.conclusionsthiopurines are an effective treatment option for patients with ibd .
management decisions should be individualized according to the risk of relapse and adverse events . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
propranolol ( 1-isopropylamino-3-(1-naphthyloxy)-2-propranolol ) is a -adrenergic blocking drug belonging to class ii of antiarrhythmics that has wide application for the treatment of cardiac arrhythmia , sinus tachycardia , angina pectoris , and hypertension . due to its therapeutic and pharmacological relevance , propranolol hydrochloride has been determined in pharmaceutical preparations by different techniques , such as spectrophotometry [ 24 ] , spectrofluorimetry , electrochemical , and liquid chromatography [ 7 , 8 ] . in addition one of the pharmacopoeia reference methods for propranolol hydrochloride assay is carried out using uv spectrometry with suitable dilution of the sample in methanol . these techniques showed some drawback as the use of high amount of reagent , time consumption , and great generation of chemical residues . in the last years , continuous increase of near and mid infrared spectrometry ( nir and mid ) applications as analytical techniques for the quantification of complex mixtures has been observed [ 1021 ] . a nir spectrometry with fiber optic probe ( ftnir / probe ) allows a fast analysis of different mixtures . in addition , this technique is promising for using in process analytical technology ( pat ) because it allows on - line quantitative monitoring and fast data acquisition . nir spectrometry combined with multivariate analysis allows improving the quality of results obtained for complex mixtures by overcoming problems related to overlapped signals . partial least - squares ( pls ) regression is the most popular multivariate calibration method for quantitative analysis because it can resolve overlapping peaks bands and broad spectral bands of nir spectra [ 23 , 24 ] . additionally , some applications have proposed the use of methods for spectral region selection with suitable algorithms to improve the performance of pls regression [ 2533 ] . in practice , these methods are based on the identification of a spectrum interval that will produce the lowest prediction error . one of these algorithms is the interval partial least squares ( ipls ) consisting in the split of the spectrum in smaller intervals . subsequent , models are constructed for each of these intervals and the root mean square error of cross validation ( rmsecv ) is calculated for each model . finally , a comparison between rmsecv value of each model obtained using ipls and rmsecv of pls using full spectrum is carried out . on the other hand , in synergy interval partial least squares ( sipls ) algorithm pls regression models using combination of different intervals of the spectrum are carried out . thus , in this study , the feasibility of ftnir / probe associated with different pls regression models was evaluated for the propranolol hydrochloride determination in pharmaceutical preparation as an alternative to the recommended method by brazilian pharmacopoeia using uv assay . models using ipls and sipls algorithms were evaluated and results obtained using ftnir / probe were compared with those obtained using the recommended procedure described in official pharmacopoeias . finally some parameters such as precision , linearity , and accuracy were also evaluated . ultrapure milli - q water ( 18.2 m cm ) and analytical - grade reagents ( merck , darmstadt , germany ) were used for preparation of standards for uv analysis . propranolol hydrochloride used in preparation of pharmaceutical preparations was purchased from local pharmacy . on the other hand , propranolol hydrochloride reference material from united states pharmacopoeia thirty - three samples were prepared in laboratory through the mixture of propranolol hydrochloride reference material and excipient mixture ( 50% of carboximetilcellulose , 48% of starch , 1% of colloidal silicon dioxide , and 1% of magnesium stearate ) . the concentration of propranolol hydrochloride samples ranged from 57.6 mg g to 222.5 mg g. samples were mixed in a cryogenic mill ( spex certiprep , model 6750 freezer mill ) using two steps : ( i ) samples were frozen in liquid argon for 2 min and ( ii ) mixed / ground for 2 min in cryogenic mill . samples with particle size less than 80 m were transferred to 15 ml polypropylene flask and used for the subsequent tests . a spectrophotometer ( shimadzu , multispec-1501 ) was used as reference technique for assay of propranolol hydrochloride in pharmaceutical preparation according to brazilian pharmacopoeia monograph . sample mass containing up to 20 mg of propranolol hydrochloride was shaken with 20 ml of water during 10 minutes . then , 50 ml of methanol was added and shaken for more 10 minutes . samples were diluted up to 100 ml with methanol , homogenized , and filtered . samples were suitably diluted and analyzed by uv spectrometry at wavelength of 290 nm . calibration curve for propranolol hydrochloride was constructed with standard concentration ranging from 0.01 to 0.1 mg ml . spectra of sample in powder form were collected in the range from 10000 to 4000 cm using a nir / mid spectrometer ( spectrum 400 ftnir / mid model , perkinelmer ) with calcium fluoride beam splitter . this instrument was equipped with a nir fiber optic probe with 2-meter length ( flexir nir fiber optic accessory , pike technologies ) operated in standard diffuse reflectance sampling tip with inert sapphire window for solid samples . in addition , the nir fiber optic probe was equipped with low - noise indium - gallium - arsenide detector . the probe was placed inside the polypropylene flask containing the mixtures of propranolol hydrochloride and excipients in order to allow the contact between the sample and the infrared beam . matlab software 7.0 version ( the math works , natick , usa ) was used for constructing of pls multivariate calibration models . pls itoolbox 2.0 version was used for variables selection and constructing of multivariate models using ipls and sipls algorithms . initially , pls models for propranolol hydrochloride determination in pharmaceutical preparations by ftnir / probe were built using treatment of multiplicative scatter correction ( msc ) or first derivative with savitzky - golay filter ( d ) . autoscalling ( a ) or mean centered data ( mc ) were used as preprocessing tools for the multivariate calibration models . in order to improve the predictive ability of the models , pls regression can be carried out using selected spectral regions containing structural information of propranolol hydrochloride molecule . in this work , the selection of variables for the pls calibration was carried out using ipls and sipls . interval pls and synergy interval pls models were built using spectra division in 10 , 20 , 30 , and 40 intervals . the subinterval or the combined subintervals presenting the minor rmsecv values were selected using itoolbox software . root mean square error ( rmse ) and correlation coefficient ( r ) were used to characterize and compare predictive abilities of the developed calibration models . root mean square error of cross validation ( rmsecv ) was used to select the number of latent variables . mean square error of prediction ( rmsep ) was employed to evaluate the prediction ability of different pls models . comparison between the prediction errors of constructed models was carried out using f test ( 95% confidence level ) . in addition , accuracy was evaluated using paired t - test ( 95% confidence level ) . accuracy was also evaluated using the weighted least square ( wls ) which takes into account that the comparable errors in the both axes should fit a straight line where the intercept is not significantly different from 1 . anova ( 95% confidence level ) was used in comparison of results obtained in experiments carried out for precision evaluation . systematic error ( bias ) and standard deviation of validation ( sdv ) were calculated and systematic error was considered not significant for t systematic ( tsist ) values lower than critical value ( tcrit) = 5% and n 1 degrees of freedom . coefficient of determination better than 0.99 was obtained and relative standard deviation ( rsd ) lower than 5% was obtained for determination of propranolol hydrochloride in pharmaceutical preparations . about 300 ml of methanol was required for each sample and sample throughput of 5 samples ( in triplicate ) per hour was achieved . these samples were randomly divided into calibration set ( 24 samples ) and prediction set ( 9 samples ) . the concentration of calibration and prediction set ranged from 57.6 to 222.5 mg g and from 64.2 to 211.7 mg g of propranolol hydrochloride , respectively . calibration set was used to build the models and prediction set was used to evaluate the predictive ability of the each model . initially , pls models were built with objective to evaluate different treatments and preprocessing . in table 1 it is possible to observe the rmsecv values of the pls models obtained using all variables , different treatment , and preprocessing . the rmsecv values obtained for the different pls models showed that there were no significant differences in these values ( f test , 95% confidence level ) . moreover , the coefficients of correlation of the pls models were quite similar ( table 1 ) . although the models have not showed significant differences in the rmsecv values , the model that used msc and mean centered data ( m ) was selected for posterior construction of ipls and sipls models , because mean centered data is preferred and most common in literature for construction of models containing spectra data . different applications found in the literature have shown that the use of ipls can improve the predictive ability of the calibration models [ 26 , 27 , 29 ] . the results obtained using ipls algorithm were presented in a typical figure in order to facilitate the comparison among the rmsecv values obtained for each subinterval model and rmsecv of pls full model ( with all variables ) ( figure 2 ) . in figure 2 the rmsecv value of the pls full model was plotted in figure 2 as horizontal black line in order to facilitate the comparison with rmsecv value of each interval . in addition , models built with interval numbers 4 , 5 , 6 , 12 , 13 , 14 , 15 , 18 , and 19 showed rmsecv values lower than rmsecv value of pls full model . however , improvement in predictive ability was not observed for ipls models constructed with spectra split in 10 , 20 , 30 , and 40 intervals because significant differences ( f test , 95% confidence level ) was not observed in rmsep values obtained for this models . in order to decrease the rmsep value sipls models table 2 shows results for propranolol hydrochloride sipls calibration models . comparing the different sipls models obtained it is possible to observe that all sipls models showed lower rmsecv and rmsep values when compared with pls full model . however , significant difference was not observed when rmsep values of pls full model and rmsep values of different sipls models were compared ( f test , confidence level 95% ) . although no significant differences were observed in rmsep values for the different sipls models , model s2i20pls showed the highest decrease in rmsep value , that is , about 2 times lower than the rmsep value of pls full model . in addition , this model provided correlation coefficient of 0.990 between reference and predicted values ( figure 3 ) when spectra were divided into 20 intervals and combination of intervals numbers 5 and 16 was used . the selected intervals included regions of 8501 to 8801 cm ( interval 5 ) and of 5201 to 5501 cm ( interval 16 ) as can be viewed in figure 2 . h stretching of second overtone probably related to groups c h of aromatic ring and ch3 that are present in propranolol hydrochloride structure . h stretching of first overtone and oh stretching + 2x c o stretching related to structure present in propranolol hydrochloride structure . systematic error calculated for the s2i20pls model was not significant ( bias = 3.1 and tsist < tcrit ) . the individual error provided by the s2i20pls model was smaller than 7% in comparison between propranolol hydrochloride concentration values for proposed method and pharmacopeical methods . the validation based on traditional chemometric parameters such as r and rmsep is insufficient towards pharmaceutical regulatory requirements . the s2i20pls model was also validated in accordance with the international conference on harmonisation ( ich ) using parameters usually recommended : precision ( repetitively and intermediate precision ) , linearity , and accuracy . the precision of an analytical procedure is usually expressed as the variance , standard deviation , or coefficient of variation of a series of measurements and can be presented as repeatability , intermediate precision , and reproducibility . in this work only repeatability and intermediate precision were evaluated . repeatability of the proposed method was performed by the same analyst and applying the method to the same samples six times on the same day . in this work the repeatability was calculated for three different samples with low , medium , and high concentrations of propranolol hydrochloride . values for repeatability of the proposed method were lower than 3.63% in concentration of 75.9 , 112.9 , and 211.7 mg g. the widely accepted rsd value for this type of determination is 5% . intermediate precision expresses within - laboratories variations like different equipment , different days , and different analysts . in this work , this parameter was determined by having two analysts for three samples of different concentration and on three different days . the first analyst performed the measurement in the morning while the second analyst measured the sample in the afternoon . in this case , relative standard deviation was lower than 5% . significant differences between results obtained for different analyst in different days were not observed ( two - way anova , 95% confidence level ) . finally , relative standard deviation was lower than 4.5% for precision evaluated in different days . b should be zero and one , respectively , if there was no systematic error in the calibration equation . for propranolol hydrochloride determination using s2i20pls model the regression equation was y = 0.96x + 6.36 . the confidence interval for the slope [ 0.87 ; 1.04 ] and for the intercept [ 5.60 ; 18.32 ] included 1 and zero , respectively . therefore , the proposed method for propranolol hydrochloride allowed a suitable linearity when it is compared to the reference method . accuracy was evaluated by comparison of results obtained using the s2i20pls model with those obtained by uv reference method . paired t - test was performed between s2i20pls model values and uv reference values . the experimental t value ( t = 0.86 ; 95% confidence level ) was smaller than the critical t value ( t = 2.31 ; 95% confidence level ) and the s2i20pls model using ftnir / probe data additionally , accuracy was also evaluated using the weighted least square ( wls ) to obtain the elliptical joint confidence region ( ejcr ) . the wls method was used with results obtained for s2i20pls model ( prediction set ) to verify the accuracy and to check if the proposed methodology is free of systematic error . the wls results include the theoretical expected value ( 1 , 0 ) , indicating that the proposed methodology is accurate and free of systematic errors . results obtained demonstrated that ftnir / probe associated with multivariate analysis is a convenient method for propranolol hydrochloride determination in pharmaceutical preparations . results obtained using sipls models for determination of propranolol hydrochloride in powder pharmaceutical preparations showed suitable prediction capacity ( lower rmsep ) . variable selection methods were able to produce better models in comparison to the pls full - spectrum model . results obtained for propranolol hydrochloride using the better sipls ( s2i20pls ) model were in agreement with results obtained by reference method . the proposed procedure using ftnir / probe and pls algorithms is faster , less expensive , accurate , and precise and minimizes solvent use when compared to pharmacopoeia uv method . | a method for determination of propranolol hydrochloride in pharmaceutical preparation using near infrared spectrometry with fiber optic probe ( ftnir / probe ) and combined with chemometric methods was developed .
calibration models were developed using two variable selection models : interval partial least squares ( ipls ) and synergy interval partial least squares ( sipls ) .
the treatments based on the mean centered data and multiplicative scatter correction ( msc ) were selected for models construction .
a root mean square error of prediction ( rmsep ) of 8.2 mg g1 was achieved using sipls ( s2i20pls ) algorithm with spectra divided into 20 intervals and combination of 2 intervals ( 8501 to 8801 and 5201 to 5501 cm1 ) .
results obtained by the proposed method were compared with those using the pharmacopoeia reference method and significant difference was not observed .
therefore , proposed method allowed a fast , precise , and accurate determination of propranolol hydrochloride in pharmaceutical preparations .
furthermore , it is possible to carry out on - line analysis of this active principle in pharmaceutical formulations with use of fiber optic probe . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
endodontic treatment aims to eliminate infection of the root canal and to completely fill the root canal space in three - dimension , in order to prevent apical and coronal penetration of liquids and microorganisms . most root canals are filled with gutta - percha points in combination with an endodontic sealer which are essential components of root canal obturation to establish a fluid - tight seal . the main function of a sealer is to fill the spaces between the core material and the walls of root canal and between the gutta - percha cones , in an attempt to form a coherent mass of obturating material without voids . the sealer is expected to fill irregularities and minor discrepancies between the filling and canal walls , accessory canals , and multiple foramina . by its germicidal action , it is also expected to destroy the remaining bacteria left after cleaning and shaping of the root canal . although all efforts are concentrated to confine the sealer within the root canal space , some extrusion inadvertently occurs during obturation procedure . when the sealer cement comes in contact with soft and hard tissues apically , it can cause persistent inflammation of periradicular tissues and may result in delayed wound healing manifesting as pain , tenderness , and swelling of the affected area ; hence , biocompatibility of sealers is an important issue in selecting the right type of sealer for different types of endodontic cases . all contemporary endodontic sealers are known to have some toxic properties , but meta - analysis on their toxicity has not been reported ; hence , the present study was undertaken to determine relative toxicity of the most commonly used root canal sealers like calcium hydroxide , zinc - oxide eugenol , and resin - based sealers . the commonly used sealers were classified into following three groups : group 1 : zinc oxide eugenol based sealers , for example , pulp canal sealer , tubli - seal . group 3 : resin - based sealers , for example , ah26 , ah plus , diaket . a comprehensive search was initiated to identify studies on sealer biotoxicity published in english language , on pubmed , cochrane , ebsco , and indmed databases between 2000 and 2012 . the keywords used in search queries on mesh were biotoxicity , root canal sealers , and biocompatibility . the reference section of each of these articles was also analyzed to determine the articles which pertain to our search criteria . all in vitro and in vivo ( animal ) experimental studies ( original research ) between 2000 and 2012 were included.only those studies comparing minimum of two of the three groups of sealers , that is , resin , calcium hydroxide , and zinc oxide eugenol.the studies should compare cytotoxicity / biotoxicity / biocompatibility of sealers at day 1 ( 24h ) and between 3 and 7 days.only those studies were finally selected in which systematic analysis and statistical tests were applied . all in vitro and in vivo ( animal ) experimental studies ( original research ) between 2000 and 2012 were included . only those studies comparing minimum of two of the three groups of sealers , that is , resin , calcium hydroxide , and zinc oxide eugenol . the studies should compare cytotoxicity / biotoxicity / biocompatibility of sealers at day 1 ( 24h ) and between 3 and 7 days . only those studies were finally selected in which systematic analysis and statistical tests were applied . ; 58 articles were found in the search , out of which 31 article abstracts , that is , six animal studies and 25 in vitro studies were reviewed by two endodontists from which only 20 studies ( 18 in vitro and two animal studies ) met the inclusion criteria and were finally selected for statistical analysis [ table 1 ] . research articles selected for final statistical meta - analysis statistical analysis was carried out using analysis of variance ( anova ) followed by post - hoc comparison by bonferroni method . the comparison between 24 h and after 3 day was done by using paired t - test for each sealer . the quantitative data was compared using student 's t - test and paired data by using paired t - test . since the data was skewed , log transformation was also applied . all in vitro and in vivo ( animal ) experimental studies ( original research ) between 2000 and 2012 were included.only those studies comparing minimum of two of the three groups of sealers , that is , resin , calcium hydroxide , and zinc oxide eugenol.the studies should compare cytotoxicity / biotoxicity / biocompatibility of sealers at day 1 ( 24h ) and between 3 and 7 days.only those studies were finally selected in which systematic analysis and statistical tests were applied . all in vitro and in vivo ( animal ) experimental studies ( original research ) between 2000 and 2012 were included . only those studies comparing minimum of two of the three groups of sealers , that is , resin , calcium hydroxide , and zinc oxide eugenol . the studies should compare cytotoxicity / biotoxicity / biocompatibility of sealers at day 1 ( 24h ) and between 3 and 7 days . only those studies were finally selected in which systematic analysis and statistical tests were applied . ; 58 articles were found in the search , out of which 31 article abstracts , that is , six animal studies and 25 in vitro studies were reviewed by two endodontists from which only 20 studies ( 18 in vitro and two animal studies ) met the inclusion criteria and were finally selected for statistical analysis [ table 1 ] . research articles selected for final statistical meta - analysis statistical analysis was carried out using analysis of variance ( anova ) followed by post - hoc comparison by bonferroni method . the comparison between 24 h and after 3 day was done by using paired t - test for each sealer . the quantitative data was compared using student 's t - test and paired data by using paired t - test . since the data was skewed , log transformation was also applied . at 24 h , the relative biotoxicity of the three sealers reported was insignificant ( p- value 0.29 ) , but the difference in toxicity was found significant ( p < 0.001 ) after 3 days . calcium hydroxide and zinc oxide eugenol sealers were found to be significantly biotoxic as compared to resin - based sealers after 3 days . it was also observed that toxicity of calcium hydroxide was slightly more than zinc oxide eugenol after 3 days , although the results were statistically insignificant . the meta - analysis also revealed the heterogeneity of various studies and their results included . summary statistics at 24h and between 3 and 7 days ( n = 20 ) for zinc oxide eugenol , calcium hydroxide , and resin revealed the values for means as 49.05 , 64.10 , and 63.35 and 51.00 , 80.45 , and 75.00 ; standard deviation as 30.45 , 21.05 , and 22.21 and 18.67 , 14.78 , and 16.63 ; and median as 41.50 , 64.00 , and 63.50 and 48.00 , 84.00 , and 77.50 with range as 5 - 95 , 23 - 92 , and 17 - 94 and 21 - 87 , 39 - 95 , and 32 - 92 , respectively . comparison of biotoxicity ( log values ) of three sealers at 24 h and between 3 and 7 days shows that there is no significant difference at 24 h between the three sealers ( p- value 0.29 ) . average log value of zinc oxide eugenol sealer at 24 h is 4.07 0.44 , calcium hydroxide is 4.09 0.39 , and resin - based sealers is 3.66 0.77 . when compared between 3 and 7 day , statistically significant difference was observed between the three sealers with p - value < 0.001 and the bonferroni correction showed that resin sealers had significant lower values [ table 2 ] . biotoxicity of three sealers at 24 h and between 3 and 7days based on paired data , no significant difference was observed in zinc oxide eugenol and resin - based sealers with p = 0.81 and 0.067 , respectively . the significant increase of 16.35 ( 25% ) was observed after 3 days in calcium hydroxide sealer ( p = 0.01 ) . based on meta - analysis , the random pooled estimates were 49 , 64 , and 63% for zinc oxide eugenol , calcium hydroxide , and resin - based sealers , respectively . the pooled estimate was found to be 0.56 with 95% confidence interval ( ci ) as 0.55 - 0.58 . root canal treatment involves a thorough cleaning and shaping , followed by three - dimensional obturation of the root canal system . presently , root canals are obturated with a core material in combination with an endodontic sealer . there are a variety of sealers to choose from , and the clinician must be careful to evaluate all characteristics of a sealer before selecting . it should be tacky when mixed to provide good adhesion between it and the canal wall , and when set a fluid tight seal should be obtained , while also having ample setting time for the clinician to make necessary adjustments to the filling material . the particles of powder should be very fine so that they can mix easily with the liquid and ideally should not shrink upon setting , although all currently available sealers shrink slightly upon setting . although during routine endodontic therapy , it is desired that the endodontic sealers remain inside the root canal , they may inadvertently be pushed beyond the apical constriction sometimes . in fact , they remain in intimate contact with the surrounding soft and hard tissues for an extended period of time . hence it is of utmost importance that they have an acceptable biocompatibility , that is , they should be nontoxic , nonmutagenic , and noncarcinogenic . all sealers exhibit toxicity when freshly mixed ; however , their toxicity is greatly reduced on setting . breakdown products from the sealers may have an adverse effect on the proliferative capability of periradicular cell populations . , that the intensity of the reaction diminishes by 60 day and the reduction progresses through to 120 day . as a result , adequate precaution should be taken not to extrude sealer in to the periapical tissue during obturation . zinc oxide eugenol sealers have a history of successful use in root canal obturation for over 100 years . it has prolonged setting time , shrinkage on setting , high solubility , and can stain the tooth structure . the advantage of zinc oxide eugenol sealer is its antimicrobial activity and popularity among clinicians , especially when used with thermoplasticized obturation technique . but eugenol is found to leak from zinc oxide eugenol sealers , which is known to induce toxic effect and decrease the transmission in nerve cells . localized inflammation with zinc oxide eugenol sealers has been seen , both in soft tissue and in the bone . the rationale for the addition of calcium hydroxide to root canal sealers is from observations of bases and liners containing the material and their antibacterial and tissue regenerating ability , exerted via the leaching of calcium and hydroxyl ions to surrounding tissues . solubility is required for release of calcium hydroxide and sustained activity , hence it is not consistent with the purpose of an ideal sealer . soares et al . , observed that canals overfilled with calcium hydroxide sealers caused chronic inflammatory reaction in periapical tissues in dog 's teeth . sealapex is primarily made of calcium hydroxide and has been shown to be cytotoxic in various studies , which probably resulted from components / additives such as polymethylene methyl salicylate resin and isobutyl salicylate present in sealapex . another possible explanation for the cytotoxicity of sealapex may come from the calcium hydroxide itself , which produces high ph ( leonardo et al . , 2000 ) . ah26 is a slow setting epoxy resin that was found to release formaldehyde when setting . ah plus is a modified formulation of ah26 in which formaldehyde is not released . endorez is a methacrylate resin - based sealer with hydrophilic properties . when used with gutta - percha cones , the dual cure endorez sealer bonds to both the canal walls and the core material . epiphany and realseal are other resin - based sealers introduced for use with a new core material . advocates of these sealers propose that they bond to the canal wall and to the core material to create a monobloc . but toxicity of epiphany might be explained by the presence of unpolymerized hydrophilic monomers ( such as hydroxyethyl methacrylate ( hema ) ) , which show diffusabilty into the surrounding tissues and exhibit toxicity . consequently , extrusion of a methacrylate resin - based sealer through the periapical foramen would create an uncured surface layer for extended time periods . this might alter the toxicity profile of resin - based sealers because the incompletely polymerized monomers present in the exposed sealer are more toxic . the meta - analysis of various studies revealed toxic potential of unset endodontic sealers , which may result in a localized inflammation and influence the healing of an apical periodontitis . these results disagree with the previous popular notion that called calcium hydroxide - based sealers as biological sealers. the use of biological sealers based on calcium hydroxide has been proposed for the permanent obturation of the root canal system . however , it exhibited increasing toxicity when set to confirm the results of previous studies that reported considerable leakage of cytotoxic substances from the disintegrating sealer ( gerosa et al . , 1995 , this instability in an aqueous environment might enhance the release of substances from set sealapex . ( 2012 ) , sealapex was strongly cytotoxic at days 1 , 7 , and 14 . in another animal study , the severity of the inflammation produced by the ca ( oh)2 sealer , sealapex , increased at the 3 day of evaluation , with no resolution until the 14 day . inflammatory cells were observed at the 7 day and foreign - body multinucleated giant cell reaction occurred after 14 days . using sealapex , leonardo et al . , ( 2007 ) also observed an intense inflammatory infiltrate in dogs , suggesting that the alterations in the original formulation might have affected negatively the tissue compatibility of this material . in case of zinc oxide it was suggested by lindqvist and otteskog ( 1981 ) that the cytotoxicity of zinc oxide eugenol root canal sealers was attributable to free eugenol liberated from the set material . a previous study has shown that these sealers can release formaldehyde after setting ( leonardo et al . , 1999 ) . the combined effects of eugenol and formaldehyde might explain why n2 and endomethasone were highly toxic . apart from formaldehyde and eugenol , n2 also contains a variety of aromatic oils that are cytotoxic . the cytotoxicity of resin - based sealers may be related to the release of formaldehyde ( spngberg et al . , 1993 ) . in addition , bisphenol a diglycidyl ether was identified as a mutagenic component of resin - based materials , which may also be cytotoxic . these results also agree with previous reports ( arenholt - bindslev and hrsted - bindslev 1989 , gerosa et al . , 1995 , cohen et al . , 2000 , leonardo et al . , 2000 ) that all materials tested showed some cytotoxicity cytotoxicity testing of freshly mixed sealers is relevant as they are placed into the root canal system in a freshly mixed and incompletely polymerized stage . changes in cytotoxicity levels may be observed after diffusion of toxic components from the materials into the surrounding environment . this could be attributed to the release of small amounts of toxic substances present in the sealers . probably as a result of the diminished leaching of these toxic substances , cytotoxicity of the tested sealers diminished over a period of time . we also observed in our meta - analysis that different studies showed varied results and data were skewed . there are reports of divergent responses and sensibilities for the xtt ( 2 , 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2h - tetrazolium-5-carboxanilide ) and mtt ( 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ) assays on a cell - type - dependent basis ( scudiero et al . , 1988 ) . it was also reported that these methods , in reality , do not evaluate the same enzyme systems , because evidence was found that mtt reduction is associated not only with mitochondria but also with the cytoplasm and with nonmitochondrial membranes including the endosome / lysosome compartment and the plasma membrane ( berridge et al . , 2005 ) . camps et al . , ( 2003 ) state that iso standards lead us to accept the cytotoxicity of endodontic sealers that were rated noncytotoxic when tested with the root - dipping technique . for example , sealapex showed a high cytotoxicity ranging from 91 to 96% of cell death when evaluated with iso standards , whereas a cytotoxicity ranging from 0 to 9% was observed when working with the other technique . this discrepancy between the results could be even more pronounced if the ratio between the surface of the sample and the volume of culture medium increases . among the reviewed articles , exploration of p- values of the selected comparisons indicated that most of them had suggested that the commonly used root canal sealers show biological risks inducing potential tissue biotoxicity leading to damage and inflammatory response in the periapical area . in cases where the sealer does not penetrate into the periradicular area directly , the elutable substances or the degradable products leach through the dentinal tubules , lateral and accessory canals , and the apical foramina . from the reviewed articles , meta - analysis revealed :
at 24 h , the relative biotoxicity of the three sealers was insignificant ( p- value 0.29 ) , but toxicity was significantly more ( p < 0.001 ) after 3days.calcium hydroxide sealer and zinc oxide eugenol were found to be significantly biotoxic as compared to resin - based sealers after 3days . it was also observed that toxicity of calcium hydroxide was slightly more than zinc oxide eugenol after 3days , although results were statistically insignificant.the meta - analysis also revealed the heterogeneity of various studies and their results included .
at 24 h , the relative biotoxicity of the three sealers was insignificant ( p- value 0.29 ) , but toxicity was significantly more ( p < 0.001 ) after 3days . calcium hydroxide sealer and zinc oxide eugenol were found to be significantly biotoxic as compared to resin - based sealers after 3days . it was also observed that toxicity of calcium hydroxide was slightly more than zinc oxide eugenol after 3days , although results were statistically insignificant . the meta - analysis also revealed the heterogeneity of various studies and their results included . exploration of p values of the selected comparisons indicated that most of them had suggested that the commonly used root canal sealers exhibited some degree of cytotoxicity . | introduction : the main objective of a root canal sealer is to provide a fluid tight seal . the purpose of this systematic meta - analysis was to determine the relative toxicity of commonly used root canal sealers like zinc oxide eugenol , calcium hydroxide , and resin - based sealers.materials and methods : an online search was conducted in peer - reviewed journals listed in pubmed , cochrane , ebsco , and indmed databases between 2000 and 2012 ) .
statistical analysis was carried out by using analysis of variance ( anova ) followed by post - hoc comparison by bonferroni method .
the comparison between toxicity at 24 h and between 3 and 7 days was done by using paired t - test for each sealer.results:at 24 h , the relative biotoxicity of the three sealers reported was insignificant ( p- value 0.29 ) , but the difference in toxicity was found significant ( p < 0.001 ) after 3 days.conclusion:calcium hydroxide sealer and zinc oxide eugenol were found to be significantly biotoxic as compared to resin - based sealers after 3 days . |
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the vulnerability of elderly hospital patients is characterised by simultaneously occurring somatic , psychological , and social problems , which may result in problems in cognitive functioning , mood , behaviour , activities of daily life , and , consequently , in declining quality of life . first , the choices of nursing interventions are substantially influenced by the patient 's cognitive abilities . the patient 's cognitive abilities determine the provision of nursing care to a large extent as they influence communication , the support to be given in daily life activities , the recognition and treatment of other nursing problems ( e.g. , pain , behavioural problems ) , and discharge policy [ 13 ] . the nurse 's approach to individual patients is also largely influenced by the type of cognitive problem . in case of memory problems , for example , information is repeated or written down ; in case of problems in sustaining attention , a quiet environment is offered ; and in case of executive problems , information is kept simple . for example , memory problems are often the first sign of alzheimer 's disease , whereas loss of awareness may indicate frontotemporal dementia , and hallucinations suggest delirium and dementia with lewy bodies . cognitive functioning is a term used to address the wide area of human information processing . the concept of cognitive functioning is operationalized by breaking it down into several cognitive domains . however , there is no uniform way to classify the cognitive domains ; different authors organise the cognitive domains in different ways [ 46 ] . cognitive decline may occur due to age - related factors , depression , delirium , dementia , and in combination with serious somatic health problems . recognition of delirium is most important because of its high incidence rate and reversible character , for example , by means of the confusion assessment method ( cam ; ) and the delirium observation scale ( dos ; ) . however , comprehensive cognitive assessment still needs to be carried out as other types of brain dysfunction might occur , such as dementia or brain injury . a diagnosis is then based on medical history , neurological assessment , neuropsychological assessment , neuroimaging , complaints of patient , and behavioural symptoms . direct observation of the patient 's cognitively mediated abilities complements cognitive assessment [ 2 , 9 ] . daily observation of the patient covers 24 hours a day and may last for several days . it is based on informal interactions between the patient and nurse , for example , when taking a bath , having breakfast , during transfers , or when interacting with other patients . the patient 's cooperation is not required , and observation can be conducted even when patients are too ill to undergo neuropsychological testing . unlike testing , which assesses cognitive abilities under optimal experimental conditions , direct observation assesses a person 's cognitive abilities in daily life . results of daily observation are therefore of high ecological validity as they are linked to the natural setting of daily life and do not depend on one specific test moment [ 9 , 10 ] . observation may improve the reliability of cognitive assessment if serial observations are recorded on several consecutive days . furthermore , observation fits very well into the nursing practice , because information is directly accessible during patient care encounters . one problem with direct observation is its standardisation . yet , well - validated observation scales are rather scarce , although some good examples exist in geriatrics , for example , for depression , pain , agitation , dementia , and as mentioned above , for delirium . we searched the literature for direct observation scales for cognition abilities , excluding , however , delirium screening instruments . we found instruments which assess cognitive functioning in a limited way ( not divided into subscales of cognitive domains : e.g. , the old ) , facilitate just one or two cognitive domains ( e.g. , the nosger ) , or are too specific for nurses ( the a - one ) . furthermore , many of them also involved issues such as mood or behavioural problems ( e.g. , moses and nosger ) . we therefore concluded that there is no valid observation scale available for nurses to comprehensively assess cognitive functioning in relation to possible dementia or brain injury . in daily practice , this means that individual nurses observe cognitive functioning in patients in a nonmethodological way . this undermines the reliability and validity of the information obtained , as demonstrated in two studies on dutch geriatric hospital wards [ 3 , 19 ] . the nurses in these studies assessed different cognitive domains per patient , aimed at different goals . the aim of this study was to develop an observation scale with an acceptable level of content validity which assesses elderly patients ' cognitive abilities in a comprehensive manner , including a wide range of cognitive domains . the resulting nurses observation scale for cognitive abilities ( nosca ) had to fulfil certain preconditions : it should be suitable for observation of the population of geriatric patients admitted in acute care hospitals ; it should structure around - the - clock observations by nurses and possibly include all interactions naturally occurring between patient and nurse ( e.g. , bathing , meal times or transfers , small talk , informing , educating ) ; it should serve goals important in daily practice , that is , it should enable tailoring to individual ( nursing ) care plans , contribute to the diagnostic process , or facilitate further neuropsychological examination . the works by haynes , polit and beck , streiner and norman , and foreman et al . on the development of ( cognitive ) measurement scales constituted the point of departure for our study . they all underpin the necessity to explicitly decide on the aim and context of the measurement instrument to be developed . content validity concerns the conceptualisation of the content and the degree to which the scale represents the concept . content validity is the degree to which an instrument has an appropriate number of items for the construct being measured . as no uniform way to classify cognitive domains was found in the literature , a panel of experts was required to evaluate the content validity of the scale developed . we obtained a written judgment from the experts concerning the nosca construct and items by means of the delphi technique . in several rounds during which experts ' preferences were integrated , consensus was achieved concerning the nosca construct : the cognitive domains ( phase 1 ) and items ( phase 2 ) . in phase 3 we proceeded from the definition of cognitive function as cited by a nursing protocol found in the literature , which was based on the work of lezak : cognitive functioning encompasses the processes by which an individual perceives , registers , stores , retrieves and uses information . as the scale had to have a firm theoretical basis and no consensus was found in the geriatric , psychiatric , or neuropsychological field , we selected the more general health - based international classification of functioning ( icf ) . the icf includes a classification system of functions , in which chapter 1 states the mental functions . some of these mental functions relate to cognitive functions . to enhance reliability , items had to be written for observable patient activities or behaviour , noticeable for all observers , so that bias in the observer 's interpretation would be minimised . as the patient 's cognitive functioning varies depending on the moment of the day , type of activity , or interaction with others , we preferred more than one observation . the observation should take place twice a day during two consecutive days in order to obtain sufficiently reliable outcome and should also account for inter - daily variation . to improve reliability , it was required that the scale be completed at the end of every shift . the multidisciplinary panel consisted of 16 experts and was composed of geriatric nurses ( 2x ) , advanced nurses in geriatrics ( 5x ) , one nurse lecturer in geriatrics , geriatricians ( 3x ) , neuropsychologists ( 4x ) , and one occupational therapist ( see table 2 ) . all of them had many years of clinical practice experience with elderly people and were in some way experts in assessing cognitive functioning . of the advanced nurses , two had additional experience in developing a measurement instrument for assessing elderly people , two nurses were familiar with the icf , and four of them had published ( internationally ) on cognitive functioning . three out of the four neuropsychologists had experience in developing a measurement instrument and had published in international journals . the geriatricians were selected because of their clinical expertise and the research they had conducted . of the originally invited experts , only one refused because the delphi rounds would be too time - consuming . all the experts received information about the objective of the observation scale , the setting , and the theoretical framework . phase 1 : domains includedin this phase , the construction of the scale was established by means of the 1st and 2nd delphi rounds . the aim was to determine the cognitive domains to be included in the scale . in the 1st delphi round , nine of the cognitive domains mentioned in chapter 1 of the icf this information also included the icf definition of the cognitive domain and 19 icf subdomains . furthermore , all functions as described in this chapter were presented , so experts were able to judge the representativeness of the nine domains proposed . the experts were requested to respond on the following : the relevance of the icf domain ( also including the formulation of the icf label and icf definition ) and the relevance of the subdomain ( also including the formulation of the icf label and icf definition ) , representing all important cognitive domains . it was required that at least 70% of the experts agree that a proposed cognitive domain should be included . suggestions from individual experts to rephrase or alter certain parts or to include new ( sub)domains were presented to the panel in the second round .
in this phase , the construction of the scale was established by means of the 1st and 2nd delphi rounds . the aim was to determine the cognitive domains to be included in the scale . in the 1st delphi round , nine of the cognitive domains mentioned in chapter 1 of the icf this information also included the icf definition of the cognitive domain and 19 icf subdomains . furthermore , all functions as described in this chapter were presented , so experts were able to judge the representativeness of the nine domains proposed . the experts were requested to respond on the following : the relevance of the icf domain ( also including the formulation of the icf label and icf definition ) and the relevance of the subdomain ( also including the formulation of the icf label and icf definition ) , representing all important cognitive domains . it was required that at least 70% of the experts agree that a proposed cognitive domain should be included . suggestions from individual experts to rephrase or alter certain parts or to include new ( sub)domains were presented to the panel in the second round . phase 2 : item selectionin this phase , the items of the scale were determined by means of the 3rd and 4th delphi rounds . central in this phase was the question as to whether an item was relevant to a domain or subdomain and whether the items sufficiently represented the domain . the items presented to the panel were mainly derived from other observation scales ( see table 1 ) . all the items were reformulated using the same sentence structure , for example , the patient is able to locate his / her own bed . furthermore , we placed the items into matching domains and subdomains . in the 3rd round , we presented the panel with the 173 items , divided across the domains and subdomains . the experts were requested to respond on the following : the relevance of the item and the priority of the item , representing all important items . then , the items approved were checked for interdependency . when it turned out that too many items were approved , we selected the best observable behaviour or activity . subsequently , the approved items were checked again in order to assess whether they differentiated from items in the other cognitive domains . if they did not , we selected the items which seemed to be most appropriate for certain domains . finally , the suggestions made by the experts concerning the rephrasing of items or inclusion of new items were incorporated.in the 4th delphi round , items which were still under discussion , newly suggested , or located in another domain or subdomain were reevaluated by the experts .
in this phase , the items of the scale were determined by means of the 3rd and 4th delphi rounds . central in this phase was the question as to whether an item was relevant to a domain or subdomain and whether the items sufficiently represented the domain . the items presented to the panel were mainly derived from other observation scales ( see table 1 ) . all the items were reformulated using the same sentence structure , for example , the patient is able to locate his / her own bed . furthermore , we placed the items into matching domains and subdomains . in the 3rd round , we presented the panel with the 173 items , divided across the domains and subdomains . the experts were requested to respond on the following : the relevance of the item and the priority of the item , representing all important items . then , the items approved were checked for interdependency . when it turned out that too many items were approved , we selected the best observable behaviour or activity . subsequently , the approved items were checked again in order to assess whether they differentiated from items in the other cognitive domains . if they did not , we selected the items which seemed to be most appropriate for certain domains . finally , the suggestions made by the experts concerning the rephrasing of items or inclusion of new items were incorporated . in the 4th delphi round , items which were still under discussion , newly suggested , or located in another domain or subdomain were reevaluated by the experts . phase 3 : pretestin this phase , the feasibility of the observation scale was tested in a small study . over a period of two weeks , nurses from one geriatric ward were asked to complete the observation scale on the basis of their observation of a single patient during their shift . the nurses were asked for comments on the instructions and on the observation scale . some of these comments were subsequently processed . after seven nurses had provided their comments , no further suggestions for adjustments were made .
in this phase , the feasibility of the observation scale was tested in a small study . over a period of two weeks , nurses from one geriatric ward were asked to complete the observation scale on the basis of their observation of a single patient during their shift . the nurses were asked for comments on the instructions and on the observation scale . some of these comments were subsequently processed . after seven nurses had provided their comments , no further suggestions for adjustments were made . phase 1 : domains includedin the first two delphi rounds , the response from the 16 panel members was 100% . table 3 presents an overview of the panel 's acceptance of the domains and subdomains and the suggestions they made . after the 1st delphi round , all nine proposed domains were accepted ( > 80% agreement ) , as well as 16 out of the 19 proposed subdomains . the response on the consciousness domain showed insufficient consensus : although 81% of the experts saw this domain as part of the cognitive function scale , several experts also suggested rephrasing because consciousness is only a prerequisite for cognitive functioning and should therefore have another position in the observation scale compared to the other cognitive domains . after the 2nd delphi round , five out of the nine newly suggested subdomains were accepted by the panel ( > 80% agreement ) , see table 3 . there was 81% agreement that consciousness should have a distinct place in the observation scale . next , as suggested by experts in the first round , the subdomain of content of thoughts was added to the 2nd round , and although 81% of the panel agreed to include this subdomain , 64% wished to rephrase the items concerned . the panel was therefore consulted by email on the question of whether or not these two subdomains should be included . thirteen out of the 16 experts responded , and it was concluded that the subdomain of dividing attention should not be included ( 56% agreed ) . it was also concluded that the subdomain of content of thoughts should be reformulated ( 85% agreed).in sum , after two delphi rounds and one follow - up email , the panel reached consensus on the construct of cognitive function by means of 8 domains and 17 subdomains ( see table 4 ) . furthermore , the domain of consciousness ( consisting of two subdomains ) was considered a prerequisite for cognitive functioning , which should therefore be assessed beforehand .
in the first two delphi rounds , the response from the 16 panel members was 100% . table 3 presents an overview of the panel 's acceptance of the domains and subdomains and the suggestions they made . after the 1st delphi round , all nine proposed domains were accepted ( > 80% agreement ) , as well as 16 out of the 19 proposed subdomains . the response on the consciousness domain showed insufficient consensus : although 81% of the experts saw this domain as part of the cognitive function scale , several experts also suggested rephrasing because consciousness is only a prerequisite for cognitive functioning and should therefore have another position in the observation scale compared to the other cognitive domains . after the 2nd delphi round , five out of the nine newly suggested subdomains were accepted by the panel ( > 80% agreement ) , see table 3 . there was 81% agreement that consciousness should have a distinct place in the observation scale . next , as suggested by experts in the first round , the subdomain of content of thoughts was added to the 2nd round , and although 81% of the panel agreed to include this subdomain , 64% wished to rephrase the items concerned . the panel was therefore consulted by email on the question of whether or not these two subdomains should be included . thirteen out of the 16 experts responded , and it was concluded that the subdomain of dividing attention should not be included ( 56% agreed ) . it was also concluded that the subdomain of content of thoughts should be reformulated ( 85% agreed ) . in sum , after two delphi rounds and one follow - up email , the panel reached consensus on the construct of cognitive function by means of 8 domains and 17 subdomains ( see table 4 ) . furthermore , the domain of consciousness ( consisting of two subdomains ) was considered a prerequisite for cognitive functioning , which should therefore be assessed beforehand . phase 2 : item selectionin the 3rd and 4th delphi rounds , 15 and 16 out of the 16 panel members responded , respectively . after the 3rd round , it turned out that 58 of the 173 items were deemed to be relevant ( > 70% agreement ) . 16 items were excluded , which left us with a selection of 42 items . although these 42 items were accepted for the observation scale , the experts suggested rephrasing of seven items and relocating one item to another domain . all in all , in the 4th round , 45 items were presented to the panel of which eleven were to be judged again on relevance , rephrasing , and representativeness . after the 4th round , the panel accepted nine out of the eleven items as being relevant to the new observation scale and well - formulated ( > 70% agreement ) .
in the 3rd and 4th delphi rounds , 15 and 16 out of the 16 panel members responded , respectively . after the 3rd round , it turned out that 58 of the 173 items were deemed to be relevant ( > 70% agreement ) . 16 items were excluded , which left us with a selection of 42 items . although these 42 items were accepted for the observation scale , the experts suggested rephrasing of seven items and relocating one item to another domain . all in all , in the 4th round , 45 items were presented to the panel of which eleven were to be judged again on relevance , rephrasing , and representativeness . after the 4th round , the panel accepted nine out of the eleven items as being relevant to the new observation scale and well - formulated ( > 70% agreement ) . in sum , after the 3rd and 4th rounds , the observation scale consisted of 39 items , divided across 8 domains and 17 subdomains , preceded by 4 items for the domain of consciousness as a condition for cognitive functioning ( see table 5 ) . please read the following information about the nosca
aim :
with this observation list , nurses can gain an impression of whether a patient has cognitive problems and if so , in which domains.these observations will be used to ( a ) contribute to the diagnostics at the multidisciplinary meeting and ( b ) to help determine the nursing interventions ( approach form , information , family education ) . instructions :
before starting your shift , read the nosca items so that you can make targeted observations and if necessary , induce behaviour ( e.g. , start a conversation , read a text , get dressed , etc.).create the most optimal conditions for the patient ( glasses on , hearing aid working).make observations over a period of two consecutive days , in the day shifts and evening shifts . research has shown that more than four observation periods do not lead to better information.record the observations per shift , so that the report is as reliable as possible .
filling in the form :
put a circle around the correct answer in accordance with your observations during one shift . repeatedly means that the behaviour occurred repeatedly during your shift.put a circle around the question mark ? if the behaviour could not be observed because the situation did not arise ( e.g. , because the patient did not read anything ) . also put a circle around the question mark ? if the patient could not display certain behaviour ( e.g. , the patient could not put on his / her clothes in the correct order , because he / she can not dress independently due to a physical disability ) . calculate the average score per subscale , representing a cognitive domain , and note it on the summary sheet ( range 03 points ) . the nosca overall score is calculated by the sum of the eight domains ( range 024 points).norm values of the subscales : lower scores indicate less cognitive abilities :
3 means that no problems were observed;2 means that problems sometimes arose;1 means that problems usually arose;0 means that problems arose repeatedly . aim :
with this observation list , nurses can gain an impression of whether a patient has cognitive problems and if so , in which domains.these observations will be used to ( a ) contribute to the diagnostics at the multidisciplinary meeting and ( b ) to help determine the nursing interventions ( approach form , information , family education ) . instructions :
before starting your shift , read the nosca items so that you can make targeted observations and if necessary , induce behaviour ( e.g. , start a conversation , read a text , get dressed , etc.).create the most optimal conditions for the patient ( glasses on , hearing aid working).make observations over a period of two consecutive days , in the day shifts and evening shifts . research has shown that more than four observation periods do not lead to better information.record the observations per shift , so that the report is as reliable as possible .
filling in the form :
put a circle around the correct answer in accordance with your observations during one shift . repeatedly means that the behaviour occurred repeatedly during your shift.put a circle around the question mark ? if the behaviour could not be observed because the situation did not arise ( e.g. , because the patient did not read anything ) if the patient could not display certain behaviour ( e.g. , the patient could not put on his / her clothes in the correct order , because he / she can not dress independently due to a physical disability ) . calculate the average score per subscale , representing a cognitive domain , and note it on the summary sheet ( range 03 points ) . the nosca overall score is calculated by the sum of the eight domains ( range 024 points).norm values of the subscales : lower scores indicate less cognitive abilities :
3 means that no problems were observed;2 means that problems sometimes arose;1 means that problems usually arose;0 means that problems arose repeatedly .
with this observation list , nurses can gain an impression of whether a patient has cognitive problems and if so , in which domains.these observations will be used to ( a ) contribute to the diagnostics at the multidisciplinary meeting and ( b ) to help determine the nursing interventions ( approach form , information , family education ) .
with this observation list , nurses can gain an impression of whether a patient has cognitive problems and if so , in which domains . these observations will be used to ( a ) contribute to the diagnostics at the multidisciplinary meeting and ( b ) to help determine the nursing interventions ( approach form , information , family education ) .
before starting your shift , read the nosca items so that you can make targeted observations and if necessary , induce behaviour ( e.g. , start a conversation , read a text , get dressed , etc.).create the most optimal conditions for the patient ( glasses on , hearing aid working).make observations over a period of two consecutive days , in the day shifts and evening shifts . research has shown that more than four observation periods do not lead to better information.record the observations per shift , so that the report is as reliable as possible .
before starting your shift , read the nosca items so that you can make targeted observations and if necessary , induce behaviour ( e.g. , start a conversation , read a text , get dressed , etc . ) . create the most optimal conditions for the patient ( glasses on , hearing aid working ) . make observations over a period of two consecutive days , in the day shifts and evening shifts . research has shown that more than four observation periods do not lead to better information . record the observations per shift , so that the report is as reliable as possible . put a circle around the correct answer in accordance with your observations during one shift . repeatedly means that the behaviour occurred repeatedly during your shift.put a circle around the question mark ? if the behaviour could not be observed because the situation did not arise ( e.g. , because the patient did not read anything ) if the patient could not display certain behaviour ( e.g. , the patient could not put on his / her clothes in the correct order , because he / she can not dress independently due to a physical disability ) . put a circle around the correct answer in accordance with your observations during one shift . if the behaviour could not be observed because the situation did not arise ( e.g. , because the patient did not read anything ) if the patient could not display certain behaviour ( e.g. , the patient could not put on his / her clothes in the correct order , because he / she can not dress independently due to a physical disability ) . the observations over four shifts lead to one conclusion . calculate the average score per subscale , representing a cognitive domain , and the nosca overall score is calculated by the sum of the eight domains ( range 024 points).norm values of the subscales : lower scores indicate less cognitive abilities :
3 means that no problems were observed;2 means that problems sometimes arose;1 means that problems usually arose;0 means that problems arose repeatedly . the observations over four shifts lead to one conclusion . calculate the average score per subscale , representing a cognitive domain , and the nosca overall score is calculated by the sum of the eight domains ( range 024 points ) . norm values of the subscales : lower scores indicate less cognitive abilities :
3 means that no problems were observed;2 means that problems sometimes arose;1 means that problems usually arose;0 means that problems arose repeatedly . 3 means that no problems were observed ; 2 means that problems sometimes arose ; 1 means that problems usually arose ; 0 means that problems arose repeatedly . phase 3 : pretestthe feasibility of the concept of the observation scale was tested seven times consecutively . five times improvements were made for the sake of clarity ; the last two tests showed no need for adjustments . in general , nurses had no difficulties filling in the form and it took them only a few minutes per patient per shift . the instructions were changed several times , and the layout of the items was improved , resulting in a more concise and clear text . five times improvements were made for the sake of clarity ; the last two tests showed no need for adjustments . in general , nurses had no difficulties filling in the form and it took them only a few minutes per patient per shift . the instructions were changed several times , and the layout of the items was improved , resulting in a more concise and clear text . the object of this study was to develop an observation scale in which cognitive abilities are assessed in a comprehensive manner , and which has an acceptable level of content validity . we succeeded in designing the nurses ' observation scale cognitive abilities ( nosca ) , in which cognitive functioning is classified into eight domains , 17 subdomains , and 39 items , preceded by 4 items on the domain of consciousness . the content validity of the nosca , as a measurement of the degree to which the scale represents the concept of cognitive functioning , is high for two reasons . first , the minimum agreement between the panel members was 70% , and often higher on certain domains and items . second , after the fourth delphi round , consensus was reached that no domains or items were lacking . in the preceding delphi rounds , the panel had made suggestions for including new domains and items , and some were accepted in the next delphi round . the strength of our procedure was that the panel members represented four disciplines ( nursing , neuropsychology , geriatrics and speech , therapy ) . thus , the quality of the panel was enhanced because each discipline with its specific focus and knowledge of the concept of cognitive functioning provided specific input for the observation scale . an equally strong point of the nosca and its development is that the scale is based on the icf 's theoretical framework . although the icf is not a cognitive concept but a general classification of functioning , it proved to be very suitable for the purpose of our study and probably will increase acceptability in the field . the method we used had only one drawback : in designing the nosca , we were required , due to lack of consensus on the concept of cognitive functioning in the literature , to develop consensus by means of the delphi technique . the disadvantage of this technique is that consensus depends on the current state of the art in the professional disciplines and that , through the years , the state of art will further develop . particularly as a result of the new technique of neuroimaging , it is expected that the knowledge of brain functions will increase in the coming years , and consequently the observation scale may have to be revised after some time . now that the content validity has been described internal consistency , interrater reliability , and intrarater reliability will be examined , as well as construct validity . for the latter , results of the nosca will be related to clinical diagnoses , severity of dementia , and results of neuropsychological tests . discriminant validity will be studied by comparing the results of the nosca with scores on depression . expecting positive results on the psychometric qualities , we are convinced that the implications of the nosca for the nursing practice will prove important . furthermore , behavioural observation within a clinical environment provides invaluable information regarding underlying cognitive function . the value of observation of daily behaviour for assessing cognitive functioning was the reason for miller et al . to develop the batch ( after we had finished our literature search ) . this is an assessment tool to record observations of patients ' daily functioning under subheading that reflect cognitive domains . they were interested in an observation scale for patients in a psychiatric setting ( mean age 50 years ) who refuse or can not undertake cognitive assessments . the batch may well be interesting if more data are collected on validity , reliability , distinction between the subscale scores and if the scale were to be applied in a geriatric patient group . for geriatric patients , several methods for assessing their cognitive functioning need to be combined , and daily observation of cognitive functioning remains important because of its high ecological value . we expect that the nosca will be useful at hospital medical and surgical wards as well , because the items all cover behaviour which is easy to observe , and thus the scoring does not require specific geriatric expertise or knowledge . in other settings , such as home care and homes for the elderly , it will also be useful in assessing cognitive functioning . for these different settings , separate validation studies will be required . for the time being , we recommend that nurses closely observe elderly patients and report cognitive functioning and dysfunctioning , and associated behaviour . improving these systematic observations will enhance the ability of nurses to truly tailor their interventions to the patients ' abilities . the nosca is a promising tool that will help nurses to perform the challenging observations of cognitive functioning , and thereby improve the quality of care provided to the fast growing number of older patients . this research received no specific grant from any funding agency in public , commercial , or not - for - profit sectors . | background . to assess a patient 's cognitive functioning is an important issue because nurses tailor their nursing interventions to the patient 's cognitive abilities .
although some observation scales exist concerning one or more cognitive domains , so far , no scale has been available which assesses cognitive functioning in a comprehensive way .
objectives . to develop an observation scale with an accepted level of content validity and which assesses elderly patients ' cognitive functioning in a comprehensive way . methods .
delphi technique , a multidisciplinary panel developed the scale by consensus through four delphi rounds ( > 70% agreement ) .
the international classification of functioning / icf was used as theoretical framework . results .
after the first two delphi rounds , the panel reached consensus about 8 cognitive domains and 17 sub domains .
after two other rounds , 39 items were selected , divided over 8 domains and 17 sub domains .
discussion .
the nurses ' observation scale cognitive abilities ( nosca ) was successfully designed .
the content validity of the scale is high because the scale sufficiently represents the concept of cognitive functioning : the experts reached a consensus of 70% or higher on all domains and items included ; and no domains or items were lacking . as a next step , the psychometric qualities of the nosca will have to be tested . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
atopic dermatitis , also known as atopic eczema or just eczema , is a very common skin condition characterized by skin inflammation and itching that typically starts in early life . atopic dermatitis is associated with dry skin and a tendency towards asthma and hay fever and now affects around 20% of children in developed and developing countries . data from the international study of asthma and allergies in childhood ( isaac ) suggests that atopic dermatitis is increasing ( figure 1 ) , especially in younger children . genetic factors that determine skin barrier integrity and skin inflammation seem to be important , but so too is the environment , given the rapid rise in prevalence , and the observation that atopic dermatitis seems to be more common in wealthier , smaller and more educated families . lack of stimulation of the immune system by microbes at an early age because our children are too clean ( the hygiene hypothesis ) or failure of a balanced gut flora to mature may also be important . whilst some people with atopic dermatitis are clearly allergic to foods and house dust mites , recent studies suggest that the role of allergy in atopic dermatitis might have been overemphasized . current treatments , although quite effective , are still geared towards treatment of symptoms and visible signs rather than fundamentally altering the course of disease . world maps depicting flexural eczema symptoms in the last year showing changes in the prevalence of eczema symptoms for 13 - 14 year olds and 6 - 7 year olds in consecutive prevalence surveys conducted 5 - 10 years apart . whilst some levelling off or even a decrease in eczema symptoms are noted in some developed countries in the 13 - 14 year old group , the trend is for eczema to be increasing throughout the world for the 6 - 7 year old groups . in the absence of disease - modifying treatment , attention needs to focus on disease prevention , especially as this might also prevent progression to asthma and hay fever ( the atopic march ) . yet an overview of seven systematic reviews , which included 39 trials on 11,897 participants , concluded that none of the previously tested interventions could be shown to conclusively prevent atopic dermatitis . the possibility that atopic dermatitis may be prevented in a subgroup of infants at high risk of disease by exclusive breastfeeding or the introduction of prebiotics ( nutrients that encourage beneficial gut bacteria ) was based on limited evidence with potential flaws , although more recent evidence for some probiotics ( beneficial live gut bacteria ) during and after pregnancy show some promise . instead of continuing to examine strategies that have been tried for disease prevention over the last 40 years , fresh approaches for disease prevention , stimulated by recent advances in our understanding of atopic dermatitis , are needed . although there is little doubt that some individuals with atopic dermatitis are truly allergic to substances such as house dust mite protein , longitudinal studies reveal allergen sensitization is probably a consequence , not a cause , of atopic dermatitis . this idea is bolstered by findings that genetic syndromes defined by a loss of immune tolerance have eczema as a feature . consequently , instead of trying to rid the environment of allergens , maybe a better approach would be to try and induce tolerance by exposure to allergens or endotoxins at an early stage of immune development . peanut allergy might serve as a good model in this regard . in israel for example , where peanut consumption is much higher than the uk , peanut allergy seems to be much less common , an observation that has stimulated a research group to test the hypothesis that introduction of peanuts into the diet in early life can reduce peanut allergy . gut parasites are another potentially interesting area to explore given that our immune system lived with them for millions of years before their relatively recent eradication . it is possible that this eradication of gut parasites has contributed to the increase in allergic disease due to insufficient immune stimulation of the right sort , and that the reintroduction of extracts from gut parasites in early life might tip the abnormally tilted atopic dermatitis immune response back into one of allergen tolerance . research into the causes of atopic dermatitis has been dominated over the last 40 years by a preoccupation with allergy and the immune responses of the skin , gut , lungs and blood . yet one of the hallmarks of atopic dermatitis is the presence of generally dry skin even in the absence of inflammation , an observation that stimulated researchers in dundee to look at the genes that may be responsible for other dry skin conditions such as ichthyosis . they found that mutations of genes that code for filaggrin a key protein in the outer skin that maintains skin barrier function are a strong predictor of atopic dermatitis , especially more severe , chronic atopic dermatitis and associated asthma . so the world of atopic dermatitis research is turning its attention away from the inside of the body to the dry skin on the outside . we speculate that exposing children born with atopic dermatitis and defective skin barriers to frequent cleansing with alkaline soaps , bubble baths and shampoos leads to a breach of the skin barrier and low grade inflammation , which can then develop a life of its own through the process of autoimmunity , ( figure 2 ) . the defective skin barrier might also permit allergic sensitization to occur as allergens are introduced more easily to antigen presenting cells in the skin . if this is true , then enhancing the skin barrier function in babies born to parents with allergic disease by limiting the assault of skin cleansers coupled with liberal use of emollients could prevent the development of atopic dermatitis and even the progression to asthma . our barrier enhancement for eczema prevention ( beep ) pilot study has provided an encouraging signal , which our group now plans to test on a much larger scale . only large scale randomized controlled trials will definitely show whether they do any good , and whether they are any better than simple , cheaper emollients . scratching results in cellular damage and release of membranous and intracellular compounds from keratinocytes ( kc ) and possibly other skin cells . the local dendritic cells ( dc ) subjected to thymic stromal lymphopoietin ( tslp ) will induce a th2 response and the generation of specific ige directed against these self - proteins . ige will bind to fc ri on dendritic cells in the skin and thereby amplify the immune response and inflammatory reaction in the skin . even if atopic dermatitis can not be prevented in the primary sense , it may still be possible to reduce disease severity through a strategy of early aggressive treatment when atopic dermatitis first appears . it is known that the constant scratching associated with atopic dermatitis may expose parts of skin to cells that can trigger an autoimmune response , which could result in a more widespread and persistent disease , ( figure 3 ) . so interrupting that chain of events by clamping down early on atopic dermatitis is an avenue worth exploring . others have also suggested exploring disease modifying strategies in atopic dermatitis to break , stop or reverse atopic dermatitis and associated asthma , in a way that is heavily informed by biomarkers . other forms of secondary prevention such as weekend therapy ( applying topical treatments for two consecutive days each week once the disease is under control ) had been shown to have a large impact on reducing disease flares . whether the length and intensity of inducing the initial remission is important for long term control needs to be tested . the emergent theme seems to be one of not chasing atopic dermatitis but instead one of getting control then keeping control . in healthy skin the corneodesmosomes ( iron rods ) are intact throughout the stratum corneum . at the surface , the corneodesmosomes start to break down as part of the normal desquamation process , analogous to iron rods rusting ( a ) . in an individual genetically predisposed to atopic dermatitis , premature breakdown of the corneodesmosomes leads to enhanced desquamation , analogous to having rusty iron rods all the way down through the brick wall ( b ) . if the iron rods are already weakened , an environmental agent , such as soap , can corrode them much more easily . the brick wall starts falling apart ( c ) and allows the penetration of allergens ( d ) . ( reproduced with permission from cork mj , robinson da , vasilopoulos y , ferguson a , moustafa m , macgowan a , duff gw , ward sj , tazi - ahnini r. new perspectives on epidermal barrier dysfunction in atopic dermatitis : gene - environment interactions . although there is little doubt that some individuals with atopic dermatitis are truly allergic to substances such as house dust mite protein , longitudinal studies reveal allergen sensitization is probably a consequence , not a cause , of atopic dermatitis . this idea is bolstered by findings that genetic syndromes defined by a loss of immune tolerance have eczema as a feature . consequently , instead of trying to rid the environment of allergens , maybe a better approach would be to try and induce tolerance by exposure to allergens or endotoxins at an early stage of immune development . peanut allergy might serve as a good model in this regard . in israel for example , where peanut consumption is much higher than the uk , peanut allergy seems to be much less common , an observation that has stimulated a research group to test the hypothesis that introduction of peanuts into the diet in early life can reduce peanut allergy . gut parasites are another potentially interesting area to explore given that our immune system lived with them for millions of years before their relatively recent eradication . it is possible that this eradication of gut parasites has contributed to the increase in allergic disease due to insufficient immune stimulation of the right sort , and that the reintroduction of extracts from gut parasites in early life might tip the abnormally tilted atopic dermatitis immune response back into one of allergen tolerance . research into the causes of atopic dermatitis has been dominated over the last 40 years by a preoccupation with allergy and the immune responses of the skin , gut , lungs and blood . yet one of the hallmarks of atopic dermatitis is the presence of generally dry skin even in the absence of inflammation , an observation that stimulated researchers in dundee to look at the genes that may be responsible for other dry skin conditions such as ichthyosis . they found that mutations of genes that code for filaggrin a key protein in the outer skin that maintains skin barrier function are a strong predictor of atopic dermatitis , especially more severe , chronic atopic dermatitis and associated asthma . so the world of atopic dermatitis research is turning its attention away from the inside of the body to the dry skin on the outside . we speculate that exposing children born with atopic dermatitis and defective skin barriers to frequent cleansing with alkaline soaps , bubble baths and shampoos leads to a breach of the skin barrier and low grade inflammation , which can then develop a life of its own through the process of autoimmunity , ( figure 2 ) . the defective skin barrier might also permit allergic sensitization to occur as allergens are introduced more easily to antigen presenting cells in the skin . if this is true , then enhancing the skin barrier function in babies born to parents with allergic disease by limiting the assault of skin cleansers coupled with liberal use of emollients could prevent the development of atopic dermatitis and even the progression to asthma . our barrier enhancement for eczema prevention ( beep ) pilot study has provided an encouraging signal , which our group now plans to test on a much larger scale . only large scale randomized controlled trials will definitely show whether they do any good , and whether they are any better than simple , cheaper emollients . scratching results in cellular damage and release of membranous and intracellular compounds from keratinocytes ( kc ) and possibly other skin cells . the local dendritic cells ( dc ) subjected to thymic stromal lymphopoietin ( tslp ) will induce a th2 response and the generation of specific ige directed against these self - proteins . ige will bind to fc ri on dendritic cells in the skin and thereby amplify the immune response and inflammatory reaction in the skin . ( reproduced with permission from tang ts , bieber t , williams hc . does autoreactivity even if atopic dermatitis can not be prevented in the primary sense , it may still be possible to reduce disease severity through a strategy of early aggressive treatment when atopic dermatitis first appears . it is known that the constant scratching associated with atopic dermatitis may expose parts of skin to cells that can trigger an autoimmune response , which could result in a more widespread and persistent disease , ( figure 3 ) . so interrupting that chain of events by clamping down early on atopic dermatitis is an avenue worth exploring . others have also suggested exploring disease modifying strategies in atopic dermatitis to break , stop or reverse atopic dermatitis and associated asthma , in a way that is heavily informed by biomarkers . other forms of secondary prevention such as weekend therapy ( applying topical treatments for two consecutive days each week once the disease is under control ) had been shown to have a large impact on reducing disease flares . whether the length and intensity of inducing the initial remission is important for long term control needs to be tested . the emergent theme seems to be one of not chasing atopic dermatitis but instead one of getting control then keeping control . in healthy skin the corneodesmosomes ( iron rods ) are intact throughout the stratum corneum . at the surface , the corneodesmosomes start to break down as part of the normal desquamation process , analogous to iron rods rusting ( a ) . in an individual genetically predisposed to atopic dermatitis , premature breakdown of the corneodesmosomes leads to enhanced desquamation , analogous to having rusty iron rods all the way down through the brick wall ( b ) . if the iron rods are already weakened , an environmental agent , such as soap , can corrode them much more easily . the brick wall starts falling apart ( c ) and allows the penetration of allergens ( d ) . ( reproduced with permission from cork mj , robinson da , vasilopoulos y , ferguson a , moustafa m , macgowan a , duff gw , ward sj , tazi - ahnini r. new perspectives on epidermal barrier dysfunction in atopic dermatitis : gene - environment interactions . although atopic dermatitis prevention has not been a very fruitful area of research in the sense that the same interventions have been explored repeatedly and inconclusively , recent gene discoveries coupled with different ways of thinking about disease progression has produced new ideas for primary and secondary disease prevention . the dry skin seen in atopic dermatitis , which has been recognized clinically yet largely ignored by science for many years , has undergone a sharp revival of interest with the discovery of mutations in the filaggrin gene . we predict this will result in studies that evaluate the potential protective effect of skin barrier enhancement in high - risk and even low - risk infants . such intervention studies are not without their challenges . for example , defining a new case of atopic dermatitis as opposed to transient irritant eczema ( which commonly occurs in infants ) is tricky , as will be the problem of how to avoid contamination of a control group if word gets out that emollients from birth might prevent eczema . selecting which emollient(s ) have true barrier enhancement properties and those that are also acceptable for long term use for families whose babies do not appear to have a problem in the first place is also challenging , especially as , paradoxically , some emollients can impair rather than repair the skin barrier . with regards to those studies that focus on inducing allergen tolerance at a young age , attention needs to be paid to the timing , amount and type of exposures to induce such tolerance and whether such tolerance leads to clinical benefit in terms of less atopic dermatitis and associated diseases . it is also unclear whether the conventional strategy of targeting a high risk population in atopic dermatitis prevention ( children of parents who have atopic disease ) , is correct as such a policy could miss around 40% of potentially preventable cases if the same risk factors operate . perhaps it would be more cost - effective to adopt a whole population - based approach in countries with high levels of atopic dermatitis , although a much stronger evidence base would be required before such a public health approach could be entertained . or perhaps the way forward is in the opposite direction , i.e. personalized medicine , so that prevention strategies for childhood disease are governed by genetic tests and biomarkers that determine disease risk . one thing is for certain , the whole field of atopic dermatitis prevention is exploding with new ideas . the biggest challenge will be to evaluate these ideas properly in large prospective randomized controlled trials . eric l simpson has received a research grant from galderma , who is the manufacturer of one of the emollients in the beep study . | atopic dermatitis now affects one in five children , and may progress to asthma and hay fever . in the absence of effective treatments that influence disease progression , prevention is a highly desirable goal . the evidence for most existing disease prevention strategies , such as avoidance of allergens and dietary interventions , has been unconvincing and inconsistent .
fresh approaches to prevention include trying to induce tolerance to allergens in early life , and enhancing the defective skin barrier to reduce skin inflammation , sensitisation and subsequent allergic disease .
early and aggressive treatment of atopic dermatitis represents another possible secondary prevention strategy that could interrupt the development of autoimmunity , which may account for atopic dermatitis persistence .
large scale and long term randomized controlled trials are needed to demonstrate that these ideas result in clinical benefit . |
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tooth agenesis is defined as congenital absence of one or a few permanent teeth without any systemic disorders and the prevalence varies from 2.6% to 11.3% ( excluding third molars ) . peg - shaped is defined as a developmental alteration in dental shape , which incisal mesio - distal width of the tooth crown is narrower than the cervical . upper lateral incisor agenesis is commonly associated to a peg - shaped in the contralateral . a previous study suggested that tooth agenesis and peg - shaped or strongly mesio - distally reduced upper lateral incisors could share the same genetic background . the etiology of tooth alterations in human is still unclear and has been the subject of numerous investigations to determine the patterns of development of different teeth , associations within and between the dental alterations and the relative degrees of influence of genetic and environmental factors . tooth development may fail due to both environmental and genetic factors . irradiation and chemotherapeutic agents can arrest tooth development . severe forms of tooth agenesis ( oligodontia ) have been linked to mutations or deletions in msh homeobox 1 , paired box 9 , axin2 , bone morphogenetic protein 4 and transforming growth factor beta 3 . familial clustering and higher concordance in monozygotic ( mz ) than in dizygotic twins also show the importance of genetic factors . the present case report aimed to study an uncommon case of mz twins , confirmed by deoxyribonucleic acid ( dna ) chip technology , which showed a discordant dental phenotype in primary and permanent dentition . mz twins caucasian girls were examined in a pediatric dental clinic at age 11 months . during intra - oral clinical and periapical radiograph examination , the twin a ( ta ) revealed a congenital absence of left primary upper lateral incisor [ figure 1a ] and the twin b ( tb ) had all the primary incisors . clinical examination of others tissues of ectodermal origin was also carried out and showed no abnormalities . dental development in twin a ( a = 11 months of age ; b = 3 years of age ; c = 7 years of age ; d = 9 years of age ) during the anamnesis , the mother reported no previous history of dental anomalies and/or others congenital alterations on neither side of the family . according to maternal reports , the twins share the same placenta , being mz ( identical ) , monochorionic . patients had follow - up twice a year and the dental development was clinically and radiographically observed [ figure 1b d ] . by the age 12 years , an orthopantomography confirmed that the left upper permanent lateral incisor was also absent and the right permanent upper lateral incisor was peg - shaped in the ta while tb had normal shaped permanent dentition [ figures 2 and 3 ] . the panoramic radiograph of twin a at 12 years of age the panoramic radiograph of twin b at 12 years of age the treatment of ta demands a multidisciplinary approach since it causes poor esthetics and functional problems . a prosthetic treatment using the esthetic space maintenance was carried out to the absent lateral . an implant procedure will be carried out in the future [ figures 4 and 5 ] . intra - oral pictures after peg - shaped upper lateral incisor restoration and prosthetic treatment of the twin a intra - oral pictures of twin b genomic dna of ta and tb were isolated from buccal epithelial cells following established protocol . the dna extraction was achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution . polymerase chain reaction amplified the purified dna sample from both twins that presented a 260/280 ratio higher than 1.8 . monozigotic was evaluated by comparisons of markers in the blood ( abo , rh ) , together with several dna variations . we used the affymetrix genechip human mapping 500k array set provides consistently high coverage across different populations . it is comprised of two arrays , each capable of genotyping on average 250,000 snps ( approximately 262,000 for nsp arrays and 238,000 for sty arrays ) . after that , we were able to verify that both girls presented strong similarities that confirmed the monozigosity . this investigation ( dental casts analysis ) was undertaken to compare morphological aspects and mesio - distal and vestibulo - lingual crown dimensions of the permanent dentition in ta and tb . based on dental features , except for the upper lateral incisors , there were no differences in the crown diameters of the two twins . genomic dna of ta and tb were isolated from buccal epithelial cells following established protocol . the dna extraction was achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution . polymerase chain reaction amplified the purified dna sample from both twins that presented a 260/280 ratio higher than 1.8 . monozigotic was evaluated by comparisons of markers in the blood ( abo , rh ) , together with several dna variations . we used the affymetrix genechip human mapping 500k array set provides consistently high coverage across different populations . it is comprised of two arrays , each capable of genotyping on average 250,000 snps ( approximately 262,000 for nsp arrays and 238,000 for sty arrays ) . after that , we were able to verify that both girls presented strong similarities that confirmed the monozigosity . this investigation ( dental casts analysis ) was undertaken to compare morphological aspects and mesio - distal and vestibulo - lingual crown dimensions of the permanent dentition in ta and tb . based on dental features , except for the upper lateral incisors , there were no differences in the crown diameters of the two twins . mz share similar dna sequences ; however , they are often discordant for some phenotypes . these discordances in mz twins are of great interest to the human genetics research , which wishes to gain further insight into the complexities of genetic - environmental interaction in development . in fact , tooth agenesis and peg - shaped seem to have a genetic determination . dental anomalies may represent a complex multifactorial trait , influenced by a combination of gene function , environmental factors and developmental timing . , that highlight some factors such as jaw fractures , surgical procedures , extraction of the preceding primary tooth , chemotherapy , irradiation , thalidomide during mother 's pregnancy , nutrient deprivation and serious illness . in our case report , the etiology of tooth agenesis could not be clearly associated with any of these environmental factors . displayed discordance for agenesis of permanent maxillary lateral incisor in five pair of mz twins and suggested a possible link with disparate birth weights of the twins . however , we do not suggest this relationship since the twin with the lower birth weight ( tb ) had all the teeth formed . it was expected that the twin who was born with lower weight had the absence of the missing tooth to prove the relationship between birth weight and agenesis . assuming that genetic information is identical for each mz twin , differences between them can be interpreted as a consequence of environmental factors . it is possible that epigenetic events may be responsible for discordant expression in genetically identical individuals . however , during pregnancy , the mother is the environment of the children , so this pair of twins were exposed to the same environmental factors . we can propose that differences between mz twins could be a consequence of gene mutation . in addition , the fact that the same individual ( ta ) , with no known family history for dental anomaly , was affected by both tooth agenesis and peg - shaped suggested that the same mutated gene could cause both anomalies . the inheritance pattern of tooth agenesis and peg - shaped upper lateral incisors demonstrated that they are different expressions of one gene with reduced penetrance . tooth agenesis is a complex condition affecting several parameters of oral development that may include the association of reducing in dental crown size dimension . in addition , in the dental casts analysis , we were able to note that only upper lateral incisor group were affected in ta and there was not differences between the others teeth dimensions . this finding reflected the possibility that different types of teeth may have independent developmental mechanisms and different genetic factors may be involved . in this case , contrary to previous beliefs , nowadays , it is assumed that mz twins are not genetically identical . previous studies have induced a paradigm shift to twin research , focusing on the genetic or epigenetic difference between mz discordant twins . ( 2005 ) studied dental alterations in mz twins and reinforce the point that using the term identical when referring to mz twin pairs can be misleading . we believe that a widely molecular investigation in mz twins with dental phenotype discordances are needed to allow the determination of genes involved with dental alterations . the clinicians need to recognize that mz twins may have different clinical characteristics , becoming of great importance the early diagnosis and implementation of appropriate treatment and monitoring , as performed in this case , minimizing the problems that can cause such a difference to the esthetic , functional and psychological aspects . | the present report aimed to study an uncommon case of a pair of twins that presented a discordant dental phenotype .
family investigation , clinical , radiographic examination and molecular analysis were performed in both girls .
molecular analysis confirmed the monozygosity by deoxyribonucleic acid chip technology .
one twin presented tooth agenesis in left upper lateral incisor and peg - shaped on the contra lateral side while the other twin had no dental alterations .
the dental casts study employed digital caliper to compare morphological dimensions and showed alteration only in peg - shaped tooth . in conclusion , this study provide support that one or more mutated gene could cause discordances in dental phenotype in these monozygotic twins . |
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an i. javanica isolate , which was isolated from a fungus - infected aleyrodidae adult in korea and showed high pathogenicity against the b. tabaci biotype q , was kept in 10% ( v / v ) glycerol at -20. the isolate was initially cultivated on potato dextrose agar ( pda ) at 25 1 for 14 days . for the mass production of this i. javanica isolate , brown rice and barley were purchased and stored in a refrigerator at 4 until use . to use grains as a substrate for conidia production , the 2 grains were soaked in tap water for 2 hr and placed into a plastic basket for 1 hr to remove water . one hundred grams of grain was put into an autoclavable plastic bag and autoclaved at 121 for 60 min . to understand the effects of different additives on conidia production by solid - state cultivation using grains as a substrate , a carbon or nitrogen source or trace minerals were added into 15ml conidia suspension ( 1 10 conidia / ml ) and dissolved . the additives investigated were as follows : 2% ( w / v ) soluble starch ( daejung co. , busan , korea ) , 2% ( w / v ) mannose ( sigma - aldrich co. , st . louis , mo , usa ) , 2% ( w / v ) caco3 and 2% ( w / v ) caso4 ( sigma - aldrich co. ) , 1.32% and 3% ( w / v ) of hydrolyzed casein ( sigma - aldrich co. ) , 1.32% and 3% ( w / v ) of difco yeast extract ( becton dickinson co. , franklin lakes , nj , usa ) , 0.6% and 1% ( w / v ) of kno3 ( sigma - aldrich co. ) , and 1.32% and 3% ( w / v ) of hydrolyzed wheat gluten protein ( sigma - aldrich co. ) . the conidia suspension was then inoculated onto barley or brown rice in a plastic bag , mixed well , and incubated at 25 1 for 15 days . the grain was added to 27 ml 0.05% sterilized tween 80 , vortexed for 1 min , and then the suspension was filtered through 4 layers of sterilized cheesecloth . the number of conidia was counted using an improved neubauer haemocytometer ( sigma - aldrich co. ) . in each trial , each cultivation condition was replicated in 3 different bags , and the trial was repeated 4 times . to test the virulence of conidia produced using solid substrates and different additives , each conidia suspension was sprayed onto second instar nymphs of tobacco whitefly , as previously described . briefly , to prepare synchronized nymphs , fifteen pairs of adult whiteflies were infested overnight on a potted eggplant seedling with 1 leaf , which was prepared by removing the other leaves from a 30-day - old pruned whole eggplant seedling , and then the adult whiteflies were removed . the seedlings were grown in a plexiglas cage ( 35 40 40 cm ) at a constant temperature of 25 1 with a 16l : 8d lighting schedule for 11 days until the whiteflies reached their second instar . for virulence tests , conidia were harvested as described above from the barley or brown rice cultures amended with different additives after 15 days cultivation . the eggplant leaf discs infested with second instar nymphs of b. tabaci were sprayed with each conidia suspension at 3 different concentrations ( 10 , 10 , and 10 conidia / ml ) . one milliliter of each suspension was applied to leaf discs using a spray box ( 90 90 90 cm ) fitted with a polyvinyl acetal cone nozzle ( 1.5 mm ) . the spray tower was a plexiglas cylinder and the spray nozzle was installed through the top layer and connected to a vacuum pump , which was fixed at the pressure setting of 100 kpa . the number of conidia applied to each leaf disc was estimated by counting the spores that landed on a petri dish ( 3.5 cm ) containing 2 ml of 0.05% tween 80 that was placed beside the eggplant seedlings during the spore application . the total number of conidia sprayed within the petri dish area was determined using a hemocytometer . the average conidia deposition on each leaf disc was calculated to be 2.8~3.2 conidia / mm for the application of 10 conidia / ml and 10.7~13.6 conidia / mm for the application of 10 conidia / ml . spore viability was determined by germination tests of the conidia suspensions after 24 hr incubation at 25 1 on pda medium . one - way analysis of variance was used to compare conidia production and mortality among the different additives used for cultivation on each grain substrate . median lethal time ( lt50 ) was estimated using the lifereg procedure and data were fitted to a weibull distribution . the differences in conidia yield , mortality and lt50 at 2 different time points ( 4 and 6 days after treatment ) among different additives in each grain were compared using the least significant difference test ( < 0.05 ) . the production of i. javanica conidia differed among the substrate and additive combinations investigated after 15 days cultivation ( brown rice , f = 8.48 ; df = 11 , 47 ; p < 0.0001 and barley , f = 3.78 ; df = 11 , 47 ; p = 0.0012 ) . barley ( 3.43 10 conidia / g ) was a higher - yielding solid substrate for conidia production than brown rice ( 3.05 10 conidia / g ) . on barley , the addition of yeast extract , casein , or gluten to barley improved conidia production compared with cultivation on barley without additives . when starch or mannose was mixed with barley as the solid substrate , the number of conidia produced was lower ( 2.33 10 and 2.15 10 conidia / g , respectively ) than the number produced by cultivation without additives ( 3.43 10 conidia / g ) . the addition of gluten ( 3% and 1.32% ) to brown rice improved the conidia production yield by 14 and 6 times , respectively , compared with that of brown rice without additives ( 3.05 10 conidia / g ) . the addition of potassium nitrate ( kno3 ) , yeast extract , 2% caco3 and 2% caso4 , or mannose to brown rice did not increase conidia production compared with cultivation on brown rice without additives . the addition of gluten stimulated conidia production on both solid substrates , but the addition of mannose and kno3 suppressed the production of conidia of the i. javanica isolate following cultivation on barley and brown rice . the mortality of sweet potato whitefly treated with the conidia suspension produced on barley was generally higher ( 13% at 10 conidia / ml , 36% at 10 conidia / ml , and 62% at 10 conidia / ml ) than that of whitefly treated with the conidia suspension produced on brown rice ( 14% , 23% , and 53% , respectively ) at 4 days after treatment ( pooled t tests : 10 conidia / ml , t = 0.16 , p = 0.8880 ; 10 conidia / ml , t = -0.55 , p = 0.6374 ; 10 conidia / ml , t = -0.84 , p = 0.4889 ) . however , these changes were not significant , and conidia virulence showed non - significant trends toward differences between the solid substrates . differences in mortality at the sixth day after application showed trends similar to those on the fourth day , with mortality rates of 57% , 81% , and 100% for barley - cultivated conidia and 46% , 74% , and 100% for brown rice at the 3 concentrations of conidia applied , respectively . the inclusion of additives in the solid - state cultivation procedure changed conidia virulence ( fig . conidia produced on barley with different additives did not statistically increase whitefly mortality at 10 , 10 , and 10 conidia / ml treatment ( anova : 10 conidia / ml , f = 0.41 , df = 11 , 23 , p = 0.9236 ; 10 conidia / ml , f = 1.04 , df = 11 , 23 , p = 0.4681 ; 10 conidia / ml , f = 4.19 , df = 11 , 23 , p = 0.0103 ) . the median lethal time ( lt50 ) was shorter for conidia produced with the addition of mannose and starch at 10 ( f = 0.58 ; df = 11 , 23 ; p = 0.8096 ) , 3% gluten and 1% kno3 at 10 ( f = 0.41 ; df = 11 , 23 ; p = 0.9228 ) , and 1.32% and 3% yeast extract , 0.6% and 1% kno3 , and 1.32% and 3% gluten at 107 conidia / ml treatment ( f = 30.80 ; df = 11 , 23 ; p < 0.0001 ) . mortality of whitefly treated with conidia that were cultivated on brown rice with additives was lower than that of the conidia produced on brown rice without additives although this difference was not significant ( table 2 ) . the lt50 differed among additives ; at 10 conidia / ml , the addition of 1.32% gluten and 1.32% casein resulted in lower lt50 values ( 6.2 days for both additives , compared with 6.3 days for substrate without additives ) ( f = 1.00 ; df = 11 , 23 ; p = 0.4962 ) , while at 10 conidia / ml , the addition of 0.6% and 1% kno3 , 1.32% gluten , and 1.32% yeast extract resulted in lt50 values of 4.6 days , 4.4 days , 4.5 days , and 4.6 days , respectively , compared with 4.7 days for conidia produced by cultivation without additives ( f = 0.50 ; df = 11 , 23 ; p = 0.8716 ) , and at 10 conidia / ml , the addition of 1.32% and 3% gluten , 0.6% and 1% kno3 and 3% yeast extract led to lt50 values of 3.1 days , 3.3 days , 3.5 days , 3.4 days , and 3.5 days , which were significantly decreased compared with the lt50 of 3.8 days for conidia produced by cultivation without additives ( f = 11.73 ; df = 11 , 23 ; p < 0.0001 ) . the production yield and virulence of i. javanica conidia varied among different combinations of additives and solid substrates used for cultivation . gluten and kno3 addition commonly increased the conidia production and virulence of the korean isolate of i. javanica under the different conditions investigated . a p. fumosoroseus isolate produced more blastospores in liquid medium that was supplemented with casein or yeast extract than in unsupplemented medium . similarly , tobacco whitefly pathogenic p. fumosoroseus produced more blastospores in liquid culture media supplemented with glucose and casamino acid . the addition of casein , yeast extract , or corn gluten as nitrogen sources , and of glucose or casein as carbon sources into liquid culture media increased the production yield of p. fumosoroseus spores , and the spores also showed enhanced tolerance to dessication . casein and yeast extract supplementation commonly increased the production of p. fumosoroseus blastospores in different liquid media . while many studies have previously investigated the effects of different liquid media and additives on the production and virulence of entomopathogenic fungal spores , few studies p. fumosoroseus , which was isolated from the cadavers of lepidopteran caterpillars in india , showed higher conidia production when cultivated on sorghum ( 10.4 10 spores/100 g ) than when cultivated on corn , rice , pearl millet , or wheat . sporulation of m. anisopliae was stimulated on agar medium by the addition of starch , mannose , or kno3 . soluble starch and mannose were the most effective stimulators of m. anisopliae sporulation . in this study , when the i. javanica isolate was cultivated on brown rice or barley with the addition of starch or mannose , conidia production was decreased . conidia of m. anisopliae cultured in sabouraud dextrose agar with added kcl showed a decreased germination rate and the strength of spore attachment onto the host cuticle differed from that of conidia produced in liquid culture media . nomuraea rileyi produced more conidia when cultivated on wheat with added yeast extract than when cultivated on wheat with added brewer 's yeast or whole milk powder . rice was the best solid substrate for the production of v. lecanii spores , compared with sorghum , corn , ragi , and wheat . the sporulation of p. lilacinus , which was isolated from nematodes , was stimulated when it was cultivated on agar medium containing added mannose , sucrose , or starch . the addition of 2% caco3 and 2% caso4 into grain cultivation bags prevented grains from sticking together and thus provided an increased surface area for fungal growth . in our study , the i. javanica isolate showed good sporulation on barley . when the isolate was cultivated on barley with added caco3 plus caso4 or gluten , conidia production was significantly increased , but mannose , starch , or kno3 addition inhibited conidia production . thus , the use of additives for the solid phase cultivation of entomopathogenic fungi led to differences in the yields of conidia production . for the mass production of fungal conidia , each isolate needs to be studied to determine the optimal solid substrate and additive ingredient(s ) . spore attachment , germination , and the activity of enzymes , including proteases such as pr1 , are important factors for fungal virulence . these characteristics may be influenced by spore production conditions such as nutritional ingredients in the cultivation media . the influence of additives such as casamino acid on the spore production and virulence of various fungal isolates has been reported to vary among different media . for example , a m. anisopliae isolate cultured in medium with added casamino acids showed increased 40% mortality against a. aegypti compared with the same isolate cultured in medium without added casamino acids . according to shah et al . , m. anisopliae produced highly virulent conidia when cultivated in osmotic stress medium , and the conidia showed higher pr1 enzyme activity . the addition of yeast extract and peptone to cultivation media also increased the pr1 activity of m. anisopliae conidia , but the relationship between virulence and pr1 activity was not clear in that study . the addition of lecithin , collagen , or lactic acid into culture broth increased the production of m. anisopliae blastospores , but the virulence of the spores was similar or lower than that of spores produced in media without additives . when conidia of lecanicillium sp . were produced by cultivation in media with the addition of 2% glucose and sprayed at low concentration ( 10 conidia / ml ) , the infection rate of whitefly was higher than that of conidia produced by cultivation in media with the addition of maltose and peptone . however , at a higher dosage ( 10 conidia / ml ) , there was no significant difference between the infection rates of the conidia produced by cultivation with different additives . the addition of trace minerals such as kj , znso4 , or kbr to agar medium increased the pathogenicity of b. bassiana conidia . for i. javanica , which is a sweet potato whitefly pathogen , the virulence of conidia was increased when the isolate was cultured with the addition of gluten on both brown rice and barley solid substrates . conidia cultivated on barley with added kno3 or yeast extract also showed improved mortality against tobacco whitefly , compared with conidia cultivated on barley without additives . we expected that when the isolate was cultured with additives , highly virulent conidia would be produced , allowing us to spray a low concentration of spores onto crops but still achieve a high control efficacy against whitefly . however , against our expectations , the virulence of the conidia was only improved at a high spraying concentration ( 10 conidia / ml ) . at spraying concentrations of 10 and 10 conidia / ml , the lt50 did not significantly differ among the conidia produced by cultivation on solid substrates with different additives . , whose study found that the addition of glucose to cultivation media led to the production of v. lecanii conidia that produced a higher mortality rate in their host ( t. vaporariorum ) . we will conduct future studies to investigate why virulence was increased by the addition of gluten , kno3 , or yeast extract at the highest spraying concentration of conidia . in conclusion , the production of sweet potato whitefly pathogenic i. javanica conidia was increased on barley by the addition of caco3+caso4 and on brown rice by the addition of gluten . gluten addition also improved the virulence of conidia cultivated on both substrates . compared with conidia produced on barley without additives , conidia produced on barley with added kno3 or yeast extract also showed improved virulence against tobacco whitefly . | recently , the q biotype of tobacco whitefly has been recognized as the most hazardous strain of bemisia tabaci worldwide , because of its increased resistance to some insecticide groups . as an alternative control agent , we selected an isaria javanica isolate as a candidate for the development of a mycopesticide against the q biotype of sweet potato whitefly . to select optimal mass production media for solid - state fermentation , we compared the production yield and virulence of conidia between 2 substrates ( barley and brown rice ) , and we also compared the effects of various additives on conidia production and virulence .
barley was a better substrate for conidia production , producing 3.43 1010 conidia / g , compared with 3.05 1010 conidia / g for brown rice .
the addition of 2% caco3 + 2% caso4 to barley significantly increased conidia production .
addition of yeast extract , casein , or gluten also improved conidia production on barley .
gluten addition ( 3% and 1.32% ) to brown rice improved conidia production by 14 and 6 times , respectively , relative to brown rice without additives .
conidia cultivated on barley produced a mortality rate of 62% in the sweet potato whitefly after 4-day treatment , compared with 53% for conidia cultivated on brown rice .
the amendment of solid substrate cultivation with additives changed the virulence of the conidia produced ; the median lethal time ( lt50 ) was shorter for conidia produced on barley and brown rice with added yeast extract ( 1.32% and 3% , respectively ) , kno3 ( 0.6% and 1% ) , or gluten ( 1.32% and 3% ) compared with conidia produced on substrates without additives . |
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invasive mycoses represent a major cause of morbidity and mortality in patients with malignancy or undergoing hematopoietic stem cell transplantation ( hsct ) ( 1 , 2 ) . patients with hematologic malignancies are currently at higher risk of invasive fungal infections ( ifi ) caused by molds than yeasts , and the incidence of ifi is the highest among patients with acute myeloid leukemia . invasive aspergillosis ( ia ) occurs more often in patients with acute leukemia than in patients with chronic leukemia , lymphomas or multiple myeloma ( 4 ) . in most reports , prognosis of the established aspergillosis is dismal ; therefore , its prevention is of utmost importance . a meta - analysis of 50 studies on aspergillosis revealed mortality rates of approximately 60% in patients with acute leukemia and lymphoma and up to 90% in allogeneic stem cell recipients ( 5 ) . definitive diagnosis of ia requires histopathological evidences of deep - tissue invasion or a positive culture from normally sterile sites ( 6 ) . to diagnose the invasive pulmonary aspergillosis ( ipa ) in high - risk patients , bronchoalveolar lavage ( bal ) specimen early and precise diagnosis of ipa is important to start antifungal treatment in time and reduce the unnecessary use of toxic antifungal agents . although traditional approaches such as direct microscopic examination , histopathological evaluation and cultivation are still the gold standard , the diagnosis of ipa is generally difficult because of their inadequate sensitivity and specificity ( 7 ) . the current study aimed to evaluate the incidence of ipa in hsct and hematological malignancies patients by using bal specimens , and also the diagnostic potential of nested polymerase chain reaction ( pcr ) and real - time pcr were compared with conventional methods . during a 16 month period ( june 2009 to october 2010 ) , 46 consecutive bal fluid specimens were obtained from patients with hematological malignancies and hsct , at shariati hospital and medical mycology laboratory in the school of public health , tehran , iran . a portion of bal specimens ( 4 - 7 ml ) were obtained by specialist physicians under standardized techniques ( 8) , and then collected in sterile vessels free of preservatives , and transferred to the laboratory within one hour . the pellet was vortexed vigorously and resuspended in a small volume of supernatant , with the final volume of 400 to 600 microlitter . seventy - five - microliter aliquots of the specimens were planted on sabouraud glucose agar ( 4% ) ( difco , usa ) and brain heart infusion agar ( difco , usa ) plates equally ( total 150 l ) ; then were incubated for three to seven days at 30c . a wet mount was prepared with 150 l of the sediment with a drop of 20% potassium hydroxide ( koh ) , and examined by 100 and 400 magnification of microscope . for cell lysis of bal fluid , each specimen was subjected to four times freezing in liquid nitrogen and thawing in boiling water , and then was crushed with a conical grinder for one minute . for sticky and viscous specimens , 2% dithiothreitol ( wako , japan ) was added , then sonicated for two to five minutes ( 9 ) . finally , dna was extracted from each bal specimen by phenol - chloroform method as described by makimura et al . with a little modification ( dna of four specimens the homogenized bal sediment was added to an equal volume of the lysis buffer ( 100 mm tris - hcl , ph = 8 , 10 mm edta , 0.1% w / v sds , 100 mm nacl , 2% v / v triton 100x ) and an equal volume of phenol chloroform isoamyl alcohol ( 25 : 24 : 1 ) , and was centrifuged at 5'000 g for 10 minutes . chloroform was added to the supernatant and was centrifuged at 5'000 g for five minutes . sodium acetate ( 3 m ; 0.1 volume ) and 2-propanol ( equal volume ) were added to the supernatant and kept at -20c for 10 minutes , and then was centrifuged at 12'000 g for 12 minutes . the precipitated dna was washed with 300 l of 70% ethanol and re - suspended in 30 l of ultrapure water and was kept at -20c . furthermore , the analytical specificity of the pcr was assessed by testing the following fourteen genomic dna of standard fungal strains , which were provided by teikyo university institute of medical mycology ( timm ) , tokyo , japan : a. flavus ( jcm 2061 ) , a. fumigatus ( atcc 26430 , timm 3968 ) , a. niger ( timm 0113 ) , candida tropicalis ( atcc 750 ) , c. albicans ( timm 3313 ) , c. glabrata ( atcc 90030 ) , c. parapsilosis ( atcc 22019 ) , penicillium expansum ( timm 1293 ) , p. notatum ( timm 0883 ) , p. marneffei ( cbs 334.59 ) , fusarium solani ( tsy 0403 ) , trichosporon asahii ( cbs 2479 ) and sporobolomyces koalae ( jcm 18063 ) . all the primers used for the nested pcr had been designed by sugita et al . ( 11 ) . the pcr mixture for contained 12.5 l 2 premix ( ampliqon , denmark ) , 0.5 l ( 30 pmol ) of each primer , and 2 l of the dna template solution , and enough distilled water up to 25 l . the mixture was heated to 95c for five minutes followed by 30 cycles of 94c for 45 seconds ; 60c for one minute ; and 72c for one minute and a final step at 72c for seven minutes . for the species specific primer sets of a. flavus and a. fumigatus , 1 l of the first pcr product ( diluted 1/100 ) was used as a template . each mixture was heated to 95c for five minutes and pcr was performed in cycles of 94c for 50 seconds ; 58c for 40 seconds and 72c for 45 seconds for 25 cycles and a final extention at 72c for eight minutes . the reactions were run with a thermal cycler ( takara pcr thermal cycler dice mini , tp100 . real - time pcr was performed with a 7500 fast real - time pcr system ( applied biosystems ) based on taqman chemistry . the real - time pcr primers and probes were designed based on multiple alignments of various sequences of partial -tubulin gene by zarrinfar et al . as described previously all of the real - time pcr primers and probes were obtained from applied biosystems ( usa ) . reactions were performed using eagle taq mastermix with rox ( roche , basel , switzerland ) according to the manufacturer s recommended protocol . each reaction mixture contained 2 l of template dna solution , 0.2 l ( 10.4 m ) of probes a. fumi - p , 0.16 l ( 12.9 m ) of probe a. flavus - p , 0.4 l ( 10 m ) of primer a. fumi - f , 0.4 l ( 10 m ) of primer a. fumi - r , 0.4 l ( 10 m ) of primer a. flavus - f , 0.4 l ( 10 m ) of primer a. flavus - r , 10 l of master mix and enough distilled water up to the final volume of 20 l . following an initial denaturation step at 95c for 10 minutes , pcr amplification was performed for 45 cycles consisting of 95c for three seconds and 60c for 30 seconds dna extracted from a. fumigatus ( timm 3968 ) and a. flavus ( timm 2912 ) were used as positive and three tubes containing water instead of dna were used as negative controls in each run . a. fumigatus and a. flavus pcr products were cloned using a ta cloning kit and pcr 2.1 vector ( invitrogen corp ) . the pcr 2.1 plasmid containing the dna target was sequenced to confirm the insertion of a single copy of amplicon . the quantitation , accuracy , and precision of the real - time pcr assay were validated via preparation of serial dilutions of the plasmid containing the cloned target . the dna concentration was determined according to the absorbance at 260 nm and molecular weight of the plasmid . in addition , the plasmid was used as the positive control in all reaction runs . quantitative results were expressed by determination of the threshold cycle ( ct ) of detection , or crossing point ( cp ) , which marked the cycle at which fluorescence of the specimen became significantly different from the baseline signal . the analysis of the results for a positive specimen was designated when at least one replicate had a ct value of 38 cycles . the efficacy of the dna isolation system in releasing aspergillus dna from the fungal cells for pcr were evaluated by analyzing serial dilutions of a. fumigatus conidia in seven concentrations ( 10 , 10 , 10 , 10 , 10 , 10 and 10 conidia / ml saline ) . seventy - five - microliter aliquots of the specimens were planted on sabouraud glucose agar ( 4% ) ( difco , usa ) and brain heart infusion agar ( difco , usa ) plates equally ( total 150 l ) ; then were incubated for three to seven days at 30c . a wet mount was prepared with 150 l of the sediment with a drop of 20% potassium hydroxide ( koh ) , and examined by 100 and 400 magnification of microscope . for cell lysis of bal fluid , each specimen was subjected to four times freezing in liquid nitrogen and thawing in boiling water , and then was crushed with a conical grinder for one minute . for sticky and viscous specimens , 2% dithiothreitol ( wako , japan ) was added , then sonicated for two to five minutes ( 9 ) . finally , dna was extracted from each bal specimen by phenol - chloroform method as described by makimura et al . with a little modification ( dna of four specimens the homogenized bal sediment was added to an equal volume of the lysis buffer ( 100 mm tris - hcl , ph = 8 , 10 mm edta , 0.1% w / v sds , 100 mm nacl , 2% v / v triton 100x ) and an equal volume of phenol chloroform isoamyl alcohol ( 25 : 24 : 1 ) , and was centrifuged at 5'000 g for 10 minutes . chloroform was added to the supernatant and was centrifuged at 5'000 g for five minutes . sodium acetate ( 3 m ; 0.1 volume ) and 2-propanol ( equal volume ) were added to the supernatant and kept at -20c for 10 minutes , and then was centrifuged at 12'000 g for 12 minutes . the precipitated dna was washed with 300 l of 70% ethanol and re - suspended in 30 l of ultrapure water and was kept at -20c . furthermore , the analytical specificity of the pcr was assessed by testing the following fourteen genomic dna of standard fungal strains , which were provided by teikyo university institute of medical mycology ( timm ) , tokyo , japan : a. flavus ( jcm 2061 ) , a. fumigatus ( atcc 26430 , timm 3968 ) , a. niger ( timm 0113 ) , candida tropicalis ( atcc 750 ) , c. albicans ( timm 3313 ) , c. glabrata ( atcc 90030 ) , c. parapsilosis ( atcc 22019 ) , penicillium expansum ( timm 1293 ) , p. notatum ( timm 0883 ) , p. marneffei ( cbs 334.59 ) , fusarium solani ( tsy 0403 ) , trichosporon asahii ( cbs 2479 ) and sporobolomyces koalae ( jcm 18063 ) . all the primers used for the nested pcr had been designed by sugita et al . ( 11 ) . the pcr mixture for contained 12.5 l 2 premix ( ampliqon , denmark ) , 0.5 l ( 30 pmol ) of each primer , and 2 l of the dna template solution , and enough distilled water up to 25 l . the mixture was heated to 95c for five minutes followed by 30 cycles of 94c for 45 seconds ; 60c for one minute ; and 72c for one minute and a final step at 72c for seven minutes . for the species specific primer sets of a. flavus and a. fumigatus , 1 l of the first pcr product ( diluted 1/100 ) was used as a template . each mixture was heated to 95c for five minutes and pcr was performed in cycles of 94c for 50 seconds ; 58c for 40 seconds and 72c for 45 seconds for 25 cycles and a final extention at 72c for eight minutes . the reactions were run with a thermal cycler ( takara pcr thermal cycler dice mini , tp100 . real - time pcr was performed with a 7500 fast real - time pcr system ( applied biosystems ) based on taqman chemistry . the real - time pcr primers and probes were designed based on multiple alignments of various sequences of partial -tubulin gene by zarrinfar et al . as described previously ( 12 ) . all of the real - time pcr primers and probes were obtained from applied biosystems ( usa ) . reactions were performed using eagle taq mastermix with rox ( roche , basel , switzerland ) according to the manufacturer s recommended protocol . each reaction mixture contained 2 l of template dna solution , 0.2 l ( 10.4 m ) of probes a. fumi - p , 0.16 l ( 12.9 m ) of probe a. flavus - p , 0.4 l ( 10 m ) of primer a. fumi - f , 0.4 l ( 10 m ) of primer a. fumi - r , 0.4 l ( 10 m ) of primer a. flavus - f , 0.4 l ( 10 m ) of primer a. flavus - r , 10 l of master mix and enough distilled water up to the final volume of 20 l . following an initial denaturation step at 95c for 10 minutes , pcr amplification was performed for 45 cycles consisting of 95c for three seconds and 60c for 30 seconds dna extracted from a. fumigatus ( timm 3968 ) and a. flavus ( timm 2912 ) were used as positive and three tubes containing water instead of dna were used as negative controls in each run . a. fumigatus and a. flavus pcr products were cloned using a ta cloning kit and pcr 2.1 vector ( invitrogen corp ) . the pcr 2.1 plasmid containing the dna target was sequenced to confirm the insertion of a single copy of amplicon . the quantitation , accuracy , and precision of the real - time pcr assay were validated via preparation of serial dilutions of the plasmid containing the cloned target . the dna concentration was determined according to the absorbance at 260 nm and molecular weight of the plasmid . in addition , the plasmid was used as the positive control in all reaction runs . quantitative results were expressed by determination of the threshold cycle ( ct ) of detection , or crossing point ( cp ) , which marked the cycle at which fluorescence of the specimen became significantly different from the baseline signal . the analysis of the results for a positive specimen was designated when at least one replicate had a ct value of 38 cycles . the efficacy of the dna isolation system in releasing aspergillus dna from the fungal cells for pcr were evaluated by analyzing serial dilutions of a. fumigatus conidia in seven concentrations ( 10 , 10 , 10 , 10 , 10 , 10 and 10 conidia / ml saline ) . forty - six bal specimens were obtained from allogeneic hsct ( n = 18 ) and patients with hematological malignancies ( n = 28 ) , 70% being men ( n = 32 ) with an average age of 38 and 30% being women ( n = 14 ) with an average age of 35 years . among the patients with hematological malignancies , six were all ( acute lymphoblastic leukemia ) ; 11 aml ( acute myeloid leukemia ) ; five had cll ( chronic lymphocytic leukemia ) ; one had nhl ( non - hodgkin lymphoma ) and also five had lymphoma ( table 1 ) . according to the eortc / msg 2008 criteria ( 13 ) , a diagnosis of proven ia can be established by the culture from a sterile tissue biopsy specimen , or the needle aspiration , or the microscopic detection of branched septate hyphae in such specimens with histopathological evidence of the associated tissue damage . is regarded as evidence for probable infection in a high - risk patient with relevant clinical and radiological findings . the radiological findings include typical pulmonary high resolution computed tomographic findings , such as the halo sign , air - crescent sign , or a cavity with a consolidated area , also findings in other tissues suggestive of fungal infection . if the radiological and mycological evidence for ia are not obtained but include the host factor and clinical criteria consistent with the infection , the diagnosis can only be classified as possible . = 1 ) were classified as proven ipa , 21.7% ( n = 10 ) as probable ipa , 41.3% ( n = 19 ) as possible ipa , and 34.8% ( n = 16 ) as not ipa ( table 2 ) . abbreviations : bal , bronchoalveolar lavage fluid ; all , acute lymphoblastic leukemia ; aml , acute myeloid leukemia ; cll , chronic lymphocytic leukemia ; nhl , non - hodgkin lymphoma . abbreviations : ipa , invasive pulmonary aspergillosis ; all , acute lymphoblastic leukemia ; aml , acute myeloid leukemia ; cll , chronic lymphocytic leukemia ; nhl , non - hodgkin lymphoma ; hsct , hematopoietic stem cell transplants . some of bal specimens had positive results in nested pcr for a. flavus and a. fumigatus , and in real - time pcr for a. flavus and a. fumigatus , together . branched septated hyphae were observed in wet mount preparitons of seven ( 15.2% ) bal specimens , and one tissue biopsy specimen in the direct microscopic examination . eleven ( 23.9% ) specimens were positive in the culture including eight ( 72.7% ) a. flavus and three ( 27.3% ) a. fumigatus . of the 46 specimens tested , 22 ( 47.8% ) were positive by the nested pcr including 13 a. flavus and one a. fumigates specimens , and eight specimens were positive for both a. flavus and a. fumigatus . among the 10 probable ipa specimens , two ( 20% ) showed positive results for a. flavus and three ( 30% ) for both a. flavus and a. fumigatus . among the 19 specimens with possible ipa , five ( 26% ) showed positive results of the nested pcr for a. flavus , 1 ( 5% ) for a. fumigatus and three ( 16% ) for both a. flavus and a. fumigatus . of the 16 specimens with not ipa , five ( 31% ) had positive nested pcr for a. flavus and two ( 12.5% ) were positive for both a. flavus and a. fumigatus . eight specimens showed positive results by real - time pcr , including five for a. flavus and three for a. fumigatus . one of the specimens showed positive results for both a. flavus and a. fumigatus . between the 10 specimens with probable ipa , two ( 20% ) of the , two ( 10.5% ) out of 19 specimens with possible ipa had positive real - time pcr results , which one of them was positive for a. flavus and the other one for both a. flavus and a. fumigatus . two ( 12.5% ) out of the 16 specimens without ipa showed positive real - time pcr results for a. flavus and one ( 6.25% ) showed positive real - time pcr result for a. fumigatus . in the current study , the lower limit of detection ( lod ) of used taqman real - time pcr method was 35 copies per assays for a. fumigatus and 40 copies per assays for a. flavus . specificity assays did not show cross - reaction with dna of the other fungal standard strains . in addition , the lowest detectable number of conidia in 1 ml saline was 10 , which would be detectable by the molecular methods . branched septated hyphae were observed in wet mount preparitons of seven ( 15.2% ) bal specimens , and one tissue biopsy specimen in the direct microscopic examination . eleven ( 23.9% ) specimens were positive in the culture including eight ( 72.7% ) a. flavus and three ( 27.3% ) a. fumigatus . of the 46 specimens tested , 22 ( 47.8% ) were positive by the nested pcr including 13 a. flavus and one a. fumigates specimens , and eight specimens were positive for both a. flavus and a. fumigatus . among the 10 probable ipa specimens , two ( 20% ) showed positive results for a. flavus and three ( 30% ) for both a. flavus and a. fumigatus . among the 19 specimens with possible ipa , five ( 26% ) showed positive results of the nested pcr for a. flavus , 1 ( 5% ) for a. fumigatus and three ( 16% ) for both a. flavus and a. fumigatus . of the 16 specimens with not ipa , five ( 31% ) had positive nested pcr for a. flavus and two ( 12.5% ) were positive for both a. flavus and a. fumigatus . eight specimens showed positive results by real - time pcr , including five for a. flavus and three for a. fumigatus . one of the specimens showed positive results for both a. flavus and a. fumigatus . between the 10 specimens with probable ipa , two ( 20% ) were positive for a. fumigatus . of the , two ( 10.5% ) out of 19 specimens with possible ipa had positive real - time pcr results , which one of them was positive for a. flavus and the other one for both a. flavus and a. fumigatus . two ( 12.5% ) out of the 16 specimens without ipa showed positive real - time pcr results for a. flavus and one ( 6.25% ) showed positive real - time pcr result for a. fumigatus . in the current study , the lower limit of detection ( lod ) of used taqman real - time pcr method was 35 copies per assays for a. fumigatus and 40 copies per assays for a. flavus . specificity assays did not show cross - reaction with dna of the other fungal standard strains . in addition , the lowest detectable number of conidia in 1 ml saline was 10 , which would be detectable by the molecular methods . as a reliable and rapid diagnosis would improve the survival rate in high - risk patients especially among hsct and patients with hematological malignancies , the current study evaluated routine laboratory methods , nested pcr and real - time pcr to diagnose pa due to a. fumigatus and a. flavus . the assay was carefully validated and applied to an analysis of bal specimens . to define ipa , the study used the diagnostic criteria described by the european organization for research and treatment of cancer / invasive fungal infections cooperative group and the national institute of allergy and infectious diseases , mycoses study group ( eortc / msg ) ( 13 ) . infection is one of the main causes of death in hsct ( 14 ) and patients with hematological malignancies ( 15 ) . on time although conventional mycological and histopathological methods are still useful for a definite diagnosis of ipa , new non - invasive diagnostic methods including molecular biomarkers are now available ( 16 ) . these new diagnostic methods facilitate an early diagnosis of invasive fungal disease and allow for utilization of a pre - emptive treatment approach , which may ultimately lead to improved treatment outcomes in hsct and patients with hematological malignancies . although microscopic examination and cultivation of clinical specimens for pa diagnosis are still gold - standard methods ; in general , they do not have enough sensitivity and specificity . furthermore these methods show positive results only in the end stages of infection where an increased fungal burden exists . on the other hand , furthermore , fungal culture is often confounded by antifungal treatment , since the initiation of empirical treatment is a common practice in hsct and patients with hematological malignancies . although there are many published articles on the application of pcr to detect aspergillus dna , to date , a standard commercially developed molecular diagnostic test is not available . real - time pcr assay is widely used to detect fungal pathogens in the molecular studies , and considerably rapid and highly sensitive results are obtained in pediatric patients ( 7 ) . according to the definition provided by eortc / msg ( 13 ) , the ipa incidence of patients examined in the current study was 28% and 21% in hsct and patients with hematological malignancies , respectively . while in the other reported studies ( 3 , 7 , 17 ) , the incidence of ipa was lower than that of the current study . in the current study , 7 ( 15.2% ) specimens were positive in direct examination while the positive results of culture ( 23.9% ) , real - time pcr ( 17.4% ) and nested pcr ( 47.8% ) were higher than it . only two specimens had positive results by all the four methods ; however , two other specimens showed positive results in direct examination , culture and nested pcr together . the reasons why there were mismatches between the various methods is not quite clear . potential explanation includes the possibility of the empirical therapies in these patients ( 18 ) , lower sensitivity of traditional methods and specimen contamination ( 7 ) . moreover , isolation of the fungus from non - sterile specimens such as bal specimen may also reflect colonization of the airway instead of invasive infection ( 19 ) . in the present study , unlike most reports that show a. fumigatus as a common cause of pa , in this study , a. flavus was the most frequent species isolated by culture and pcr while a. fumigatus was the second etiologic agent ( 20 ) . these findings may be consistent with the increase of non - fumigatus aspergillus spp . and or other studies conducted in iran have shown that a. flavus is the most frequent species isolated from patients and air ( 12 , 21 - 23 ) . in the 16 specimens with not ipa , none of them had a positive direct examination and culture but seven and three of the specimens had positive nested pcr and positive real - time pcr , respectively . this may be due contamination and or a hidden suppressed immune system in the patients ( 24 ) . however , in immunocompromised patients with characteristic clinical presentation , observation of aspergillus in culture or pcr assay , even if obtained from sputum or bal , has a high diagnostic value for ipa ( 25 ) . although this conclusion is based on a limited number of patients with hematological malignancies and hsct for whom pa were suspected , the performance was promising . despite this seemingly small number of subjects , the study reports a high number of proven / probable ipa cases in terms of evaluating diagnostic tests or surrogate parameter performance in bal specimens for pa . although there was a weak correlation between the results of several methods that may not increase the diagnosis power , it is useful to make diagnostic decisions especially in patients with hematological malignancies and hsct with clinical signs suspicion to pa . in conclusion , incidence of ipa in allogeneic hsct and patients with hematological malignancies was relatively high and a. flavus was the most common cause of pa . as molecular methods had higher sensitivity , it may be useful as the screening methods in hsct and patients with hematological malignancies , or to determine when empirical antifungal therapy can be withheld . | background : pulmonary aspergillosis ( pa ) is one of the most serious complications in immunocompromised patients , in particular among hematopoietic stem cell transplants ( hsct ) and patients with hematological malignancies.objectives:the current study aimed to evaluate the incidence of pa and utility of molecular methods in hsct and patients with hematological malignancies , four methods including direct examination , culture , nested polymerase chain reaction ( pcr ) and real - time pcr were performed on bronchoalveolar lavage ( bal ) specimens in tehran , iran.patients and methods : during 16 months , 46 bal specimens were obtained from individuals with allogeneic hsct ( n = 18 ) and patients with hematological malignancies ( n = 28 ) .
direct wet mounts with 20% potassium hydroxide ( koh ) and culture on mycological media were performed .
the molecular detection of aspergillus fumigatus and a. flavus was done by amplifying the conserved sequences of internal transcribed spacer 1 ( its1 ) ribosomal dna by nested - pcr and the -tubulin gene by taqman real - time pcr.results:seven ( 15.2% ) out of 46 specimens were positive in direct examination and showed branched septate hyphae ; 11 ( 23.9% ) had positive culture including eight ( 72.7% ) a. flavus and three ( 27.3% ) a. fumigatus ; 22 ( 47.8% ) had positive nested - pcr and eight ( 17.4% ) had positive real - time pcr .
the incidence of invasive pulmonary aspergillosis ( ipa ) in these patients included proven ipa in 1 ( 2.2% ) , probable ipa in 10 ( 21.7% ) , possible ipa in 19 ( 41.3% ) and not ipa in 16 cases ( 34.8%).conclusions : the incidence of ipa in allogeneic hsct and patients with hematological malignancies was relatively high and a. flavus was the most common cause of pa .
as molecular methods had higher sensitivity , it may be useful as screening methods in hsct and patients with hematological malignancies , or to determine when empirical antifungal therapy can be withheld . |
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vertical transmission of human immunodeficiency virus ( hiv ) is still a major challenge in the world , especially in the developing countries ( usaid ) . it is estimated that 90% of hiv infections in children result from mother - to - child - transmission ( who ( switzerland ) ) . in the absence of any intervention to prevent mtct ( pmtct ) , the mtct rate varies between 13% and 48% [ 3 , 4 ] . maternal combination antiretroviral therapy ( art ) together with postnatal interventions has demonstrated its efficacy in substantially reducing the risk of mtct in african breastfed children to less than 5% ( usaid 2013 ) [ 3 , 5 ] . however , access to art and the uptake of pmtct programs remain limited and children continue to be hiv infected ( abidjan , cote d'ivoire ) . the world health organisation ( who ) guidelines recommend that all pregnant women should be tested for hiv in the first trimester and that a second test be considered in the third trimester by 34 weeks of gestational age [ 1 , 68 ] . guidelines in resource - limited settings are increasingly recommending hiv testing as early as possible during pregnancy and repeat testing towards the end of pregnancy or during labour , a strategy that has proven to be cost effective . despite these recommendations , recent studies report hiv incidence rates of 10.8% in serodiscordant couples in kenya whenever a pregnancy occurred . more recently , a meta - analysis published in 2014 reports an aggregate seroconversion rate of 3.8 per 100 person - years in african countries by drake et al . in washington usa . hiv testing during labour has remained a challenge over the years in cameroon . in 2009 , of the 94,406 women with a previous negative result who presented in the labour rooms of the clinics carrying out pmtct activities , only 2,643 were retested , giving a proportion of 2.8% . in cameroon , the prevalence of hiv was estimated to be 4.3% in the general population ; a serosurveillance survey among pregnant women showed an hiv prevalence of 7.6% in 2010 . as a result , the number of new pediatric infections continues to grow in cameroon and there are still thousands of new infections each year . in 2011 , the unaids launched the global plan towards eliminating new hiv infections among children and keeping their mothers alive , making cameroon , where overall mtct risk was reported to be around 24% , one of the 21 priority countries . since 2011 , cameroon has tripled its coverage of pmtct prophylaxis , ranging from 6.9% to 36.5% in 2011 , leading to 30% fewer new hiv infections among children . in 2011 , continuing access of pregnant women living with hiv to prenatal hiv services and increasing access to hiv treatment for eligible children and pregnant women will reduce maternal and child mortality . cameroon has focused on strengthening pmtct services and caring of pediatric hiv cases for the 20112015 period ; 99.4% of health districts were equipped to provide hiv treatment services for pregnant women and children living with hiv in 2011 . however , even where the most effective pmtct interventions are available , many women and infants are lost at different steps of the pmtct cascade ; and the low cumulative uptake of pmtct services does not allow controlling the extent of mtct in cameroon . the hiv seroprevalence in the swr is 11.9% . this is one of the highest in cameroon , closely followed by the east region with 9.3% . there is a high rate of mtct of hiv with seroconversion in pregnancy [ 10 , 18 ] . in cameroon , particularly in the south west region , there are no reports regarding the incidence of hiv seroconversion during pregnancy . there is a probability that many cases that seroconvert in pregnancy go without appropriate management , resulting in high mtct as reported by muffih in 2011 . data on the seroconversion rate after initial negative hiv test result in pregnancy would be useful in improving the management of hiv in pregnancy in fako division , swr , cameroon . the aim of this study was to determine the incidence of hiv seroconversion during the second and third trimesters of pregnancy and ad hoc potential cofactors associated with hiv seroconversion after having an hiv - negative test result in the booking visit . we also studied knowledge of pmtct among pregnant women in seven health facilities in fako division , south west region ( swr ) , cameroon . this was a hospital based cohort study of women attending antenatal care ( anc ) clinics and labour rooms of the maternity units of seven healthcare facilities in the fako division , south west region , cameroon , during the period between september 12 and december 4 , 2011 . study participants were women who attended their booking or first antenatal care visit in any of the seven selected health facilities in the last six months and for whom an hiv test was done using the determine test strips on this booking ( first ) anc visit between 16 and 20 weeks of gestation . hiv seroconversion in pregnancy ( hsp ) was defined as maternal self - report of an hiv - negative test during the first antenatal care visit during this pregnancy , no documented use of antiretroviral drugs , and a positive hiv rapid test ( determine ) done 3 months after the first antenatal care ( booking ) test . the gestational age of the pregnancy was calculated from the last normal menstrual period ( lnmp ) . all the women who were found to be hiv - negative at this first visit consented ( written consent after study procedure and objectives had been explained to them ) to participate in the study ( table 1 ) . all study participants were counselled to repeat the hiv test within an interval of 3 to 6 months following the test of the booking anc visit . all participants were those residing within a 10 km radius from health facility for easy follow - up . the main outcome of interest was hiv seroconversion at second test during the ongoing pregnancy . sample size was calculated by using the who - steps approach with the assumptions of 95% confidence limits , 5% proportion of seroconverting women , and 2% margin of error . the minimum sample size was calculated to be 457 participants , but considering a nonresponse rate of 4% , we enrolled 477 participants for study . a total of seven health facilities were selected by simple random sampling ( balloting ) for study . participants who met the inclusion criteria were then selected by cluster sampling ; and in each cluster , participants were included individually and consecutively to maximize confidentiality . data collection was done during a period of 12 weeks , from september 12 to december 4 , 2011 . during this period , the study participants were met at their various antenatal clinic sites on specific days of the week when these activities were carried out . a total of 4 weeks were spent per health facility with at least two facilities targeted at once depending on their anc days ( table 2 ) . sociodemographic information ( maternal age , gravidity , marital status and marital type , employment status , level of education , and residence ) , knowledge of pmtct , and methods , if any , of hiv prevention were obtained from the study population . furthermore , voluntary counselling and testing ( vct ) for hiv was done on each study participant , whereby pretest counselling was done followed by the collections of 2 - 3 ml of venous blood by venipuncture into a dry vacutainer tube which was then allowed to settle for about 20 minutes to obtain serum from whole blood . the supernatant serum from the dry tube was then tested using determine hiv-1/2 rapid test ( abbott laboratories , abbott park , illinois , usa ) in the laboratory of the health facilities by trained laboratory personnel , experienced in hiv testing , according to manufacturer 's test procedure . the same brand of test kits was used across all the health facilities that participated in the study . following testing , a posttest counselling was done and results were delivered about one hour after testing . for participants who were positive for determine hiv 1/2 rapid test , a second - line test , sd bioline hiv 1/2 3.0 ( standard diagnostics , inc . ) , was done according to manufacturer 's instructions to differentiate between hiv-1 and hiv-2 . a participant was only considered positive if both tests were positive , negative if determine was negative , and indeterminate if determine was positive and then bioline test was negative [ 2022 ] . all the participants who were diagnosed hiv - positive were treated according to cameroon 's national guidelines for the pmtct , which at the time recommended option b involving either azt + 3tc + nvp or d4 t + 3tc + nvp . numerical variables like age , parity , and gestational age were classified into groups and their frequencies expressed in percentage were presented ; meanwhile , categorical variables like marital status , educational level , and occupation were expressed as frequencies . comparison of seroconversion incidence to other variables was done using fisher 's exact test reported with p values that were considered significant if p was less than 0.05 . univariable analysis was done using logistic regression to identify the potential factors associated with seroconversion in pregnancy , and then those with a p value less than 0.2 were included in the final model for multivariable logistic regression . results were reported as adjusted odds ratios ( or ) together with their 95% confidence intervals ( ci ) . ethical clearance was obtained firstly from the faculty of health sciences institutional review board ( fhs / irb ) as well as the cameroon baptist convention ( cbc ) institutional review board before patient enrolment . then , an authorisation was obtained from the regional delegation of public health for the south west region . permission was obtained from the health districts and the various health facilities for the study to be carried out in the desired health facilities . a total of 1954 antenatal women in seven healthcare facilities were tested for hiv to provide annual prevalence data in their first antenatal ( booking ) visit . among this sample , 201 ( 10.3% ) women were tested hiv - positive and were excluded from study . amongst the remaining 1753 hiv - seronegative pregnant women , 477 ( 27.2% ) were enrolled into the study in the second and third trimesters to study the incidence of seroconversion . the majority , 308 participants ( 64.65% ) , were in the age group 2130 and 364 ( 76.32% ) were married . about 350 ( 96.2% ) of those who were married were monogamously married . of the study population , 418 ( 87.6% ) were urban dwellers , 394 ( 83.12% ) had less than two children , three hundred and ninety ( 81.8% ) were in the third trimester of pregnancy , and 388 ( 81.3% ) had done secondary level of education and above . the incidence of participants who seroconverted was 2.1% ( 147.25 woman - years ) , giving an incidence rate of hiv seroconversion during pregnancy of 6.8 per 100 woman - years . the majority , 3% ( 8/262 ) , of the patients who seroconverted did the second test 3 months after the first test . a minority , 31.9% , of participants did not know their hiv status prior to the first antenatal care ( booking ) visit while 33.0% of participants did not also know the hiv status of their partners / husbands . it shows that no statistically significant relationship was found between sociodemographic factors and hiv seroconversion in pregnancy . had a repeat test 3 months after the first negative ( booking ) test result while 25.8% , 13.2% , and 6.1% had their repeat test done at 4 , 5 , and 6 months , respectively . eighty percent ( n = 8) of those who seroconverted did so by the fourth month after the booking or first antenatal care visit hiv testing ( figure 2 ) . six ( 60% ) of those who seroconverted were in a monogamous regime and 3 ( 30% ) were single , while 1 ( 10% ) was in a polygamous regime ( one husband and two wives ) . , 8 knew about mtct and pmtct of hiv including when transmission could occur ( either during pregnancy , labour / delivery , and breastfeeding or when the mother does not know she is hiv - positive ) . all the eight participants also knew it was possible to prevent mtct ( p = 0.07 ) and 7 out of 8 knew at least one correct method of pmtct ( avoiding breastfeeding , taking antiretroviral treatment , caesarean delivery , or mother being aware of her hiv serologic status before engaging in a pregnancy ) ( p = 0.78 ) . all the participants who seroconverted were having sexual intercourse during the current pregnancy ( p = 0.37 ) and 9 ( 90% ) were not users of the condom . all the participants who were hiv - positive had only one sexual partner throughout pregnancy ( p = 0.34 ) . the odds of hiv seroconversion during pregnancy were 5 times higher among pregnant women who did not know about pmtct ( aor 5.4 ; 95% ci 1.0627.56 ) . also , pregnant women who were employed were at a higher risk of seroconversion than those who were unemployed ( aor 3.2 ; 95% ci 0.6815.4 ) , though this was not statistically significant . the majority , 407 ( 85.3% ) , of the participants who knew about pmtct got the information from the hospital / health personnel mainly during antenatal consultations , while 36 ( 7.5% ) got informed through the media . hiv incidence during pregnancy and postpartum significantly increases risk of mtct and is an important public health problem in africa . understanding maternal hiv incidence during this time period can be helpful to guide prevention and repeat testing strategies and policies , and little data on hiv incidence in pregnancy from west africa exist . this study measures hiv incidence during pregnancy in seven healthcare facilities in fako division , south region of cameroon , by repeat testing later in pregnancy . women in the south west region , like those in most other low - income countries , come to health facilities for antenatal care very late in pregnancy , usually in the second trimester . at the same time , they prefer to give birth in a health facility because they perceive labour and delivery as a time of significant health risks that require biomedical attention . for this reason we sometimes find it difficult to have women tested for hiv in the first trimester of pregnancy , especially in the rural areas . the incidence of hiv seroconversion during pregnancy in this study was 2.1% ( 147.25 woman - years ) , yielding an incidence rate of 6.8/100 woman - years . these results are lower than that reported by moodley et al . in 2009 in uganda who showed that , among 2377 hiv - negative women retested , 1099 ( 46.2% ) and 1278 ( 53.4% ) were tested at urban and rural health facilities , respectively . seventy - two women ( 3% ) were hiv - positive ( 679 woman - years of exposure ) , yielding an hiv incidence rate of 10.7/100 woman - years ( 95% confidence interval ( ci ) 8.213.1 ) . hiv incidence in pregnancy was higher but not statistically significant at the urban facilities ( 12.4/100 woman - years versus 9.1/100 woman - years ) and at least two - fold higher among the 2529- and 3034-year age groups ( 3.8 and 4.5% , resp . ) as compared with the less than 20-year age group ( 1.9% ) . single women were at 2.5 times higher risk of seroconverting during pregnancy ( p = 0.017 ) . in another study , humphrey et al . reported that breastfeeding associated transmission for mothers who seroconverted postnatally ( n = 334 ) averaged 34.56 infant infections per 100 child - years ( 95% ci 26.60 to 44.91 ) during the first nine months after maternal infection , declined to 9.50 ( 95% ci 3.07 to 29.47 ) during the next three months , and was zero thereafter . among women who seroconverted postnatally and in whom the precise timing of infection was known ( 90 days between last negative and first positive test ) ( n = 51 ) , 62% ( 8/13 ) of transmissions occurred in the first three months after maternal infection and breastfeeding associated transmission was 4.6 times higher than in mothers who tested hiv - positive at baseline and whose infant tested hiv - negative with pcr at six weeks . our results conform with the 2.2% seroconversion rate reported by qolohle et al . in 1995 at the king edward viii hospital in durban , south africa ( 2.6% ) and the 2.9% reported by lockman and creek in francistown , botswana , at 62 weeks postpartum . with the 2.1% incidence of hiv or 6.8/100 woman - years incidence rate of hiv seroconversion during pregnancy in fako division , there would be a correspondingly increasing rate of mtct of hiv , thereby increasing the pediatric hiv burden . it has been estimated that 19% of infants born to hiv - positive mothers are hiv infected when tested at 19 months . this high rate of infant infection can be attributed to the fact that maternal hiv infection during pregnancy and breastfeeding occasion equally high rates of mtct of hiv-1 . all participants who seroconverted did so in the third trimester of pregnancy , when most of the study participants ( 81.8% ) were recruited . all the participants who seroconverted in our study had hiv-1 which is the type of hiv mainly responsible for mtct of the virus . therefore , all cases of hiv seroconversion during pregnancy should be considered as potential risks for more pediatric hiv . seventy percent of the participants who seroconverted were married ; therefore , marriage does not protect women from hiv infection in pregnancy . eighty percent of the participants who seroconverted had 3 - 4-month interval between the two tests , while there was no case of seroconversion among women who had 6-month interval . these results differ from those obtained in the tygerberg hospital of cape town where it was reported that women especially at risk of seroconversion are those who book much earlier , thereby increasing the interval between the initial test at first anc visit and the repeat test . to identify cases of seroconversion more early , it will be advisable for repeat hiv testing to be done to pregnant women every three months until delivery , no matter the time of first anc visit . the risk factors that were studied ( age , marital status , residence , parity and knowledge of mtct of hiv , hiv prevention , and hiv seroconversion in pregnancy ) did not show any significant statistical variations . these results are not in conformity with those reported in harare , zimbabwe , where women aged 17 and below were at higher risk of seroconverting than the general population of hiv - negative pregnant women studied . in this study , 442 ( 98.2% ) of participants who remained seronegative and 8 ( 1.8% ) of those who seroconverted had a good knowledge of pmtct techniques . this is probably because it has been a routine practice at all antenatal clinics to give the women talks on hiv and pmtct , especially during the first anc visit . familiarity level with pmtct practices in this study is similar to that obtained in yaounde , cameroon , in 2011 . the majority of study participants , though aware of mtct of hiv during pregnancy , did not take the necessary preventive measures . this was exemplified by the fact that some participants did not know their hiv status before the first antenatal care ( booking ) visit . this may be the result of inadequate sensitization of the population of the swr or negligence on the part of the participants . this fact alone could have a negative impact on the pmtct strategies put in place in fako division and based on recommendations of the who and the government of cameroon . the limitations to this study include the fact that we did not study the male partners ' or husbands ' risk factors and relied on results of hiv tests done at the booking or first antenatal care visit in the different health facilities . also , seroconversions were determined using rapid tests , not hiv rna tests , thus potentially underestimating incidence rates . furthermore , the study was underpowered to detect cofactors for risk and only measured cofactors based on demographics and pmtct knowledge . we did not assess many other potential cofactors for acquisition of hiv , such as stis . finally , the collection of data using an interviewer - administered questionnaire could have resulted in social desirability bias . the incidence of hiv seroconversion among pregnant women in the study is 2.1% , yielding an incidence rate of 6.8/100 woman - years in fako division . most of the participants seroconverted 3 months after the first test and therefore make up a potential risk for pediatric hiv . lastly , pregnancy did not stop the women from sexual activities and most of them had one sex partner . | background . we determined the incidence of hiv seroconversion during the second and third trimesters of pregnancy and ad hoc potential cofactors associated with hiv seroconversion after having an hiv - negative result antenatally .
we also studied knowledge of pmtct among pregnant women in seven health facilities in fako division , south west region , cameroon . method . during the period between september 12 and december 4 , 2011 , we recruited a cohort of 477 hiv - negative pregnant women by cluster sampling .
data collection was with a pretested interviewer - administered questionnaire .
sociodemographic information , knowledge of pmtct , and methods of hiv prevention were obtained from the study population and we did voluntary counselling and testing ( vct ) for hiv . results .
the incidence rate of hiv seroconversion during pregnancy was 6.8/100 woman - years .
ninety percent of the participants did not use condoms throughout pregnancy but had a good knowledge of pmtct of hiv .
only 31.9% of participants knew their hiv status before the booking visit and 33% did not know the hiv status of their partners .
conclusion .
the incidence rate of hiv seroconversion in the fako division , cameroon , was 6.8/100 woman - years .
no risk factors associated with hiv seroconversion were identified among the study participants because of lack of power to do so . |
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this study was performed on 1101 ambulatory patients who consulted to the obstetric care clinic of haydarpasa training and research hospital affiliated to association of istanbul northern anatolia public hospitals between september 2013 and january 2015 . hospital files of the patients were retrospectively reviewed , from computer database serologic data of torch group of infections were retrieved . toxoplasma gondii , cmv , rubella ig g and ig m antibodies were analyzed using elisa method . ages of the patients included in the study ranged between 17 and 47 years and mean age of the pregnants was 30.35.8 years . in the study , population ig g positivities for cmv ig g ( 99.5% ) , rubella ( 94.2% ) and toxo ( 31% ) were detected in respective percentages of patients . the corresponding ig m positivities for cmv and rubella were 0.5 , and 0.2% , respectively . a total of 64 patients were rubella ig g seronegative , while 2 of them were also igm positive . serologic analysis of these two patients were performed during the last trimester without causing any problems to the pregnant women and their babies . serologic tests of 4 patients with cmv ig m positivity were performed during the second trimester without causing any problem among babies born . accordingly in 5.8% of our study population rubella seronegativity was detected . ( the percentage of seropositivity of pregnant women ) seroprevalence rates for torch ig g , and igm in pregnant women cmv : cytomegalovirus ; toxo : toxoplasma . in our country within the context of national vacination program , rubella vaccines are administered . the patients were divided into 1725 , 2635 and 3644 age brackets , and re - evaluated as for rubella ig g according to these age groups . accordingly , rubella ig g seronegativities differed among age groups of 1725 ( 3.2% ) , 2635 ( 8.4% ) and 3647 ( 12% ) as indicated . prevalence of seronegativity increased with age without any statistically significant difference between age groups ( p>0.05 ) . in the study , population ig g positivities for cmv ig g ( 99.5% ) , rubella ( 94.2% ) and toxo ( 31% ) were detected in respective percentages of patients . the corresponding ig m positivities for cmv and rubella were 0.5 , and 0.2% , respectively . a total of 64 patients were rubella ig g seronegative , while 2 of them were also igm positive . serologic analysis of these two patients were performed during the last trimester without causing any problems to the pregnant women and their babies . serologic tests of 4 patients with cmv ig m positivity were performed during the second trimester without causing any problem among babies born . accordingly in 5.8% of our study population rubella seronegativity was detected . ( the percentage of seropositivity of pregnant women ) seroprevalence rates for torch ig g , and igm in pregnant women cmv : cytomegalovirus ; toxo : toxoplasma . in our country within the context of national vacination program , rubella vaccines are administered . the patients were divided into 1725 , 2635 and 3644 age brackets , and re - evaluated as for rubella ig g according to these age groups . accordingly , rubella ig g seronegativities differed among age groups of 1725 ( 3.2% ) , 2635 ( 8.4% ) and 3647 ( 12% ) as indicated . prevalence of seronegativity increased with age without any statistically significant difference between age groups ( p>0.05 ) . serologic screening tests directed at agents of maternal rubella , cmv and toxoplasma gondii infections carry importance so as to prevent related malformations and other worse neonatal outcomes . prevalence rates obtained as a result of screening tests aimed to detect infectious agents of the diseases included in the torch group can change from region to region based on geographical , socioeconomic and cultural values [ 7 , 11 ] . in this study , torch seroprevalence rates of the patients who consulted to obstetric care clinic of haydarpasa training and research hospital affiliated to association of istanbul northern anatolia public hospitals were investigated . considering the applicability of screening tests in every pregnant woman , its potential cost and necessity of performing these tests are evaluated in combination . every country can plan its antibody screening test to be performed against infection agent(s ) based on national prevalence values . for instance , in a study performed in the united kingdom which lasted for 10 years , because of fetal findings detected with detailed investigations , the health authorities have concluded that all torch screening tests in maternal serum should not be performed in every case . thus in uk , based on expert comments this screening test can be limited for the investigation of only cmv seropositivity . in a similar study in japan , it was reported that serologic screening tests for torch have not any significance apart from cmv screening in cases with fetal growth retardation . in france and austria , toxoplasma screening test is a legal necessity . in france , prevalence of congenital toxoplasmosis is 3.3 for every 10,000 live births , while prevalence of symptomatic infection is 0.34 for every 10.000 live births [ 9 , 10 ] . clinical manifestations of mild disease emerging after toxoplasma gondii infection are generally characterized by fever , lassitude and lymphadenopathy . however , congenital infections can lead to pregnancy losses and serious diseases in the newborn as mental retardation , blindness and epilepsy . infection contracted during early stage of pregnancy has a risk of transmittance to the fetus less than 6% , while during the third trimester this risk ranges between 60 , and 81 percent . on the other hand , during embryogenesis vertical transfer of toxoplasma gondii is rarely seen , an infection contracted during this period exerts more serious effects on fetus . on the contrary , babies born to mothers who contracted an infection during the third trimester are usually asymptomatic [ 9 , 10 ] . in parallel with cultural eating habits and socioeconomic status of the countries , the results of the seroprevalence studies for toxoplasma gondii demonstrate variations in the world . in france where consumption of underdone red meat is prevalent 71% seropositivity rates were detected . however in uk seropositivity rates are lower ranging between 7.7 , and 9.1 percent [ 8 , 9 , 10 ] . when other studies on torch seroprevalence are taken into consideration , seroprevalence rates for toxoplasma gondii vary between 30 and 69.5 percent [ 11 , 12 ] . anti - toxoplasma ig g seropositivity was detected as 31% which is consistent with most of the studies performed in turkey [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ] . cultural differences between regions , rich and variable cuisine culture in turkey also reflect on this type of studies . in studies conducted in southeastern anatolia region and neighbouring provincences toxoplasma gondii seropositivity were higher and ranged between 48.4 and 69.5 percent [ 7 , 13 , 14 , 15 , 16 , 17 ] . in studies conducted in turkey , higher toxoplasmosis seroprevalence rates were detected in urfa ( 69.5% ; tekay et al . ) , sivas ( 52.2% ; duran et al . ) , hatay ( 52.1% ; ocak et al . ) , adyaman ( 48.4% ; kogeliler et al . ) , and kocaeli ( 48.3% , tamer et al . toxoplasma ig g positivity rates in different provinces were observed as follows : artvin ( 30.3% ; inci et al . ) , edirne ( 30% ; varol et al . ) , afyon ( 30.7% ; yilmazer et al . ) denizli ( 37% ; karabulut et al . ) , erzurum ( 24% ; yigit et al . ) , diyarbakir ( 32.9% ; gul et al . ) , elazig ( 41% ; asci et al . ) , and van ( 41% ; efe et al . ) [ 11 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ] . previous seroprevalence studies performed in metropolitan city of istanbul , toxoplasma ig g seropositivities were detected as 43% by polat et al . and in a more recent study [ 24 , 25 ] . excluding provinces of southeastern anatolia , seroprevalence of toxoplasma changes between 30 and 43 percent . however , in pilot studies performed in various times in the same province , different results have been obtained . it is important to detect seronegative women especially in regions with higher seroprevalence rates , training people so as to ensure consumption of vegetables and fruits after vigorous washing , popularization of the habit of hand washing and refraining from intake of raw or rare cooked meat carry importance as for prevention of maternal and fetal infections . our study results are consistent with those of the studies performed in our country excluding southeastern anatolia region where the toxoplasma seropositivity ranges between 30 and 43 percent . because of lower seropositivity rates , especially during pregnancy screening for toxoplasma can be recommended . rubella courses with mild degree of rashes and fever in childhood , when it is experienced in the first months of pregnancy , it can lead to pregnancy losses and stillbirths and during the neonatal period it may result in congenital rubella syndrome ( crs ) [ 1 , 2 , 3 ] . for the possible prevention of vertical transmittance from mother to the fetus , seronegative women should be detected during preconceptional period , and their pregnancies should be planned , and postponed after immunization . in turkey rubella vaccination program was implemented firstly in the year 1995 , and in the year 2006 it was introduced into national vaccination calender [ 1 , 7 ] . national vaccination calender determined by public health agency of turkey , tr ministry of health and social welfare in the year 2014 measles - rubella - mumps ( mmr ) vaccine is administered as a single dose within the first 12 month of life , and booster dose is injected in the first grade of the primary school . when other seroprevalence studies performed in the world are taken into consideration seropositivity rates for rubella virus were reported as 87% in the united states of america ( usa ) , 98% in spain and 96.3% in iran [ 10 , 11 ] . in our study , seropositivity rates for rubella was detected as 94.2% which demonstrates similarities with the results of other relevant studies . higher seropositivity rates were detected in denizli ( 95.1% ; karabulut et al . ) , samsun ( 94.3% ) , hatay ( 95% ) , van ( 99.5% ) , and kocaeli ( 96.1% ) . rubella ig g seropositivity rates were 76.5% , and 76.6% in mardin , and edirne , respectively . these data are important in that they give an idea about whether national vaccination programs have achieved their goals and implemented all over the population , their acceptability by every section of the community and tracking the success of vaccination programs . in our study , we detected that none of 62 patients had ever encountered rubella virus and had nt developed immunity against the disease . besides , in our study population , after implementation of our national vaccination program in pregnants aged 20 years and younger , rubella seronegativity was not detected . when national data obtained were evaluated globally , higher prevalence of rubella seropositivity was detected . these data have suggested us that even though lower rubella seronegativity rates were noted , it will be appropriate to screen and immunize women over 20 years of age during preconceptional period . cmv is the most frequently encountered congenital infection agent and apart from toxoplasma gondii and rubella it may emerge as a primary infection or demonstrate recurrences . therefore , maternal serologic tests may not rule out a new strain of cmv or reactivation of latent maternal infection and congenital infection . risk of vertical transmittance after primary infection ranges between 30 , and 40% , and it emerges after a secondary infection at a rate of 1 percent [ 29 , 30 , 31 ] . therefore , in pregnant women routine screening tests for cmv is debatable and american college of obstetrician and gynecologists ( acog ) does not recommend screening tests for cmv infection . cmv infections are frequently seen in communal life conditions and populations with lower socioeconomic level . in populations with higher seropositivity rates routine screening tests for cmv may not be recommended . indeed , an effective treatment modality for congenital cmv infection has not been definitively established yet and this test is costly , and clinically useless . in developed countries , seropositivity rates were 46.8% in france , 56.8% in australia , 56.3% in finland , 78% in russia , 92.1% in saudi arabia and 100% in thailand [ 29 , 30 ] . in studies performed in turkey cmv seropositivity rates were detected as 92.6% in afyon , 98.5% in ankara , 97.3% in samsun , 98.6% in artvin , 96.4% in kocaeli , and 98.7% in denizli [ 1 , 9 , 11 ] . however , in our study cmv seroprevalence was detected as 99.5% which is one of the highest seropositivity rates . this cmv seroprevalence rates demonstrate parallelism with the relevant results of developed and developing countries . when these findings and seroprevalence rates of our country are taken into consideration , we think that cmv screening during pregnancy is not a necessary application . in conclusion , seroprevalence of torch demonstrates changes among countries or even between regions in the same geographic location . when data of our study and relevant available studies performed in our country are investigated , increased cmv and seropositivity is detected . because of its higher seropositivity , screening for cmv is not cost - effective and so it should not be recommended . if national vaccination program can be applied fully , then rubella screening tests during pregnancies of women born after the year 1995 will be useless . however , for the time being , if possible it will be appropriate to perform rubella screening tests during preconceptional period and in women over 20 years of age in addition to vaccination of women against rubella before becoming pregnant . although seroprevalence of toxoplasmaosis changes among regions , since its treatment is possible at the time of diagnosis and its higher seroprevalence ( 3043% ) outside the risky regions , it may be presumably included in antenatal screening programs . we think that perinatologists , public health specialists and health policy makers should work in collaboration and cost - effectiveness of the screening for torch group of diseases ( if any ) should be reviewed . | objective : toxoplasma gondii , rubella , cytomegalovirus and herpes simplex viruses are microorganisms that cause congenital infections and they are called briefly as torch . there is an ongoing argument for the screening of reproductive age women due to the high cost of tests . for a test to be used in screening , prevalence of disease in this population should be known .
the aim of this study was to investigate torch seroprevalence among women attending a teaching hospital in istanbul.methods:a total of 1101 patients attending outpatient clinic of obstetric care clinic of haydarpasa training and research hospital affiliated to association of istanbul northern anatolia public hospitals between september 2013 to january 2015 and their laboratory data were evaluated retrospectively .
we investigated ig g and m seropositivity rates against torch.results:the age of the patients ranged between 1747 years with a mean age of 30.35.8 years .
pregnant population had 99.5% anti - cmv ig g ( + ) , 94.2% anti - rubella ig g ( + ) , 31% anti - toxoplasma ig g ( + ) .
seroprevalence for anti igm was 0.5% for cmv , 0.2% for rubella.conclusion:the screening for toxoplasma gondii may be suggested since the prevalence is not high in our population .
the screening of cmv is not meaningful , due to high seroprevalence .
although seroprevalence of rubella is also high , it may be suggested for preconception vaccination especially in women above 20 years of age born prior to national vaccination programme . |
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complex neurovascular procedures may expose patients and , secondarily , staff to high doses of ionizing radiation . the potential consequences , such as erythema and hair loss , are of major concern . however , in accordance with the as low as reasonably achievable ( alara ) principle , radiation doses can not be reduced below the level necessary for acceptable clinical image quality . an x - ray imaging technology using advanced image noise reduction algorithms combined with optimized system settings that enable dose reduction and optimal image quality was designed for digital subtraction angiography ( dsa ) and fluoroscopy in neuroradiology , in order to reduce patient radiation dose without impairing image quality ( alluraclarity ; philips healthcare , best , netherlands ) . a previous study addressed non - inferiority of image quality in dsa at 75% radiation dose reduction . the current study aims to quantify the radiation dose reduction in a patient cohort subjected to diagnostic neuroangiography or interventional neuroradiology and to test the hypothesis that the image noise reduction algorithms with optimized system settings would significantly reduce patient radiation exposure without affecting parameters such as fluoroscopy time and number of dsa images ( the working habits of the physician ) . the study was approved by the local ethical review board at karolinska university hospital , stockholm , sweden . data acquired on a system equipped with a state - of - the - art image processing with reference system settings ( reference system ) was compared with data from the same system equipped with advanced image noise reduction algorithms combined with optimized system settings ( current system ) . procedure and dose data for the reference system and the current system were collected during january june 2010 and january june 2012 , respectively . all examinations were performed on the same biplane flat panel detector angiography system ( alluraxper fd20/20 biplane ; philips healthcare ) , equipped during the second part of the study with an image processing chain for noise reduction in dsa and fluoroscopy , combined with optimized system settings ( alluraclarity ; philips healthcare ) . the real - time automatic pixel shift feature is used to reduce the anatomical structure noise which is introduced in the subtracted image by patient motion or accidental table motion . by minimizing this undesired noise source another feature is the temporal averaging of consecutive images to create a combined mask and a combined live image . contrast detection functionality will reveal changes in the iodine bolus location and prevent this from being diluted by the averaging . in the spatial noise reduction feature , the first analysis phase aims to reveal the predominant signal structures in the image , which will be excluded from the low - pass spatial filter in the second phase . the combination of phases will smooth only the parts of the image which are considered featureless . optimization of system settings for dsa acquired with the current system included typical tube voltage 75 kvp , additional 0.1 mm cu+1 mm al filter , detector dose of 0.7 gy / fr on largest field of view and 0.4 mm focal spot size . depending on the average equivalent water thickness , the patient dose reduction for fluoroscopy , due to the low - exposure acquisition settings of the current system , can range from approximately 10% for small equivalent water thickness to approximately 50% for large equivalent water thickness , achieved , for example , with steeper projections . the average equivalent water thickness for the head is considered approximately 22 cm on the frontal plane and 18 cm for the lateral plane , over the full population range . for this average water equivalent thickness , the expected patient dose reduction is 30% . patients were subjected to neuroangiography or endovascular treatment during the study periods . interventional procedures were further divided in subgroups : ( a ) arteriovenous malformation ( avm ) , ( b ) aneurysm , ( c ) stroke and ( d ) others . patient radiation dose indicators , quantified as ( cumulative ) dose area product ( dap ) and cumulative air kerma ( cak ) , as well as acquisition parameters , such as number of dsa images , fluoroscopy time , procedure time , and number of dsa runs , were collected . the equipment displayed the updated cumulative dose - area product ( dap ) , measured by the internal transmission ionization chambers ( kermax plus ; iba dosimetry , schwarzenbruck , germany ) configured in both planes . the inherent dose - report system in the angiography equipment provided information , measured in gy cm , of dap fluoroscopy , dap exposure , and total dap ( sum of dap fluoroscopy and dap exposure ) . cak at the patient entrance reference point for frontal and lateral channels was provided in gy . this information was sent via modality performance procedure step ( mpps ) automatically to the radiology information system ( ris ; carestream , vaughan , canada ) the primary outcome of the study was radiation dose quantified as dap and cak . secondary outcomes were fluoroscopy time , number of dsa images , number of dsa runs , and procedure duration . descriptive statistics were used to describe patient and procedure characteristics , with differences between reference and current system evaluated with one - way analysis of variance ( anova ) models at a significance level of =0.05 . differences in exposure between the reference and the current systems were compared using anova with least square mean dose values , using an f - test . secondary covariance analyses were performed for dap , cak , and acquisition measures ( analysis of covariance ) to determine potential impact of demographic differences or type of intervention . descriptive statistics were used to describe patient and procedure characteristics , with differences between reference and current system evaluated with one - way analysis of variance ( anova ) models at a significance level of =0.05 . differences in exposure between the reference and the current systems were compared using anova with least square mean dose values , using an f - test . secondary covariance analyses were performed for dap , cak , and acquisition measures ( analysis of covariance ) to determine potential impact of demographic differences or type of intervention . a total of 620 patients were included in the data collection ; however , 4 patients were excluded because the radiation dose data information was incomplete , and 2 patients were excluded because they were classified as diagnostic and treatment together . therefore , 614 patients were analyzed . the reference system group included 302 patients ( 172 females and 130 males ) divided in 190 diagnostic and 112 interventional procedures . the current system group included 312 patients ( 172 females and 140 males ) divided in 174 diagnostic and 138 interventional procedures . for both groups , the number of avms is very small ; therefore , procedural dose reduction will not be calculated . the mean age for both groups was 54.117.7 years ; however , patients treated with the current system were older than those treated with the reference system ( p<0.001).table 1patient demographicscharacteristicreference system ( n=302)current system ( n=312)total ( n=614 )
p - value
age ( years)<0.001 meansd51.617.656.517.454.117.7 95% ci49.653.654.658.452.755.5 median53.059.056.0 min max091191091procedure duration ( min)0.212 meansd58.554.064.258.961.456.6 95% ci52.464.657.670.856.965.9 median35.040.039.5 min max526053255325intervention procedures ,
n ( % ) 0.232 avm9 ( 8.0)4 ( 2.9)13 ( 5.2 ) aneurysm26 ( 23.2)38 ( 27.5)64 ( 25.6 ) stroke41 ( 36.6)45 ( 32.6)86 ( 34.4 ) other36 ( 32.1)51 ( 37.0)87 ( 34.8 )
ci confidence interval , sd standard deviation
p - value ( two - sided ) from anova for continuous parameters , and from chi - squared test for categorical parameters
the number of interventions was 112 for the reference system , 138 for the current system , and 250 in total
ci confidence interval , sd standard deviation
p - value ( two - sided ) from anova for continuous parameters , and from chi - squared test for categorical parameters
the number of interventions was 112 for the reference system , 138 for the current system , and 250 in total dap values were highly skewed with large variability ( figs . 1 and 2 ) . patient exposure and number of images were higher for interventional procedures compared with diagnostic procedures . for diagnostic procedures , median total dap and median dap exposure with the current system decreased from 124 to 47 gy cm and from 113 to 36 gy cm , respectively ( table 2 ) . the patient radiation dose reduction was 62% for total dap and 69% for dap exposure.fig . the x - axis is limited to 410 gy cm , with 5% of the procedures for the reference system not being displayed on the scale ( max value is 2,997 gy cm)fig . dashed lines represent third quartile . for graph layout purposes , the x - axis is limited to 900 gy cm , with 5% of the procedures for the reference system not being displayed on the scale ( max value is 1,114 gy cm)table 2descriptive summary of dap values for diagnostic and interventional proceduresvariablediagnosticinterventionreference system ( n=190)current system ( n=174)reference system ( n=112)current system ( n=138)dap fluoroscopy , ( gy cm ) meansd17.245.714.620.885.468.555.547.3 median987042 q1q3617515331192175 min max1593117912911245 95% ci10.723.811.517.772.698.347.563.4dap exposure ( gy cm ) meansd145.2190.046.139.8304.5199.386.271.0 median1133625366 q1q372161225617238639118 min max182,4041247199527455 95% ci118.0172.440.252.1267.2341.874.298.1total dap ( gy cm ) meansd162.2231.760.451.1389.9247.0141.7106.6 median12447328109 q1q378179287622551867196 min max212,9977325281,1148635 95% ci129.0195.452.868.1343.7436.2123.7159.6
ci confidence interval , sd standard deviation , q1 first quartile , q3 third quartile distribution of dap values for diagnostic procedures . dashed lines represent third quartile ( q3 ) . for graph layout purposes , the x - axis is limited to 410 gy cm , with 5% of the procedures for the reference system not being displayed on the scale ( max value is 2,997 gy cm ) distribution of dap values for interventional procedures . the x - axis is limited to 900 gy cm , with 5% of the procedures for the reference system not being displayed on the scale ( max value is 1,114 gy cm ) descriptive summary of dap values for diagnostic and interventional procedures
ci confidence interval , sd standard deviation , q1 first quartile , q3 third quartile for interventional procedures , there was a dose reduction from 328 to 109 gy cm for total dap , from 70 to 42 gy cm for dap fluoroscopy , and from 253 to 66 gy cm for dap exposure , based on median values ( table 2 ) ; the differences were significant ( p<0.001 ) . based on geometric means , the patient radiation dose reduction was 65% , 33% , and 73% for total dap , dap fluoroscopy , and dap exposure , respectively . for both diagnostic and interventional procedures , the first quartile ( q1 ) dap for the reference system exceeded the third quartile ( q3 ) of the dap for the current system , except for dap fluoroscopy . the dose reduction ( total dap ) was approximately 70% , 60% , and 65% for aneurysm , stroke , and other categories , respectively , based on geometric means . secondary analyses were performed to evaluate the impact of age on dap values because patient age was statistically significantly different between the reference system and current system group , for interventional procedures . there was no effect of age on dap in the interventional procedure group ( p=0.2597 ) . for diagnostic angiographies the median cak values were reduced from 0.78 to 0.27 gy for frontal and lateral channel together , with reduction from 0.59 to 0.21 gy and from 0.19 to 0.06 gy for the frontal and lateral channels , respectively ( table 3 ) ; the difference was significant ( p<0.001 ) . the dose reduction for the diagnostic procedures was 62% on the frontal channel and 67% on the lateral channel based on geometric means . the median cak values for interventional procedures diminished from 2.71 to 0.89 gy for frontal and lateral channel together , with reduction from 2.06 to 0.68 gy and from 0.65 to 0.21 gy for frontal and lateral channels , respectively . the dose reduction was 66% on the frontal channel and 67% on the lateral channel based on geometric means.table 3descriptive summary of cak values for diagnostic and interventional proceduresvariablediagnosticinterventionreference systemcurrent systemreference systemcurrent systemcak frontal channel , gy
n=190
n=174
n=111
n=138meansd0.780.890.310.332.561.740.970.89median0.590.212.060.68q1q30.350.910.130.371.223.650.381.35min max0.022.880.001.490.064.860.013.1895% ci0.220.300.070.120.781.100.290.47
ci confidence interval , sd standard deviation , q1 first quartile , q3 third quartile descriptive summary of cak values for diagnostic and interventional procedures
ci confidence interval , sd standard deviation , q1 first quartile , q3 third quartile regardless of the interventional subgroup , cak values were reduced for both frontal and lateral channels . fluoroscopy time and number of exposure images obtained were substantially higher for interventional procedures ( table 4 ) . the difference in fluoroscopy time , number of dsa images , number of dsa runs , and procedure duration did not reach statistical significance.table 4descriptive summary of acquisition settings for diagnostic and interventional proceduresvariablediagnosticinterventionreference system ( n=190)current system ( n=174)reference system ( n=112)current system ( n=138)no . of acquired exposures / dsa images meansd291.7181.3310.6192.5625.8445.2635.7506.1 median266278525464 q1q3160385173402292870299845 min max301,09611,434502,3271082,986 95% ci265.8317.6281.7339.4542.5709.2550.5720.9fluoroscopy time ( min ) meansd7.14.27.24.512.56.411.06.6 median661312 q1q34949717516 min max022022124023 95% ci6.57.76.67.911.313.79.912.1
ci confidence interval , sd standard deviation , q1 first quartiler , q3 third quartile descriptive summary of acquisition settings for diagnostic and interventional procedures
ci confidence interval , sd standard deviation , q1 first quartiler , q3 third quartile a total of 620 patients were included in the data collection ; however , 4 patients were excluded because the radiation dose data information was incomplete , and 2 patients were excluded because they were classified as diagnostic and treatment together . therefore , 614 patients were analyzed . the reference system group included 302 patients ( 172 females and 130 males ) divided in 190 diagnostic and 112 interventional procedures . the current system group included 312 patients ( 172 females and 140 males ) divided in 174 diagnostic and 138 interventional procedures . for both groups , the number of avms is very small ; therefore , procedural dose reduction will not be calculated . the mean age for both groups was 54.117.7 years ; however , patients treated with the current system were older than those treated with the reference system ( p<0.001).table 1patient demographicscharacteristicreference system ( n=302)current system ( n=312)total ( n=614 )
p - value
age ( years)<0.001 meansd51.617.656.517.454.117.7 95% ci49.653.654.658.452.755.5 median53.059.056.0 min max091191091procedure duration ( min)0.212 meansd58.554.064.258.961.456.6 95% ci52.464.657.670.856.965.9 median35.040.039.5 min max526053255325intervention procedures ,
n ( % ) 0.232 avm9 ( 8.0)4 ( 2.9)13 ( 5.2 ) aneurysm26 ( 23.2)38 ( 27.5)64 ( 25.6 ) stroke41 ( 36.6)45 ( 32.6)86 ( 34.4 ) other36 ( 32.1)51 ( 37.0)87 ( 34.8 )
ci confidence interval , sd standard deviation
p - value ( two - sided ) from anova for continuous parameters , and from chi - squared test for categorical parameters
the number of interventions was 112 for the reference system , 138 for the current system , and 250 in total
ci confidence interval , sd standard deviation
p - value ( two - sided ) from anova for continuous parameters , and from chi - squared test for categorical parameters
the number of interventions was 112 for the reference system , 138 for the current system , and 250 in total dap values were highly skewed with large variability ( figs . 1 and 2 ) . patient exposure and number of images were higher for interventional procedures compared with diagnostic procedures . for diagnostic procedures , median total dap and median dap exposure with the current system decreased from 124 to 47 gy cm and from 113 to 36 gy cm , respectively ( table 2 ) . the patient radiation dose reduction was 62% for total dap and 69% for dap exposure.fig . the x - axis is limited to 410 gy cm , with 5% of the procedures for the reference system not being displayed on the scale ( max value is 2,997 gy cm)fig . the x - axis is limited to 900 gy cm , with 5% of the procedures for the reference system not being displayed on the scale ( max value is 1,114 gy cm)table 2descriptive summary of dap values for diagnostic and interventional proceduresvariablediagnosticinterventionreference system ( n=190)current system ( n=174)reference system ( n=112)current system ( n=138)dap fluoroscopy , ( gy cm ) meansd17.245.714.620.885.468.555.547.3 median987042 q1q3617515331192175 min max1593117912911245 95% ci10.723.811.517.772.698.347.563.4dap exposure ( gy cm ) meansd145.2190.046.139.8304.5199.386.271.0 median1133625366 q1q372161225617238639118 min max182,4041247199527455 95% ci118.0172.440.252.1267.2341.874.298.1total dap ( gy cm ) meansd162.2231.760.451.1389.9247.0141.7106.6 median12447328109 q1q378179287622551867196 min max212,9977325281,1148635 95% ci129.0195.452.868.1343.7436.2123.7159.6
ci confidence interval , sd standard deviation , q1 first quartile , q3 third quartile distribution of dap values for diagnostic procedures . dashed lines represent third quartile ( q3 ) . for graph layout purposes , the x - axis is limited to 410 gy cm , with 5% of the procedures for the reference system not being displayed on the scale ( max value is 2,997 gy cm ) distribution of dap values for interventional procedures . the x - axis is limited to 900 gy cm , with 5% of the procedures for the reference system not being displayed on the scale ( max value is 1,114 gy cm ) descriptive summary of dap values for diagnostic and interventional procedures
ci confidence interval , sd standard deviation , q1 first quartile , q3 third quartile for interventional procedures , there was a dose reduction from 328 to 109 gy cm for total dap , from 70 to 42 gy cm for dap fluoroscopy , and from 253 to 66 gy cm for dap exposure , based on median values ( table 2 ) ; the differences were significant ( p<0.001 ) . based on geometric means , the patient radiation dose reduction was 65% , 33% , and 73% for total dap , dap fluoroscopy , and dap exposure , respectively . for both diagnostic and interventional procedures , the first quartile ( q1 ) dap for the reference system exceeded the third quartile ( q3 ) of the dap for the current system , except for dap fluoroscopy . the dose reduction ( total dap ) was approximately 70% , 60% , and 65% for aneurysm , stroke , and other categories , respectively , based on geometric means . secondary analyses were performed to evaluate the impact of age on dap values because patient age was statistically significantly different between the reference system and current system group , for interventional procedures . there was no effect of age on dap in the interventional procedure group ( p=0.2597 ) . the median cak values were reduced from 0.78 to 0.27 gy for frontal and lateral channel together , with reduction from 0.59 to 0.21 gy and from 0.19 to 0.06 gy for the frontal and lateral channels , respectively ( table 3 ) ; the difference was significant ( p<0.001 ) . the dose reduction for the diagnostic procedures was 62% on the frontal channel and 67% on the lateral channel based on geometric means . the median cak values for interventional procedures diminished from 2.71 to 0.89 gy for frontal and lateral channel together , with reduction from 2.06 to 0.68 gy and from 0.65 to 0.21 gy for frontal and lateral channels , respectively . the dose reduction was 66% on the frontal channel and 67% on the lateral channel based on geometric means.table 3descriptive summary of cak values for diagnostic and interventional proceduresvariablediagnosticinterventionreference systemcurrent systemreference systemcurrent systemcak frontal channel , gy
n=190
n=174
n=111
n=138meansd0.780.890.310.332.561.740.970.89median0.590.212.060.68q1q30.350.910.130.371.223.650.381.35min max0.022.880.001.490.064.860.013.1895% ci0.220.300.070.120.781.100.290.47
ci confidence interval , sd standard deviation , q1 first quartile , q3 third quartile descriptive summary of cak values for diagnostic and interventional procedures
ci confidence interval , sd standard deviation , q1 first quartile , q3 third quartile regardless of the interventional subgroup , cak values were reduced for both frontal and lateral channels . fluoroscopy time and number of exposure images obtained were substantially higher for interventional procedures ( table 4 ) . the difference in fluoroscopy time , number of dsa images , number of dsa runs , and procedure duration did not reach statistical significance.table 4descriptive summary of acquisition settings for diagnostic and interventional proceduresvariablediagnosticinterventionreference system ( n=190)current system ( n=174)reference system ( n=112)current system ( n=138)no . of acquired exposures / dsa images meansd291.7181.3310.6192.5625.8445.2635.7506.1 median266278525464 q1q3160385173402292870299845 min max301,09611,434502,3271082,986 95% ci265.8317.6281.7339.4542.5709.2550.5720.9fluoroscopy time ( min ) meansd7.14.27.24.512.56.411.06.6 median661312 q1q34949717516 min max022022124023 95% ci6.57.76.67.911.313.79.912.1
ci confidence interval , sd standard deviation , q1 first quartiler , q3 third quartile descriptive summary of acquisition settings for diagnostic and interventional procedures
ci confidence interval , sd standard deviation , q1 first quartiler , q3 third quartile the aim of the study was to quantify the impact of x - ray imaging technology using image noise reduction image processing combined with optimized system settings on patient radiation dose indicators and to assess its effects on the working habits of the physician . this study confirmed dose reduction for dap exposure to be 69% and 73% for diagnostic and interventional procedures , respectively , based on geometric means . similar results were obtained in previously published work where comparison was based on two dsa acquisitions , performed with reference and current system settings , on the same patient . patient dose reduction in fluoroscopy was 33% for interventional neuroradiology based on geometric means . for diagnostic and interventional procedures together , patient radiation dose reduction for total dap ( sum of dap fluoroscopy and dap exposure ) and cak was approximately 60% . similar to published analyses of patient exposure in interventional neuroradiology [ 37 ] , this study reported wide variability in radiation exposure and acquisition parameters , with variation per procedure type and diagnostic versus interventional procedure [ 3 , 9 , 10 ] . some authors report peak skin dose , measured or estimated [ 11 , 12 ] , whereas we report dap and cak as displayed by the angiography system . the rad - ir study reports mean dap values of 320 gy cm for 382 cases including embolization of aneurysm and avm . reports a mean dap value of 305 gy cm and a median dap value of 256 gy cm for 172 embolization procedures . however , dercole et al . reports a higher median value of 352 gy cm for 82 procedures . the rad - ir study also reported cak values of 3.8 gy for interventional neuroradiology procedures among patients with avm and aneurysm . the study by vano et al . reported a cak median of 2.4 gy and a cak third quartile ( q3 ) value of 3.9 gy for cerebral embolizations . the 2009 society of interventional radiology guidelines recommend operators to be notified anytime cak values exceed 3 gy and then every 1 gy thereafter , or anytime dap exceeds 300 gy cm , and subsequently at increments of 100 gy cm . less than 4% of diagnostic procedures performed with reference system exceeded the threshold values for cak ( sum of frontal and lateral channels ) and about 9% exceeded the threshold for dap . only in 1% of the cases undergoing diagnostic procedures performed with the current system exceeded the threshold for cak , and < 1% exceeded the threshold for dap . however , about 47% of interventional procedures performed with the reference system exceeded cak values , and 58% exceeded dap threshold values . the rad - ir study reported 55% and 67% of avm and aneurysm procedures to be greater than 3 gy . the percentage of interventional procedures performed with the current system that exceeded the threshold values was reduced ; cak threshold is exceeded in 10% of cases , whereas dap in 8% of cases . in this study , dsa provides about 7080% of the total procedure dose . other studies report similar values [ 2 , 14 ] including the baseline study that this study is based on . with the patient radiation dose reduction in dsa seen with the current system , the relation fluoroscopy dsa changes making the contribution of fluoroscopy equally relevant on total procedure radiation load ( fluoroscopy contributes for about 40% and dsa for about 60% ) . interpreting data from this study is limited by those factors inherent to retrospective design including the inability to match patients . in addition , the clinical complexity of the procedure and comparative image quality has not been taken into account in the study . although data were obtained at the same facility with the same clinical staff for the two recording periods , it was not possible to determine if other patient dose reduction measures , such as further collimation , were implemented in the current system group . however , the reduction in dap exposure observed in this study is in the same range as that observed in a previously published work , where comparison was based on the same patient for dsa acquisition . thus the patient dose reduction is likely the effect of the optimized system settings interpreting data from this study is limited by those factors inherent to retrospective design including the inability to match patients . in addition , the clinical complexity of the procedure and comparative image quality has not been taken into account in the study . although data were obtained at the same facility with the same clinical staff for the two recording periods , it was not possible to determine if other patient dose reduction measures , such as further collimation , were implemented in the current system group . however , the reduction in dap exposure observed in this study is in the same range as that observed in a previously published work , where comparison was based on the same patient for dsa acquisition . the image noise reduction x - ray imaging technology in combination with optimized system settings , which enable dose reduction and optimal image quality , reduced patient radiation doses in dsa and fluoroscopy by approximately 60% ; there was no impact on physician working habits . | introductionthe purpose of this study was to quantify the reduction in patient radiation dose by x - ray imaging technology using image noise reduction and system settings for neuroangiography and to assess its impact on the working habits of the physician.methodsradiation dose data from 190 neuroangiographies and 112 interventional neuroprocedures performed with state - of - the - art image processing and reference system settings were collected for the period january
june 2010 .
the system was then configured with extra image noise reduction algorithms and system settings , which enabled radiation dose reduction without loss of image quality .
radiation dose data from 174 neuroangiographies and 138 interventional neuroprocedures were collected for the period january
june 2012 .
procedures were classified as diagnostic or interventional .
patient radiation exposure was quantified using cumulative dose area product and cumulative air kerma .
impact on working habits of the physician was quantified using fluoroscopy time and number of digital subtraction angiography ( dsa ) images.resultsthe optimized system settings provided significant reduction in dose indicators versus reference system settings ( p<0.001 ) : from 124 to 47 gy cm2 and from 0.78 to 0.27 gy for neuroangiography , and from 328 to 109 gy cm2 and from 2.71 to 0.89 gy for interventional neuroradiology .
differences were not significant between the two systems with regard to fluoroscopy time or number of dsa images.conclusionx-ray imaging technology using an image noise reduction algorithm and system settings provided approximately 60% radiation dose reduction in neuroangiography and interventional neuroradiology , without affecting the working habits of the physician . |
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neuronal degeneration , i.e. , neuronal cell death , underlies the pathology and malfunction of many different neurological diseases occurring in both animals and human beings . progressive and selective neuronal cell death in the central nervous system ( cns ) and/or the peripheral nerve network has been profoundly implicated in the pathogenesis of neurodegenerative disorders including alzheimer s disease ( ad ) , parkinson s disease , huntington s disease , lou gehrig s disease or amyotrophic lateral sclerosis , multiple sclerosis , epilepsy , stroke , traumatic injury , age - related macular degeneration , glaucoma , prion diseases , infections , and so on [ 1 , 2 ] . evidence is rapidly accumulating to suggest that selective neuronal cell death through necrosis and/or apoptosis mechanisms contributes significantly to the functional anomalies of specific neurologic disorders . changes of genetic , epigenetic , metabolic , and environmental factors might directly or indirectly cause ( 1 ) massive dna damage , ( 2 ) dysfunction of the ubiquitin - proteasomal system , ( 3 ) disruption of the axonal transport machinery , ( 4 ) abnormalities of mitochondrial structure and function , ( 4 ) disturbance of intracellular ionic homeostasis ( particularly ca2 + and zn2 + ) , and ( 5 ) accumulation of reactive oxygen species ( ros ) in the neuronal cells . accumulating intracellular stress subsequently results in ( 1 ) loss of spines and synapses , ( 2 ) fragmentation of neuronal processes and extended neuritic degeneration following demyelination , ( 3 ) global neuronal cell death following activation of signal transduction cascade for programmed cell death , ( 4 ) anomalies of microvasculature , and ( 5 ) provocated neuroinflammatory response leading to the destructive pathogenic changes ( see several representative reviews ) [ 14 ] . accordingly , the main aim of neural protection in neurodegenerative disorders is to retard progression by blocking the mechanisms that lead to neuronal cell death as well as associated neuroinflammatory events . therefore , considerable efforts have been made in recent decades to discover new potential therapeutic compounds that can help to prevent the onset or to slow down the progression of such diseases . equal attempts are also made to improve the therapeutic efficacy of known medications through chemical modification . given the evident advantages of being stable in the ambient environment , being permeable to the blood brain and/or blood eye barriers , and being convenient for administration ( fig . 1 ) , naturopathic compounds have growingly become groups of the best therapeutic candidates for neural protection from the sensory system including retina to the central nervous system ( brain ) . table 1 summarizes a group of selected herbal extracts that have demonstrated significant neuroprotective efficacy both in vivo and in vitro . taking tetramethylpyrazine ( tmp ) as an example , the neuroprotective efficacy and related issues are discussed here . 1the conventional route of most natural compounds from herbs to target organs . in the traditional chinese medicine , the herbal therapeutic ingredients are extracted and prepared as herbal tea or soup with water through regular cooking and taken orally . the effective ingredients are absorbed by the gastrointestinal tract and successfully pass through the blood brain and/or blood eye barriers to reach the targets , brain and/or retinas ( graphic clips were taken from the internet free sources)table 1list of selected naturopathic compounds that have demonstrated neuroprotective efficacy in both retina and brainnamenatural sources ( selected)structural classificationselected studiesbaicaleinradix scutellariaealkaloidsc [ 49 , 50 ] ; r [ 51 , 52 ] ; b [ 53 , 54]chlorogenic acideucommia or other plantspolyphenolicsc ; r ; b [ 57 , 58]curcumincurcuma longaalkaloidsc [ 59 , 60 ] ; r [ 59 , 61 ] ; b [ 60 , 62 , 63]emodinleguminosae seedalkaloidsc [ 64 , 65 ] ; r ; b [ 65 , 67]fisetinrhus cotinus barkpolyphenolicsc [ 68 , 69 ] ; r ; b kaempferoleuonymus alatus or impatiens balsaminaflavonoidsc [ 72 , 73 ] ; r [ 70 , 74 ] ; b ligustrazineligusticum wallichii rootsalkaloidsc ; r ; b [ 23 , 77]morinthe moraceae family , e.g. , mulberryflavonoidsc ; r ; b [ 80 , 81]myricetinmyrica rubraflavonoidsc ; r ; b naringeninsatureja obovataflavonoidsc ; r ; b paeoniflorinpaeony rootspolyphenolicsc ; r ; b [ 8890]puerarinpueraria lobata rootsalkaloidsc [ 91 , 92 ] ; r [ 93 , 94 ] ; b [ 95 , 96]pycnogenolpinus maritime barkflavonoidsc ; r [ 98 , 99 ] ; b quercetineuonymus alatusflavonoidsc [ 101 , 102 ] ; r [ 84 , 103 ] ; b [ 104 , 105].resveratrolgrapesflavonoidsc [ 106 , 107 ] ; r ; b [ 108110]rutinbuckwheatflavonoidsc ; r ; b wogoninscutellaria baicalensisflavonoidsc [ 112 , 113 ] ; r ; b [ 115 , 116]c , in vitro studies conducted in cultured cells ; r , in vivo studies showing retinal protection ; b , in vivo studies demonstrating neuroprotective efficacy in the brain the conventional route of most natural compounds from herbs to target organs . in the traditional chinese medicine , the herbal therapeutic ingredients are extracted and prepared as herbal tea or soup with water through regular cooking and taken orally . the effective ingredients are absorbed by the gastrointestinal tract and successfully pass through the blood brain and/or blood eye barriers to reach the targets , brain and/or retinas ( graphic clips were taken from the internet free sources ) list of selected naturopathic compounds that have demonstrated neuroprotective efficacy in both retina and brain c , in vitro studies conducted in cultured cells ; r , in vivo studies showing retinal protection ; b , in vivo studies demonstrating neuroprotective efficacy in the brain as listed in table 1 , tmp , also designated as ligustrazine , is an alkaloid extracted from the chinese herbal medicine , ligusticum wallichii franchat ( chuanxiong ) . for hundreds of years , chuanxiong has been used as a traditional chinese medicine for heart , kidney , and brain diseases [ 6 , 7 ] . experimental studies demonstrated that tmp treatments significantly improved cardiac and cerebral blood flow and elevated blood reperfusion as shown in the nail microcirculation [ 8 , 9 ] . in an ex vivo study , a semi - synthetic form of tmp monomer induced a dose - dependent relaxation of human pulmonary and bronchial arteries . tmp also exhibited a calcium antagonist role in vascular tissues ; functioned as a ros scavenger to deactivate cytotoxic ros such as superoxide anion ( o2 ) , hydroxyl ( oh ) , and lipid peroxyl ( loo ) free radicals [ 12 , 13 ] ; and inhibited inflammatory events in vivo possibly through modulating secretion of specific cytokines and nitric oxide - related pathways [ 1416 ] . about two decades ago , a study briefly reported that tmp alleviated ischemic retinal degeneration in vivo . recently , a different group of researchers demonstrated that systemic injection of tmp significantly protected retinal photoreceptor from loss induced by n - methyl - n - nitrosourea in rats . further , we demonstrated that tmp efficiently enhanced in vitro survival of cultured rat retinal cells and significantly attenuated cell damage in these cells exposed to hydrogen peroxide . in the cns , tmp significantly suppressed oxidative stress and attenuated neuronal cell death in neuronal cultures following iron - mediated oxidative damage and glutamate - mediated excitotoxicity [ 2022 ] . systemic administration of tmp protected neuronal cells against ischemic or traumatic brain or spinal cord injury and promoted functional recovery in rodents and rabbits [ 23 , 24 ] . interestingly , systemic administration of tmp also attenuated impairment of learning and memory performance in rodents following d - galactose- or ischemia - induced brain injury [ 25 , 26 ] . the potential therapeutic efficacy of tmp for ad is further supported by our recent observations about significant improvement of cognitive function as well as cerebral amyloid pathology in the demented alzheimer s triple transgenic ( 3xtg - ad ) mice ( tan et al . , unpublished observations ) . systemic administration of excitotoxin , kainic acid ( ka ) , a glutamate analog , causes striking neuronal cell death in rat brain following prolonged seizures . to examine further whether tmp protects brain cells from ka - induced neuronal degeneration , which is one of the most common animal models for excitotoxic neuronal cell death that is apparently involved in the pathogenesis of multiple neurodegenerative disorders [ 2729 ] , 1-month - old male sprague dawley rats were housed and treated according to the national institutes of health guidelines for the care and use of laboratory animals and a protocol approved by the uci institutional animal care and use committee . one hour after the onset of seizures following subcutaneous ( s.c . ) injection of ka ( 10.5 mg / kg ) , rats received tmp ( 50 mg / kg , s.c . ) or equal volume of vehicle . all the animals ( n = 6 each group ) were decapitated 24 h after tmp or vehicle injection under co2 gas - induced deep anesthesia . the brain was rapidly harvested , frozen , and cryosectioned in the coronal plane at 10 m . eosin ( h & e ) and in situ cell death assay kit for terminal deoxytransferase - mediated dutp nick end labeling ( tunel ; roche , indianapolis , in , usa ) , respectively , as described in our previous work [ 30 , 31 ] . systemic administration of ka resulted in a well - described pattern of behavioral seizures including wet dog shake at the beginning stage and progressed to tonic seizures typically occurred intermittently during the first 68 h and yielded about 10% or less mortality . as observed in this study , tmp treatments did not alter the pattern and the severity of the seizures . ka + vehicle demonstrated robust neuronal degeneration in ka - vulnerable regions in brain as revealed by both h & e staining and tunel labeling ( fig . 2a ( f j ) ) whereas the controls showed no eosinophilic or tunel - positive damaged cells ( fig . in contrast , given 50 mg / kg tmp following the onset of seizures , animals showed markedly fewer damaged cells in the corresponding regions in the brain ( fig . quantification of tunel - positive cells conducted as described in our previous work [ 32 , 33 ] revealed a statistically significant difference between ka + vehicle and ka + tmp , suggesting remarkable neuroprotective efficacy of tmp in the cns under excitotoxic attack . 2systemic administration of tmp attenuates neuronal degeneration in rat brains following kainate - induced seizures . a both hematoxylin eosin ( h & e ) and tunel staining demonstrate massive neuronal damage shown as pink in h & e staining ( f , g ) and green in tunel ( h , i , and j ) in both hippocampus ( f , g , h , and i ) and piriform cortex ( j ) regions . in contrast , the animals received tmp only exhibited a few sporadic eosinophilic ( l ) and tunel - positive cells in the corresponding regions ( m , n , and o ) . b quantification of tunel - positive cells according to our previous methods reveals a significant decrease in the number of damaged neurons in the hippocampal regions with tmp treatment . scale bar = 100 m in b , d , e , g , i , j , l , n , and o ; and 400 m in a , c , f , h , k , and m systemic administration of tmp attenuates neuronal degeneration in rat brains following kainate - induced seizures . a both hematoxylin eosin ( h & e ) and tunel staining demonstrate massive neuronal damage shown as pink in h & e staining ( f , g ) and green in tunel ( h , i , and j ) in both hippocampus ( f , g , h , and i ) and piriform cortex ( j ) regions . in contrast , the animals received tmp only exhibited a few sporadic eosinophilic ( l ) and tunel - positive cells in the corresponding regions ( m , n , and o ) . b quantification of tunel - positive cells according to our previous methods reveals a significant decrease in the number of damaged neurons in the hippocampal regions with tmp treatment . scale bar = 100 m in b , d , e , g , i , j , l , n , and o ; and 400 m in a , c , f , h , k , and m in cultured neural cells , tmp treatments significantly reduced generation of lipid peroxidation products , malondialdehyde , induced by hydrogen peroxide [ 19 , 20 ] . the observations are also in agreement with an increase in abundance of glutathione in 3xtg - ad mouse brains following tmp treatments ( data not shown ) . in addition , the benefits of tmp treatments preserved abundance of map2 and rattin , two molecules that play important roles in cell growth and function [ 19 , 34 , 35 ] . taken these together , tmp may target multiple levels and cell signal transduction pathways to contribute to the survival of neural cells and the normal function of the nervous system . as a naturopathic compound isolated from a chinese herbal medicine , tmp has been the subject of many pharmacological and toxicological studies . the solubility of purified tmp in crystal form is relatively low in neutral aqueous solution ( ~10 g / ml ) and dramatically increases in an acidic environment ( > 40 mg / ml , ph < 4 ) , in which tmp is stable and active . preclinical assessment of the distribution , metabolism , excretion , and toxicity ( admet ) of tmp has been performed in animals and in vitro since more than 20 years ago [ 3741 ] . without any surprise , pharmacokinetic studies demonstrated that tmp was efficiently permeable to the blood toxicity assays revealed a very low level of toxicity in animals with an oral ld50 of about 1,910 mg / kg in rats and 1,436 mg / kg in mice [ 42 , 43 ] . importantly , practitioners of traditional chinese medicine have continued to use tmp as a treatment for inflammatory or degenerative diseases , usually in combination with other medications [ 6 , 8 , 4446 ] . in this regard , both tmp tablets and tmp hcl injection solution are prescription - available for a treatment of cardiovascular diseases in china [ 47 , 48 ] . given the admet information that is already available on tmp , it would not take much preclinical data to justify bringing tmp to clinical trials in humans . this proof - of - concept proposal will provide sufficient preclinical evidence to justify studying tmp in patients with neurodegenerative disorders in a double - blind placebo - controlled manner . nevertheless , our understanding of the molecular basis for tmp - mediated pharmacological actions remains limiting . similarly , there is a lack of well - designed preclinical studies of tmp efficacy for neurological disorders . in addition to tmp , there are groups of naturopathic compounds that have been purified from related herbal medicines and identified as efficient neuroprotective ingredients as mentioned . resembling tmp , many of these compounds have demonstrated remarkable neuroprotective efficacy in experimental studies conducted in cell cultures and/or live animals . some of them are also used in clinics as treatment for specific neurological disorders . therefore , once further studies are warranted to decipher the molecular basis of related pharmacological efficacy , many of such naturopathic molecules would move to clinical assessments for neural protection in humans . | given the advantages of being stable in the ambient environment , being permeable to the blood
brain and/or blood eye barriers and being convenient for administration , naturopathic compounds have growingly become promising therapeutic candidates for neural protection . extracted from one of the most common chinese herbal medicines , tetramethylpyrazine ( tmp ) , also designated as ligustrazine , has been suggested to be neuroprotective in the central nervous system as well as the peripheral nerve network .
although the detailed molecular mechanisms of its efficacy for neural protection are understood limitedly , accumulating evidence suggests that antioxidative stress , antagonism for calcium , and suppression of pro - inflammatory factors contribute significantly to its neuroprotection . in animal studies , systemic administration of tmp ( subcutaneous injection , 50 mg / kg ) significantly blocked neuronal degeneration in hippocampus as well as the other vulnerable regions in brains of sprague dawley rats following kainate - induced prolonged seizures .
results from us and others also demonstrated potent neuroprotective efficacy of tmp for retinal cells and robust benefits for brain in alzheimer s disease or other brain injury .
these results suggest a promising prospect for tmp to be used as a treatment of specific neurodegenerative diseases .
given the assessment of the distribution , metabolism , excretion , and toxicity information that is already available on most neuroprotective naturopathic compounds such as tmp , it would not take much preclinical data to justify bringing such therapeutic compounds to clinical trials in humans . |
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hepatic portal venous gas ( hpvg ) is an uncommon radiological sign associated with numerous conditions and often portends significant underlying abdominal disease . it was first described by wolfe and evens in infants with necrotizing enterocolitis ( nec ) . a 65-year - old hypertensive diabetic female patient developed colicky left flank pain radiating to the groin . ultrasonography revealed an 8.0 mm mid - ureteric calculus for which she underwent ureterorenoscopic lithotripsy and double - j stenting . the stent was removed after 6 weeks . a week after stent removal , she developed continuous left flank pain , progressive abdominal distension , obstipation , and high - grade continuous fever . she had anemia ( hemoglobin 8 g / dl ) , polymorphonuclear leukocytosis ( total leucocyte count 42,600/mm ) and raised blood glucose ( 400 mg / dl ) , blood urea ( 55 mg / dl ) and serum creatinine ( 2.7 mg / dl ) levels . non - contrast computerized tomography ( ct ) revealed a grossly enlarged left kidney ( 15.5 cm 12.0 cm 19.0 cm ) distended with fluid and air . air was also noted in the peritoneal cavity , retroperitoneum , right inguinal canal and hepatic portal venous radicals [ figures 1 and 2 ] . emergency percutaneous nephrostomy drained 1150 ml of pus , which grew escherichia coli on culture . the patient was managed successfully with central venous pressure - guided intravenous fluids , insulin and broad - spectrum antibiotics . histopathology of the resected kidney suggested end - stage kidney with chronic pyelonephritis . computerized tomography axial section ( a ) and the coronal reformatted image ( b ) showing the obstructed left kidney ( white arrows ) distended with air and fluid . the parenchyma is markedly thinned out computerized tomography axial sections through the abdomen ( cranial to caudal ) showing air within the hepatic portal venous radicles ( white arrows ) hpvg is an uncommon radiological sign and often portends significant underlying abdominal disease . though first described in infants with nec , it may be seen in inflammatory , infective , obstructive and neoplastic abdominal conditions ; post - chemotherapy ; gastrointestinal investigations and interventions ; post - liver transplantation ; hemodialysis ; cardiopulmonary resuscitation ; severe hyperglycemic shock ; superior mesenteric artery syndrome ; cystic fibrosis ; intra - aortic balloon pulsation and bronchopneumonia . the mechanisms of porto - venous gas accumulation are : ( 1 ) passage of intestinal intraluminal or extraluminal peritoneal gas through capillaries and mesenteric venous tributaries , ( 2 ) passage of extra - peritoneal ( or retroperitoneal ) gas through the portosystemic collaterals , and ( 3 ) presence of gas - forming organisms . emphysematous pyelonephritis ( epn ) is a grave , acute gas - forming necrotizing renal and perinephric infection , commonly seen in adult diabetic females and immunocompromised patients with obstructive uropathy . e. coli is the commonest offending organism followed by proteus , klebsiella , anaerobic streptococci and candida . classified epn as type i ( dry ) and type 2 ( wet with intraparenchymal or perinephric fluid ) , the former associated with a higher mortality . huang and tseng classified epn based on ct appearance as : ( i ) class 1 : gas in the collecting system , ( ii ) class 2 : gas in the renal parenchyma without extrarenal extension , ( iii ) class 3a : perinephric extension of gas or abscess , class 3b : pararenal extension of gas or abscess , ( iv ) class 4 : bilateral epn or solitary kidney with epn . , this association probably occurred due to the passage of gas through retroperitoneal porto - systemic collaterals . however , the possibility of low - grade bowel infection in this immunocompromised state leading to hpvg exists . this may be attributed to the passage of gas from renal and perinephric tissues through portosystemic collaterals and possibly through mesenteric veins due to concomitant low - grade bowel infection in susceptible immunocompromised patients . | hepatic portal venous gas ( hpvg ) is an uncommon radiological sign and often portends significant underlying abdominal disease .
a number of conditions may produce this sign and identifying the underlying etiology is essential for management .
the advent of ultrasonography - color doppler imaging and computerized tomography has led to more frequent recognition of this condition .
this article describes the very rare association of hpvg in a patient with emphysematous pyelonephritis . |
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children < 2 years of age with ari attending al - sabeen hospital , a reference hospital in sana'a , yemen , were enrolled in a study from october 2002 to may 2003 . all children attending emergency and outpatient services between 8:00 a.m. and 1:00 a.m. with signs and symptoms of ari were recruited independent of disease severity . diagnosis was based on clinical signs , as suggested by the world health organization protocol for research on ari ( 4 ) . oxygen pressure ( po2 ) was measured by using pulse oximetry ( nonin medical , inc . , plymouth , mn , usa ) , and children were classified as having no or moderate ( po2 > 88% ) or severe ( po2<88% ) hypoxia , as suggested for high altitudes ( 5 ) , since sana'a is 2,200 m above sea level . nasopharyngeal aspirates were tested by using reverse transcription pcr , as previously reported ( 6 ) . children with severe hypoxia caused by rsv were compared with children with rsv and no or moderate hypoxia . the and student t tests were used , and values with p values < 0.20 were entered into backward logistic regressions . ethical approval was obtained from the ethics committees of the liverpool school of tropical medicine and al - sabeen hospital . a total of 325 ( 54% ) children were recruited from the emergency service , 235 ( 39% ) from outpatient clinics and 41 ( 7% ) from wards . rsv was identified in 266 ( 44% ) and hmpv in 66 ( 11% ) children , including 25 ( 4% ) coinfected with rsv and hmpv . two hundred thirty - two ( 87% ) children with rsv , 46 ( 70% ) with hmpv ( p<0.01 ) , and 22 ( 88% ) coinfected with both viruses had severe hypoxia ( table ) . among rsv - positive specimens , 171 ( 82% ) of 208 were group a and 37 ( 18% ) of 208 were group b. no association was seen between groups or genotypes and hypoxia . rsv , respiratory syncytial virus ; hmpv , human metapneumovirus ; sd , standard deviation ; ari , acute respiratory illness ; chd , congenital heart disease . children <3 months of age were more likely to have severe hypoxia if they were infected with both viruses ( figure ) . children with severe rsv hypoxia were more likely to have relatives with ari , and this factor plus a history of recurrent respiratory infections were risk factors for hmpv hypoxia . personal or family histories of atopy , prematurity , or chronic lung or congenital heart diseases were not associated with ari severity caused by rsv or hmpv . proportion of children with severe infection with respiratory syncytial virus ( rsv ) and human metapneumovirus ( hmpv ) , by age , yemen . the presence of animals ( cows and donkeys ) , a person in the house smoking , using private water sources , and cooking with fuels other than gas were more frequent for children with severe rsv hypoxia . only the presence of cows , private water , and cooking fuels other than gas were associated with severe hmpv hypoxia . multivariate analysis showed that age <3 months and using cooking fuels other than gas were risk factors for severe rsv hypoxia ( adjusted odds ratio [ aor ] 3.4 , 95% confidence interval [ ci ] 1.38.7 and aor 10.3 , 95% ci 2.248 ) and hmpv hypoxia ( aor 14.2 , 95% ci 3.165 and aor 13.1 , 95% ci 2.278 ) , while incomplete vaccinations ( aor 4.5 , 95% ci 1.712 ) and smoking ( aor 3.8 , 95% ci 1.59.8 ) were associated with severe rsv hypoxia but not hmpv hypoxia . conversely , a history of recurrent ari ( aor 13 , 95% ci 2.084 ) was associated with severe hmpv hypoxia but not with rsv hypoxia . identification of factors that increase the severity of ari may affect health policies . however , little information is available about such factors in developing countries . young children are especially susceptible to severe ari , and our findings confirm that in a hospital setting age is a factor for both severe rsv and severe hmpv ari . prematurity and congenital heart and chronic lung diseases , which have been associated with increased risk for hospitalization for rsv and hmpv infections , were not risk factors . since most yemeni children are born at home , with limited access to health services , these children are underrepresented in our sample . infection with rsv was more frequent and severe in well - nourished children in nigeria , the gambia , and chile ( 7 ) , but this finding was not confirmed by other investigators ( 8) , and it is still contentious . although no information exists about their role in infection with hmpv , increased levels of common allergens within households had no effect on rsv infection severity in the united states ( 9 ) . however , gambian mothers of children hospitalized with rsv infections reported asthma more frequently in their children than mothers of nonhospitalized children ( 10 ) . the role of atopy in development of severe rsv or hmpv infections in developing countries needs further elucidation . several reports have suggested that coinfections with rsv and hmpv increase disease severity , but we did not find such an interaction . although we can not exclude coinfection with bacterial or other viral agents , for which rsv or hmpv may increase disease severity , our findings are similar to reports from the mediterranean region ( 11,12 ) . this finding suggests that hmpv strains vary with location and time , and certain strains increase disease severity . children with severe hypoxia were less likely to be vaccinated , which is a likely indicator of poverty because poor parents in yemen only use health services if their children are ill and are less likely to have their children vaccinated . in industrialized countries , poverty is associated with a higher incidence of rsv , and risk factors for infection with rsv are more likely to occur simultaneously ( 13 ) . we also observed that other household characteristics , such as cooking fuel and water , proximity to animals and relatives with ari , or smoking increased the risk for hypoxia . although we did not quantify air pollution within households , traditional mud stoves are built at ground level , use wood or dung , and generate large amounts of fumes , which blacken adjacent walls . parental and maternal smoking during pregnancy , indoor pollution , and presence of pets are also risk factors in industrialized countries ( 14,15 ) . however , in the gambia the relationship between severe rsv infection and frequency of cooking was inverse ( 10 ) , which may reflect the lower socioeconomic status of mothers who cook frequently ; this finding needs to be explored further . finally , as a hospital - based study , our study had a selection bias toward children with severe ari , and community - based studies might find different risk factors than those reported here . in conclusion , age <3 months , incomplete vaccinations , persons smoking in the house , and using cooking fuels other than gas were associated with an increased risk for severe rsv hypoxia . similarly , age <3 months , using cooking fuels other than gas , and recurrent ari interventions to eliminate air pollution in households may reduce the severity of rsv and hmpv infections in developing countries , and further studies should be encouraged . | factors increasing the severity of respiratory infections in developing countries are poorly described .
we report factors associated with severe acute respiratory illness in yemeni children ( 266 infected with respiratory syncytial virus and 66 with human metapneumovirus ) .
age , indoor air pollution , and incomplete vaccinations were risk factors and differed from those in industrialized countries . |
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a 58-year - old woman presented to the dental hospital for prosthetic evaluation after a rhinectomy . the examination revealed a partial nasal resection leaving the bridge of the nose intact [ figure 1 ] . patient expressed dissatisfaction with her appearance and was especially concerned about attending an upcoming social event because of her facial disfigurement . the patient elected to proceed with the fabrication of an interim nasal prosthesis . patient facial photograph after surgery a facial moulage was made using standard dental impression material to permit the fabrication of a stone cast [ figure 2 ] of the face . a model of the planned nasal prosthesis was sculpted in wax on this resultant cast using the remaining normal anatomic landmarks for reference [ figure 3 ] . an interim nasal prosthesis was processed from the wax model using self - cure acrylic resin with extrinsic coloring incorporated to match the surrounding skin tones . the bridge of the nose provided enhanced support for the prosthesis and eyeglass frames . at the subsequent 4-week follow - up appointment , patient stated that she was satisfied with the cosmetic resulting [ figure 4 ] and had felt very comfortable attending the social event while wearing the prosthesis . patient profile photograph after wearing prosthesis white surgical tape was used to permit easy masking , to anticipate discrepancies in marginal fit as healing progressed and to create the illusion of a less extensive surgical procedure having been performed . retention of the prosthesis was obtained through the use of a medical - grade adhesive augmented by surgical tape , eyeglasses and the use of an eye patch over the prosthetic eye and tied behind the head . patient accommodated the prosthesis well and was discharged to return to his out - of - state home with plans to return for reconstructive surgery after an adequate disease - free period had elapsed . surgical reconstruction of facial defects may be delayed or determined to be inappropriate for some patients . a 3 - 5-month delay after resection is typically required before fabricating a definitive facial prosthesis to allow for sufficient healing and reorganization of the defect to occur to obtain an acceptable long - term fit . such delays can present a significant hardship for patients with pronounced facial defects and create the potential for serious adverse psychosocial consequences . the post - surgical fabrication of a custom - sculpted interim facial prosthesis combined with masking agents such as surgical tape and eyeglasses can provide rapid cosmetic rehabilitation , allowing the patient to more comfortably and confidently resume social interactions without the obvious stigma of facial disfigurement . this procedure introduces no trauma to the operative site ; hence , fabrication of the prosthesis can commence within several days after surgery . the problem posed in maintaining the proper esthetic fit of the interim prostheses subsequent to post - surgical marginal tissue changes was addressed by the placement of white surgical tape along the margins of the prostheses . because marginal fit is lost during healing , additional tape can rapidly be applied , eliminating the need for revising the prosthesis . in this manner , effective early rehabilitation can be achieved and easily maintained over time using a more natural - feeling and readily adaptable prosthesis . the alternative of attempting to continually revise the interim prostheses in response to loss of fit poses significant logistical and technical problems . further , it is technically difficult to effectively add new medical grade silicone material to existing silicone prostheses with the products currently available . de - bonding with separation of the newly added silicone material away from the existing prosthesis commonly occurs . because of this technical difficulty , poly ( methyl methacrylate ) resins have been recommended as an alternative material for interim prosthesis . new resin can be easily bonded as needed over time in response to post - operative marginal tissue changes . prostheses made from these resins ; however , are rigid and feel much more artificial . medical - grade silicone materials as used in the aforementioned case reports more closely approximate the viscoelasticity of the surrounding tissues . because some individuals may demonstrate hypersensitivity to tape adhesive , patients should be closely observed initially to permit early identification of adverse tissue reactions . marked , persistent contact irritations may require patient to limit the time during which the prosthesis is worn or to discontinue wearing of the prosthesis . patients need to be instructed to remove the prosthesis at least once daily to permit cleaning of the underlying tissue . the prosthesis should be removed in the evening before patient sleeps to further limit the risk of contact irritation of the skin . measurements of pre - surgical facial anatomy and planning discussions between the surgeon and prosthodontist can assist in maximizing interim prosthetic treatment esthetic outcomes . anatomic sites of value in supporting and retaining the prosthesis can be identified and taken into account during the surgery . for example , the bridge of the nose typically provides valuable support for a nasal prosthesis as well as for eyeglass frames . however , when pre - surgical planning is not possible , rehabilitation can still rapidly proceed with good results as seen in the case study presented . interim prostheses may provide one additional benefit in that they allow for easy access to clinically observe post - operative wound healing and provide additional intervention therapies as needed . final surgical revisions of facial defects or definitive prosthetic rehabilitation can then proceed at an appropriate pace without the overlay of patient concerns regarding appearance . interim facial prostheses offer an option to commence early rehabilitation for patients with significant facial defects who might otherwise be faced with extended periods of disfigurement . interim prostheses can be rapidly fabricated using soft , silicone materials and placed soon after facial surgery . they can then be easily revised with the use of surgical tape to accommodate for post - operative defect changes arising from marginal tissue healing . they provide a cosmetically acceptable interim treatment outcome , permitting patients to comfortably resume many social activities . further , they permit easy access to observe wound healing and provide additional therapy as indicated . | surgical resection of neoplasms or malformations of the face may result in defects that are not amenable to immediate surgical reconstruction .
such defects can have a severe adverse effect on patient perceptions of body image and self - esteem . in these cases ,
the use of an interim removable facial prosthesis can offer a rapid alternative treatment solution .
the patient may then resume social interactions more comfortably while permitting easy access to the facial defect to observe tissue healing while awaiting definitive rehabilitation .
this article presents a case report describing the use of interim nasal prostheses to provide rapid patient rehabilitation of facial defects . |
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thyroglobulin ( tg ) transport through the thyroid follicular cell into the follicular lumen ( and to a more limited degree across the basement membrane and into the circulation ) involves a number of plasma membrane receptors and internal transport systems that direct the tg molecule to specific intracellular and extracellular locations [ 1 , 2 ] . although internal tg transport takes place within membrane - bounded organelles ( e.g. , endosomes and golgi ) , tg transport outside the cell has heretofore been thought to encompass only the secretion of soluble tg through extracellular domains such as the thyroid follicular lumen , extracellular spaces , and the circulatory system . several independent findings [ 35 ] taken together however suggest that tg might also be secreted from thyroid cells as a constituent of membrane - delimited vesicles ( exosomes ) that originate as invaginations of late stage endosomes called multivesicular bodies ( mvbs ) . exosomes together with microvesicles that bud directly from the plasma membrane into the extracellular space contain proteins , mrnas , and micrornas that are increasingly being seen as performing important regulatory roles in both normal and abnormal ( e.g. , cancerous ) cells . no specific role for either type of vesicle in the thyroid gland has yet been defined . the production of exosomes in thyroid - derived cells was indicated in a recent study showing that three thyroid cancer cell lines ( all derived initially from thyroid follicular cells ) release vesicles with the well - defined morphological features of exosomes into the cellular environment . however , many tumor cell types have been shown to secrete large numbers of exosomes differing in both protein and nucleic acid content from exosomes released from the cell type of origin [ 6 , 8 , 9 ] , so it would be difficult to draw conclusions from the cancer cells as to what exosomes contain and what they do in the normal thyroid . however , a recent proteomic analysis of fetal bovine serum - derived exosomes listed tg as one of the 51 different proteins contained in an exosome - enriched fraction of the serum , but not in an exosome - free fraction . this finding would support the hypothesis that thyroglobulin - containing exosomes may be released from normal thyroid cells into the circulation in healthy individuals . on the other hand , a tem study of the thyroid of risso 's dolphin , grampus griseus , showing circular membrane - bound vesicles < 100 nm in size located within the lumen of thyroid follicles sufficiently distant from the epithelium so as not to represent microvilli might be evidence of vesicle secretion into the colloid follicle , similar to what has been shown to occur in the ovarian follicle . in the present study we show for the first time that a line of functional thyroid cells ( frtl-5 ) secretes tg - containing exosomes into the culture medium , suggesting that the release of tg in exosomes could be a normal physiologic process that serves as an alternative to currently understood pathways of tg secretion and processing . a continuous , diploid cell line , frtl-5 , derived from the thyroid gland of the fisher rat and retaining many of the biochemical markers of the thyroid follicular cell ( such as tsh dependence ) was employed in this study . the cells were grown in a medium ( 6h medium ) consisting of coon 's modified ham 's f-12 solution containing six critical hormones ( insulin ( 10 g / ml ) , transferrin ( 5 g / ml ) , somatostatin ( 0.01 g / ml ) , glycyl - l - histidyl - l - lysine acetate ( 0.1 g / ml ) , hydrocortisone ( 0.362 ng / ml ) , and thyroid stimulating hormone ( tsh ) ( 0.001 iu / ml ) ) plus 5% fetal bovine serum , glutamine , penicillin , and streptomycin . medium containing all of these components is referred to as 6h medium , and medium with all components except tsh is referred to as 5h medium . to collect exosomes for tem , frtl-5 cells were grown in plastic 75 cm culture flasks containing 10 ml of 6h medium at 37c in the presence of 5% co2 in a humidified incubator . medium was replaced every 3 days until cells reached confluence , at which point the cultures were trypsinized and split into four flasks containing 6h media . samples of spent 6h media were collected and stored at 80c for later exosome collection and tem . for western blot , confluent frtl-5 cultures in 6 , 75 cm flasks were washed with hbss and received 5h medium for 3 days , after which 3 cultures received a volume of 7 ml of 5h medium and the remaining 3 received 7 ml of 6h medium . for all studies , purified exosomes were collected from medium using two steps of ultracentrifugation as described previously . briefly , conditioned medium was removed from frtl-5 cultures , a protease inhibitor cocktail ( complete protease inhibitor cocktail tablets , roche diagnostics ) was added , and the medium was centrifuged at 1500 rpm for 10 min to remove cellular debris . the cellular monolayer was then washed and trypsinized , and cell counts were determined with a hemocytometer . to isolate microvesicles , the frozen medium was thawed and centrifuged at 17,000 g for 18 min to pellet larger organelles and other membrane structures out , followed by a final centrifugation at 200,000 g for 1 hour and 15 minutes and collection of the pellet ( exosomal fraction ) for either electron microscopy or protein extraction . for some studies , additional pellets and supernatants from the two centrifugation steps were collected for protein measurement and western blot . for some western blot studies , exosome collection from 5h and 6h medium , respectively , was maximized by the sequential ultracentrifugation of medium collected from each of three flasks in the same centrifuge tube , adding medium to the pellet from the previous round of ultracentrifugation . exosome pellets for western blot were resuspended in 15 l of lysis buffer containing leupeptin and phenylmethylsulfonyl fluoride ( pmsf ) and frozen at 4c . approximately 5 l of exosome sample was applied to the surface of electron microscopy sciences formvar - coated , carbon - stabilized 3 mm copper grids . each sample was left on a grid for 5 minutes before the excess liquid was wicked off using the edge of a piece of filter paper . next , 5 l of electron microscopy sciences 2% aqueous uranyl acetate stain was applied to the grid for 1 minute as a negative stain , excess solution was wicked off , and the samples were allowed to air - dry before observation . imaging was done using a jeol jem-1011 transmission electron microscope and images were collected using an advanced microscopy techniques xr50s - a digital camera . exosome samples from 5h and 6h cells were mixed with 15 l of bio - rad 2x laemmli buffer . 9.25 l supernatant samples were mixed with 3.75 l of nupage lds sample buffer ( 4x concentration ) containing lithium dodecyl sulfate and 1.5 l of nupage sample reducing agent ( 10x concentration ) containing 500 mm dithiothreitol ( dtt ) . all samples were heated to 70c for 10 minutes and 15 l of each sample was loaded into lanes in a precast polyacrylamide 412% gel alongside a bio - rad precision plus protein kaleidoscope standard ( bio - rad laboratories ) . samples on the gel were then transferred to a pdvf membrane using a dry transfer system ( iblot , thermo fisher scientific ) . the membrane was blocked with 5% fat - free milk/0.05% tween-20 in immuno - tris . the exosome marker cd9 was detected using a rabbit polyclonal antibody ( system biosciences ) and a goat anti - rabbit igg antibody conjugated to horseradish peroxidase ( hrp ) . antibodies were each applied to the membrane dissolved in 10 ml of blocking solution with the primary antibody applied overnight for approximately 24 hours and the secondary antibody applied for 1 hour . sonia q. doi as an optimization of existing sds - page and western blotting procedures . positive signal was detected with addition of a substrate ( supersignal west femto chemiluminescence reagent , fisher scientific ) and the image was captured by lcd camera system . subsequently , the membrane was stripped of antibodies using a buffer containing 3-(n - morpholino)propanesulfonic acid ( mops ) , -mercaptoethanol , and sds . a second immunostaining procedure was performed to detect thyroglobulin ( tg ) using a dako mouse anti - human - tg clone dak - tg6 as a primary antibody ( dako , agilent technologies ) and a horse anti - mouse igg conjugated with hrp as the secondary antibody . the blots were developed with a supersignal west pico chemiluminescence kit and imaged as described above . exosomes collected by ultracentrifugation of conditioned frtl-5 6h growth medium were examined using tem of resuspended pellet contents immobilized and stained on copper grids ( figure 1 ) . images show spherical structures of varying sizes primarily less than 100 nm in diameter , consistent with the size and shape of exosomes . figure 2 shows the results of a representative western blot for tg on pellet and supernatant fractions collected by the differential centrifugation of 6h medium exposed to frtl-5 cells in culture for 48 h ( lanes 14 ) and from fresh 5h medium containing 1 mg / ml exogenous bovine tg ( btg ) ( lanes 58 ) . pellets and supernatants were saved following both 17,000 g ( low - speed ) and 200,000 g ( high - speed ) centrifugations and proteins from both low - speed and high - speed ( i.e. , exosome ) pellets were extracted and subjected to electrophoresis together with samples of the low - speed and high - speed ( i.e. , soluble tg ) supernatants . the figure clearly shows the presence of immunoreactive bands at the expected size of ~330 kda for monomeric tg in each of the samples taken from medium exposed to frtl-5 cells ( lanes 14 ) . the intensity of the bands varied considerably , due to the intrinsic differences in protein concentration between supernatants and pellet extracts and also , as we demonstrate below , because of differential partitioning of tg into either a soluble state ( supernatant ) or membrane - delimited exosomes ( pellet ) after the final centrifugation . the 200,000 g exosome pellet also exhibited a less dense band of 220 kda ( figure 2 ) . in contrast to the conditioned medium collected from frtl-5 cells ( lanes 14 ) , fresh 5h medium to which 1 mg / ml btg was added did not display any 330 kda immunoreactive bands ( lanes 58 ) , confirming that the dako anti - human - tg monoclonal antibody used in this study recognizes rat but not bovine tg ( figure 2 ) . the species specificity of the antibody was advantageous for subsequent experiments to examine the effects of exogenous tg and tsh on the secretion of exosomal tg from frtl-5 cells . in order to gain a better understanding of tg localization into exosomes under high- and low - tsh conditions , the partitioning of tg into pellet and supernatant fractions from 5h- and 6h - treated cells as illustrated in figure 2 was also examined in two additional studies ( figure 3 ) , in which careful measurements of total protein were made . as noted above , the 200,000 g pellets showed strong tg immunoreactivity at 330 kda and also to a lesser degree at 220 kda , with the 6h extracts yielding bands of greater density than in the corresponding 5h extracts ( figure 3 ) . immunopositive tg bands were also observed in both 17,000 g and 200,000 g supernatants , but these were of lower density than in the final pellet due to the lower concentration of protein loaded in supernatant fractions than in pellet fractions . to ascertain the presence or absence of exosomes in each of these extracts , western blots for the exosome marker cd9 immunoreactive bands of appropriate molecular weight for cd9 ( two bands of 31,000 and 28,000 d , resp . ) were observed only in the 200,000 g pellet and were not observed in either the 200,000 g supernatant or the 17,000 g pellet . moreover , the results indicated that the amount of cd9 was greater in the 6h samples than in the 5h samples ( figure 4 ) . since the protein concentration of exosomal pellets differed between 5h- and 6h - treated cells , we normalized tg exosomal content ( measured in uncalibrated optical density ) to total protein content in order to make valid comparisons of total tg present in the final 200,000 g pellet of cells grown in the presence or absence of tsh ( table 1 ) . results showed an approximately 2-fold greater tg content in exosomes from the 6h - treated cells than from 5h - treated cells . this finding suggests that higher tg content in the 200,000 g pellet from 6h cells could represent not only a greater number of exosomes secreted from 6h exposed cells , but also selective partitioning of tg into these exosomes compared to exosomes collected from 5h exposed cells . recent studies have shown that tg exerts an autoregulatory effect on its own production in frtl-5 cells . we were therefore interested in ascertaining whether treatment of cells with a high content of bovine tg ( btg ) could markedly affect the amount of rat tg ( rtg ) partitioned into exosomes . figure 5 shows the results of such a study in which frtl-5 cells were exposed to either 6h medium , 5h medium containing 1 mg / ml btg , or 5h medium only ( control ) for 48 h prior to collection of the medium for exosome preparation . equal volumes of supernatant from the 17,000 g centrifugation , theoretically containing both soluble protein and microvesicle / exosomal fractions , produced definitive immunoreactive tg bands of ~330 kda that showed little difference in density among the three treatment groups ( figure 5 , lanes 810 ) . equal volumes of extract from the low - speed pellet , theoretically containing particles larger than exosomes and other high mw particulate matter , showed somewhat more variability in tg content than the corresponding pellets , with the tsh - treated group showing a 2-fold higher density band than the 5h group ( figure 5 , lanes 1113 ) . in contrast to the low - speed pellets , equal volumes of extract from the 200,000 g pellet from 5h - treated , 6h - treated , and bovine tg - treated cells , respectively ( figure 5 , lanes 13 ) , showed marked variability , with the tsh - treated ( 6h ) exosome extract exhibiting an approximately 10-fold greater density than the 5h extract . the relevant band from the tg - treated exosome extract was intermediate in density between the other two groups . the bovine tg - treated sample also showed funneling and band distortion of the type associated with an overload of total protein in the sample ( lane 3 ) . in contrast to the exosome fractions , tg in the 200,000 g supernatants showed less variation , but the tsh - treated group exhibited a denser tg band than the other two groups ( figure 5 , lanes 57 ) . the present study demonstrates , using transmission electron micrography and western blotting for cd9 , that exosomes are produced in vitro by cultures of thyroid - derived cells . in addition , western blotting using a monoclonal antibody against tg suggests that tg is encapsulated in these vesicles and released into the culture medium together with tg secreted via conventional exocytosis . tem images of microvesicle pellets from conditioned 6h media consistently showed spherical structures within the expected exosomal size range of 30 to 100 nm in diameter . it is likely that some of these structures are exosomes , but the presence of other possible structures in these samples with similar sizes and shapes ( including spherical protein aggregates and other membrane - delimited vesicle structures ) can not be excluded using this technique . western blotting for exosome markers supports the presence of exosomes in these samples , but the definitive identities of the observed spherical structures will require further tem imaging with immunogold staining for exosome markers . the results suggest that sequestration of tg into exosomes could represent a normal alternative pathway of tg processing in the thyroid cell , in which tg is diverted from the pathway of lysosomal degradation and production of t3 and t4 , and released intact from the cell . given the novelty of this finding , alternative trivial explanations for these results must also be considered , including the coprecipitation of insoluble tg with exosomes and the nonspecific attachment of tg to those exosomes . colloidal tg is known to be covalently linked by different enzymatic reactions in different species to form insoluble high molecular weight aggregates ( globules ) of tg that could conceivably coprecipitate with exosomes . indeed the tg obtained from bovine thyroid glands used in this study contained a substantial fraction ( 11% , according to the manufacturer ) of cross - linked , insoluble tg that probably accounted for the large yellowish pellets collected after the final 200,000 g centrifugation of either conditioned medium from btg - treated cells or fresh medium to which 1 mg / ml exogenous btg was added . since cross - linkage of tg into insoluble forms occurs only within the thyroid follicle , monolayer frtl-5 medium would have contained very little , if any , insoluble rat tg ( rtg ) to coprecipitate with exosomes . moreover , had rat tg been adsorbed to other proteins in high concentration in the incubation medium , most especially the 1 mg / ml btg that was added in some studies , then the exosome fraction from cells treated with btg ( figure 5 , exosome pellet , lane 3 ) should have contained a much greater amount of 330 kda immunoreactive rtg than the other two treatments , acquired through nonspecific adsorption to and coprecipitation of soluble rtg with btg . previous investigations from this lab and others have shown a suppressive effect of excess exogenous btg on tg production by frtl-5 cells that we have argued constitutes a local negative feedback system . however , no clear effect of that exogenous tg on exosomal rtg could be discerned from a comparison of the density of tg staining in the exosomes of control , tsh - treated , and tg - treated cells shown in figure 5 . an autocrine regulatory effect of tg on the packaging or secretion of tg in exosomes thus can not be inferred . for the reasons discussed above , however , the experiment provides support for the hypothesis that most of the tg in the exosome pellet is contained within vesicles and not attached to aggregate surfaces . unlike exogenous tg , an effect of tsh on exosomal secretion from thyroid cells has substantial support from the present western blot results which show consistently that exosome extracts from cells receiving tsh had a higher content of immunoreactive tg than cells lacking tg . moreover , our results suggest that the effect of tsh may be due to a greater content of tg per exosome ( twofold ) in addition to the secretion of a higher number of exosomes over a given time period . our current study of exosomes in frtl-5 cells in monolayer culture does not distinguish between apical and basal secretion of the vesicles . investigations of exosomes in polarized cultures of frtl-5 cells will be important in addressing this question definitively . the previous identification of membrane - bound vesicles of a size consistent with exosomes in the follicle lumen in tem studies suggests that at least some exosomes are secreted into the follicular colloid . exosome secretion across the basement membrane and into the circulation , on the other hand , has been observed in many epithelial cell types and could therefore take place in the thyroid follicular cell . there is some evidence from a proteomic analysis of exosomes in fetal bovine serum ( fbs ) that this occurs . in this study , proteomic analysis of the exosome - enriched ( eem ) and the exosome - free ( efm ) fractions of bovine serum revealed that tg was one of a total of 51 proteins found only in the eem . these results provide evidence that exosome - enclosed tg is secreted through the basolateral aspect of the thyroid cell and into the general circulation . in summary , we have demonstrated that tg is secreted from frtl-5 cells in culture in a membrane - enclosed form . future studies aimed at identifying a mechanism by which tg is diverted into an exosomal pathway , rather than transcytotic or lysosomal pathway , will be required to determine whether the tg exosomal secretion we observed in monolayer cultures of rat thyroid cells occurs as a normal process in vivo and , if so , what function(s ) tg could perform within and outside of the thyroid gland . | exosomes are 30100 nm , membrane - bound vesicles containing specific cellular proteins , mrnas , and micrornas that take part in intercellular communication between cells . a possible role for exosomes in thyroid function has not been fully explored . in the present study , frtl-5 rat thyroid cells
were grown to confluence and received medium containing either thyroid stimulating hormone ( tsh ) , exogenous bovine thyroglobulin ( btg ) , or neither additive for 24 or 48 hours followed by collection of spent medium and ultracentrifugation to isolate small vesicles .
transmission electron microscopy and western blotting for cd9 indicated the presence of exosomes .
western blotting of exosome extract using a monoclonal anti - tg antibody revealed a tg - positive band at ~330 kda ( the expected size of monomeric tg ) with a higher density in tsh - treated cells compared to that in untreated cells .
these results are the first to show that normal thyroid cells in culture produce exosomes containing undegraded tg . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the deepwater horizon
oil spill in the gulf of mexico in 2010 was
the largest release of crude oil in u.s . history . polycyclic
aromatic hydrocarbons ( pahs ) , which are suspected human carcinogens ,
are among the most toxic and persistent components of crude oil . according to their origins , pahs are classified
into pyrogenic pahs arising from fossil fuel combustion and petrogenic
pahs that are unique to crude oil . petrogenic pahs differ in structure
from that of pyrogenic pahs ( unsubstituted ) in that they are either
extensively alkylated or oxygenated to yield pah - quinones . contamination
of the food chain with petrogenic pahs is a major
hazard that would impact human health . it is widely recognized that pyrogenic pahs themselves are biologically
inert and that their carcinogenic effects require metabolic activation
to generate biologically reactive intermediates to form dna adducts
that result in mutations . however , there
is a paucity of information on the toxicological properties of petrogenic
pahs . in our previous studies , we described metabolic activation of
a representative alkylated pah 5-methylchrysene ( regioisomer of c1-chrysenes )
and a representative oxygenated pah phenanthrene-9,10-quinone in human
hepatoma ( hepg2 ) cells . alkylated pah 6-ethylchrysene
( 6-ec ) is a representative regioisomer
of c2-chrysenes detected in the crude oil released from the deepwater
horizon oil spill . to our knowledge , there are no
published accounts of the metabolic activation of 6-ec . in view of
the fact that human liver is the major target organ for exposure to
6-ec following ingestion , we studied the metabolism of 6-ec in human
hepg2 cells as a model to predict metabolism in primary human hepatocytes . four possible routes for the potential metabolic activation of
6-ec were considered to predict and identify its metabolites ( scheme 1 ) . first , we predicted
the diol - epoxide pathway , involving conversion of 6-ec to trans - dihydrodiols
followed by formation of diol - epoxides , which can form dna adducts
or be hydrolyzed to the corresponding tetraols . second , we predicted
the ortho - quinone pathway , involving conversion of trans - dihydrodiols
to catechols followed by either formation of conjugates , or their
oxidation to ortho - quinones , which could be reduced back to the catechols
to establish redox cycling that leads to oxidative dna damage . the
electrophilic ortho - quinone could also react with dna or the most
abundant cellular nucleophile , glutathione ( gsh ) , to form the gsh
conjugates , which could be further metabolized into the cysteinylglycine
( cys - gly ) conjugates , cysteine ( cys ) conjugates , and eventually n - acetyl - l - cysteine ( nac ) conjugates . third , as
6-ec contains two bay regions within its structure , a combination
of these two potential metabolic activation pathways was also predicted . fourth , we also predicted that hydroxylation on the side chain of
the 6-ethyl group followed by formation of sulfate conjugates resulting
in dna adducts could be another potential metabolic activation pathway . it was found that the metabolism of 6-ec involved
the formation
of mono - diol - epoxides , mono - ortho - quinones , and bis - electrophiles . bis - electrophiles involved the formation of a mono - diol - epoxide and
a mono - ortho - quinone on different terminal rings within the same structure ,
the formation of bis - diol - epoxides , and the formation of bis - ortho - quinones . we conclude that the diol - epoxide pathway , the ortho - quinone pathway ,
and their combination likely lead to the potential metabolic activation
of 6-ec following ingestion . 6-ec - tetraols and 6-ec - catechol conjugates
represent human exposure biomarkers for 6-ec that may result from
consuming seafood contaminated by crude oil spills . caution!all pahs are potentially hazardous
and should be handled in accordance with the national institutes of
health guidelines for the laboratory use of chemical carcinogens . cell culture media and reagents
were all obtained from invitrogen co. ( carlsbad , ca ) except for fetal
bovine serum , which was purchased from hyclone ( logan , ut ) . all other chemicals
used were of the highest grade available , and all solvents were hplc
grade . hepg2 ( human hepatocellular carcinoma )
cells were obtained from american type culture collection and maintained
as previously described . cultured cells
with a passage number of 1020 were used in the experiments
to reduce variability due to long - term culture conditions . cultured
cells were authenticated by short - terminal repeat dna analysis and
were mycoplasma - free ( dna diagnostics center medical , fairfield , oh ) . confluent hepg2 cells were plated in a 6-well plate ( 5
10 ) . the cells were washed twice and then treated
with mem ( without phenol red ) containing 10 mm glucose and 1 m
6-ec ( dmso , 0.2% v / v ) . the culture media were collected at 0 and 24
h and subsequently acidified with 0.1% formic acid before extraction
as previously described . the extract from
the cell culture media was reconstituted in 150 l of methanol . for hplc - uv - flr analysis , a 10 l aliquot of the reconstituted
extract was analyzed on a tandem waters alliance 2695 chromatographic
system with a waters 2996 photodiode array ( pda ) detector and a waters
2475 multi fluorescence ( flr ) detector ( waters corporation ,
milford , ma ) . separations were accomplished on a zorbax - ods c18 analytical
column ( 5 m , 4.6 mm 250 mm ) with a zorbax - ods analytical
guard column ( 5 m , 4.6 mm 12.5 mm ) ( dupont co. , wilmington ,
de ) at ambient temperature . the mobile phase consisted of 5 mm ammonium
acetate and 0.1% trifluoroacetic acid ( tfa ) ( v / v ) in h2o ( solvent a ) and 5 mm ammonium acetate and 0.1% tfa in acetonitrile
( solvent b ) and was delivered at a flow rate of 0.5 ml / min . the linear
gradient elution program was as follows : 5 to 95% b over 30 min , followed
by an isocratic hold at 95% b for another 10 min . at 40 min , b was
returned to 5% in 1 min , and the column was equilibrated for 19 min
before the next injection . excitation ( ex ) and emission
( em ) wavelengths for the flr detector were set at
269 and 366 nm , respectively , based on the spectral properties of
6-ec ( figure s1 ) . the optimal pair of ex and em of 6-ec was employed to detect
its metabolites based on the assumption that most 6-ec metabolites
show fluorescence signals at these wavelengths . for ion trap lc - ms / ms analysis , a 10 l aliquot of the reconstituted
extract was analyzed on a waters alliance 2690 hplc system ( waters
corporation , milford , ma ) coupled to a finnigan ltq linear ion trap
mass spectrometer ( thermo scientific , san jose , ca ) . the column , mobile
phase , flow rate , and linear gradient elution program were the same
as those described above . during lc - ms / ms analysis , up to 10 min of
the initial flow was diverted to waste before evaluation of eluants . the mass spectrometer was operated in both positive and negative ion
modes with an electrospray ionization ( esi ) source . eluants were monitored
on the ltq using product ion scan ( ms ) , subsequent ms / ms / ms
( ms ) , and pseudo selected reaction monitoring ( srm ) modes . the mass spectrometry parameters included spray voltage ( 3 kv in positive
ion mode and 5 kv in negative ion mode ) , sheath gas flow rate ( 40
arbitrary units in both ion modes ) , auxiliary gas flow rate ( 15 arbitrary
units in both ion modes ) , capillary temperature ( 275 c in both
ion modes ) , capillary voltage ( 38 v in positive ion mode and 19
v in negative ion mode ) , and tube lens ( 20 v in positive ion mode
and 22.05 v in negative ion mode ) . an isolation width of 3
bracketed around the m / z of interest ,
activation q of 0.25 , and activation time of 30 ms were used for data
acquisition . xcalibur software , version 2.0 ( thermo scientific , san
jose , ca ) , was used to control the lc - ms / ms system and to process
data . the preliminary information on metabolite structures was obtained
by interpreting the corresponding ms and ms spectra of 6-ec metabolites from ion trap lc - ms / ms . in some
instances , another 5 l aliquot of the reconstituted
extract was analyzed on a nano - acquity ultra - performance liquid chromatography
( uplc ) system ( waters corporation , milford , ma ) coupled to a ltq orbitrap
xl mass spectrometer ( thermo scientific , san jose , ca ) . separations
were accomplished on a nano - uplc c18 column ( 1.7 m beh130 ,
150 m 100 mm ) ( waters corporation , milford , ma ) at 50
c . the mobile phase consisted of 0.1% formic acid ( v / v ) in h2o ( solvent a ) and 0.1% formic acid ( v / v ) in acetonitrile ( solvent
b ) and was delivered at a flow rate of 1.6 l / min . the linear
gradient elution program was as follows : an isocratic hold at 5% b
for 5 min , 5 to 95% b over 30 min , followed by an isocratic hold at
95% b for another 10 min . at 46 min , b was returned to 5% in 2 min ,
and the column was equilibrated for 12 min before the next injection . the mass spectrometer
was operated in positive and negative ion modes with a nano - electrospray
ionization ( nano - esi ) source after accurate calibration with the manufacturer s
calibration mixture . the ionization voltage was set to 1.5 kv , and
the capillary temperature was set to 200 c . full scan spectra
were acquired with a resolving power of 60 000 full width at
half - maximum in the mass range from m / z 100 to 600 . lists of accurate masses of the potential 6-ec metabolites
were used to detect the formation of bis - electrophiles containing
both a diol - epoxide and an ortho - quinone within the same structure
in table 1 , the formation
of bis - diol - epoxides in table 2 , and the formation of bis - ortho - quinones in table 3 . xcalibur software , version
2.0 ( thermo scientific , san jose , ca ) , was used to control the orbitrap
mass spectrometer and to process data . comparison of uv chromatograms
at max 269 nm
at 0 h ( figure 1a )
and 24 h ( figure 1b )
showed that nine metabolites of 6-ec were detected in the organic
phase of the ethyl acetate - extracted acidified media from hepg2 cells . the peak attributed to 6-ec at 0 h was almost completely absent at
24 h , suggesting that 6-ec was rapidly metabolized by hepg2 cells
over this time course . the corresponding uv spectra of the nine metabolites
were extracted from the pda detector and are shown in figure s2 . the uv spectra of metabolites 48
were similar to that of 6-ec , showing that aromaticity has been retained ,
whereas metabolites 2 and 3 were predicted to have lost their aromaticity . ( a ) uv
chromatogram at max 269 nm at 0 h. ( b ) uv chromatogram
at max 269 nm at 24 h. ( c ) flr chromatogram at ex 269 nm and em 366 nm at 0 h. ( d ) flr chromatogram
at ex 269 nm and em 366 nm at
24 h. human hepg2 cells ( 5 10 ) were treated
with 6-ec ( 1 m , 0.2% ( v / v ) dmso ) in mem ( without phenol red )
containing 10 mm glucose . the cell media were collected at 0 and 24
h and subsequently acidified with 0.1% formic acid before extraction
with ethyl acetate . comparison of flr chromatograms
at ex 269 nm
and em 366 nm at 0 h ( figure 1c ) and 24 h ( figure 1d ) showed that there were seven fluorescence
peaks in figure 1d ,
corresponding to metabolites 28 in figure 1b , thus validating the presence of a fluorophore . the peaks corresponding to metabolites 1 and 9 were detected only
in the uv chromatogram ( figure 1b ) but not in the flr chromatogram ( figure 1d ) , suggesting a loss of the 6-ec ring fluorophore . identification
of o - monosulfonated-6-ec - dihydrodiol , 6-ec - dihydrodiol , and 6-ec - tetraol
indicated the occurrence of the diol - epoxide pathway as a potential
metabolic route of activation of 6-ec in hepg2 cells . a single
isomer of monodehydrated o - monosulfonated-6-ec - dihydrodiol with the
retention time of 23.42 min was detected by comparing the pseudo srm
chromatograms ( m / z 351
271 ) at 0 h ( figure 2a ) and 24 h ( figure 2b ) in the negative ion mode . the corresponding ms spectrum
( m / z 351 ) of this metabolite showed
the characteristic loss of the sulfate group ( 80 amu ) from the deprotonated
molecular ion ( figure 2c ) , and the ms spectrum ( m / z 351 271 ) of this metabolite showed the subsequent
loss of one ch3 group from the alkyl side chain ( figure 2d ) . the specific
position of the o - monosulfonated - dihydrodiol could not be assigned
using mass spectrometry . ( a ) extracted ion chromatogram of pseudo srm transition
at 0 h. ( b ) extracted ion chromatogram of pseudo srm transition at
24 h. ( c ) ms spectrum of the peak at 23.42 min . the samples were prepared
as described in the caption to figure 1 and were subsequently analyzed on an ion trap lc - ms / ms . three isomers of 6-ec - dihydrodiols
and three isomers of monodehydrated
6-ec - dihydrodiols were detected in hepg2 cells with identical retention
times by monitoring the extracted ion chromatograms of the orbitrap
full scan at 0 and 24 h in the positive ion mode ( figure s3 ) . ms spectra of these isomers provided the accurate
masses and molecular formulas of 6-ec - dihydrodiols and their monodehydrated
products with acceptable ppm values ( figure s3 ) . the presence of these isomers suggests saturation of 6-ec on both
terminal benzo rings . two isomers of bis - dehydrated 6-ec - tetraols
at 17.72 and 19.04
min were detected by monitoring the ms chromatograms ( m / z 289 ) at 0 and 24 h in the positive
ion mode ( table 4 ) . the corresponding ms spectra ( m / z 289 ) of these metabolites showed the sequential loss of
h2o ( 18 amu ) , co ( 28 amu ) , and ch3 ( 15 amu )
from the protonated molecular ion ( table 4 ) . comparison of the retention times of bis - dehydrated
6-ec - tetraols with metabolites 2 and 3 by hplc - uv - flr in figure 1 showed good agreement . a single isomer of
a monodehydrated 6-ec - tetraol and a single isomer
of a bis - dehydrated 6-ec - tetraol were detected in hepg2 cells with
different retention times by monitoring the extracted ion chromatograms
of the orbitrap full scan at 0 and 24 h in the positive ion mode ( figure s4 ) . because 6-ec is asymmetric , there
are two possible regioisomeric 6-ec - tetraols : those that come from
6-ec - trans-1,2-dihydrodiol and those that come from
6-ec - trans-7,8-dihydrodiol . the resulting tetraols
and their absolute configurations can not be assigned using mass spectrometry . identification
of 6-ec - dihydrodiol , o - monosulfonated-6-ec - catechol , o - monomethyl - o - monosulfonated-6-ec - catechol ,
monohydroxy - o - monomethyl - o - monosulfonated-6-ec - catechol , 6-ec - dione ,
monohydroxy-6-ec - dione , and nac-6-ec - ortho - quinone indicated the occurrence
of the ortho - quinone pathway as a potential route of metabolic activation
of 6-ec in hepg2 cells . a single isomer at 24.38 min corresponding
to either an o - monosulfonated-6-ec - catechol or an o - monosulfonated-6-ec - bis - phenol
was detected by monitoring the pseudo srm chromatograms ( m / z 367 287 ) at 0 h ( figure 3a ) and 24 h ( figure 3b ) in the negative ion mode . the corresponding
ms spectrum ( m / z 367 )
of this metabolite showed the characteristic loss of the sulfate group
( 80 amu ) from the deprotonated molecular ion ( figure 3c ) , and the ms spectrum ( m / z 367 287 ) of this
metabolite showed the subsequent loss of one ch3 group
( figure 3d ) . comparison
of the retention time of this peak with that of metabolite 7 by hplc - uv - flr
in figure 1 showed
that they were in agreement . however , we favor formation of the o - monosulfonated-6-ec - catechol
isomer because it would be derived from the reduction of either 6-ec-1,2-dione
or 7,8-dione . both the intermediate 6-ec - dihydrodiol and 6-ec - dione
were detected in this study . detection of either an o - monosulfonated-6-ec - catechol
or an o - monosulfonated-6-ec - bis - phenol
in human hepg2 cells . ( a ) extracted ion chromatogram of pseudo srm
transition at 0 h. ( b ) extracted ion chromatogram of pseudo srm transition
at 24 h. ( c ) ms spectrum of the peak at 24.38 min . the samples were
prepared as described in the caption to figure 1 and were subsequently analyzed on an ion
trap lc - ms / ms . two isomers of o - monomethyl - o - monosulfonated-6-ec - catechols
at
21.12 and 24.05 min were detected by monitoring the pseudo srm chromatograms
( m / z 381 301 ) at 0 h ( figure 4a ) and 24 h ( figure 4b ) in the negative
ion mode , which showed the loss of the sulfate group ( 80 amu ) from
the deprotonated molecular ion . the ms spectrum ( m / z 381 301 ) of the isomer
peak at 24.05 min showed the characteristic losses of ch3 and ch2ch3 groups ( figure 4c ) , suggesting that the ch3 group
was not cleaved from the ch2ch3 side chain of
6-ec and was due to the occurrence of o - methylation . the unique biotransformation
of o - methylation strongly indicated the formation of the catechol ,
thus confirming the potential metabolic activation of 6-ec via the
ortho - quinone pathway . although the specific position of catechol
conjugation , the methyl group , and the sulfate group on 6-ec - catechols
could not be assigned using mass spectrometry , it is likely that only
6-ec-1,2-catechols or 7,8-catechols were formed . comparison of the
retention time of the major isomer peak at 24.05 min with that of
metabolite 6 by hplc - uv - flr in figure 1 showed good concordance . detection of o - monomethyl - o - monosulfonated-6-ec - catechols
in human
hepg2 cells . ( a ) extracted ion chromatogram of pseudo srm transition
at 0 h. ( b ) extracted ion chromatogram of pseudo srm transition at
24 h. ( c ) ms spectrum of the peak at 24.05 min . the samples
were prepared as described in the caption to figure 1 and were subsequently analyzed on an ion
trap lc - ms / ms . five isomers of monohydroxy - o - monomethyl - o - monosulfonated-6-ec - catechols
at 19.51 , 19.93 , 20.47 , 24.05 , and 25.18 min were detected by monitoring
the pseudo srm chromatograms ( m / z 397 317 ) at 0 h ( figure 5a ) and 24 h ( figure 5b ) in the negative ion mode , which showed the loss
of the sulfate group ( 80 amu ) from the deprotonated molecular ion . the ms spectrum ( m / z 397 317 ) of the isomer peak at 19.93 min showed
the characteristic losses of ch3 and ch2ch3 ( figure 5c ) ,
suggesting that the ch3 group was not cleaved from the
ch2ch3 side chain of 6-ec and was due to the
occurrence of o - methylation . the unique biotransformation of o - methylation
strongly indicated the formation of the catechol , thus confirming
the potential metabolic activation of 6-ec by the ortho - quinone pathway . as the loss of ch2ch2oh was not observed , this
ruled out activation of 6-ec by a combination of the ortho - quinone
pathway and side chain hydroxylation . although the specific position
of catechol conjugation , the hydroxyl group , the methyl group , and
the sulfate group on 6-ec - catechols could not be assigned using mass
spectrometry , it is likely that only 6-ec-1,2-catechols or 7,8-catechols
were formed . comparison of the retention times of the isomer peaks
at 19.93 , 20.47 , 25.18 min with those of metabolites 4 , 5 , and 8 by
hplc - uv - flr in figure 1 showed that they were in agreement . detection of monohydroxy - o - monomethyl - o - monosulfonated-6-ec - catechol
isomers in human hepg2 cells . ( a ) extracted ion chromatogram of pseudo
srm transition at 0 h. ( b ) extracted ion chromatogram of pseudo srm
transition at 24 h. ( c ) ms spectrum of the peak at 19.93
min . the samples were prepared as described in the caption to figure 1 and were subsequently
analyzed on an ion trap lc - ms / ms . two peaks at 24.41 and 34.06 min corresponding to 6-ec - dione
were
detected by comparing the pseudo srm chromatograms ( m / z 287 259 ) at 0 h ( figure 6a ) and 24 h ( figure 6b ) in the positive ion mode . the respective
ms spectra ( m / z 287 )
of these two metabolites showed the characteristic loss of co ( 28
amu ) from the protonated molecular ion , and a representative ms spectrum of the metabolite with a retention time of 34.06
min is shown in figure 6c . the ms spectrum ( m / z 287 259 ) of this metabolite showed the characteristic
loss of a second co and the loss of ch3 ( figure 6d ) . the sequential loss of
two co groups supported the presence of either an ortho - quinone or
a remote quinone , which can not be distinguished on the basis of mass
spectrometry . ( a ) extracted
ion
chromatogram of pseudo srm transition at 0 h. ( b ) extracted ion chromatogram
of pseudo srm transition at 24 h. ( c ) ms spectrum of the
peak at 34.06 min . the samples were prepared as described in the caption to figure 1 and were subsequently
analyzed on an ion trap lc - ms / ms . another peak with a retention time
of 24.41 min with a relatively high polarity could be an isomer of
o - monosulfonated-6-ec - catechol , which could undergo cleavage of its
sulfate conjugate in the mass spectrometer , followed by auto - oxidation ,
and thus result in the detection of quinone instead . a single isomer of monohydroxy-6-ec - dione at 29.58
min was detected
by comparing the pseudo srm chromatograms ( m / z 303 274 ) at 0 h ( figure 7a ) and 24 h ( figure 7b ) in the positive ion mode . the corresponding
ms spectrum ( m / z 303 )
of this metabolite ( figure 7c ) showed the characteristic loss of co ( 28 amu ) plus one
hydrogen from the protonated molecular ion . the ms spectrum
( m / z 303 274 )
of this metabolite ( figure 7d ) showed another characteristic loss of co. monohydroxy-6-ec - dione
could be derived from either an ortho - quinone or a remote quinone ,
and these alternatives can not be distinguished on the basis of mass
spectrometry . as the loss of ch2ch2oh was not
observed , this ruled out activation of 6-ec by a combination of the
ortho - quinone pathway and side chain hydroxylation . ( a ) extracted
ion chromatogram of pseudo srm transition at 0 h. ( b ) extracted ion
chromatogram of pseudo srm transition at 24 h. ( c ) ms spectrum
of the peak at 29.58 min . the samples were prepared as described in the caption
to figure 1 and were
subsequently analyzed on an ion trap lc - ms / ms . a single isomer of nac-6-ec - ortho - quinone at 32.99 min was
detected
by monitoring the ms chromatograms ( m / z 446 ) at 0 and 24 h in the negative ion mode ( table 4 ) . the corresponding
ms spectrum ( m / z 446 )
of this metabolite showed the characteristic loss of co ( 28 amu ) from
the deprotonated molecular ion and the characteristic loss of 129
amu resulting from a cleavage of the thioether bond , followed by the loss of ch2ch3 side
chain ( table 4 ) . the
unique biotransformation of nac conjugation strongly indicated the
formation of the ortho - quinone , thus confirming a potential route
of metabolic activation of 6-ec via the ortho - quinone pathway . the
specific position of nac conjugation on 6-ec - ortho - quinone could not
be assigned using mass spectrometry . evidence
for potential dual metabolic activation of 6-ec in hepg2 cells to
form a bis - electrophile containing a diol - epoxide and an ortho - quinone
within the same structure was obtained . two isomers of tetrahydroxy - o - monomethyl-6-ec - catechol
were detected by comparing the extracted ion chromatograms of the
orbitrap full scan at 0 and 24 h in the negative ion mode ( figure 8) . in particular ,
the unique biotransformation of o - methylation strongly indicated the
formation of the catechol , thus confirming the occurrence of ortho - quinone
pathway . the specific positions of the hydroxy groups of the tetraol
and o - monomethyl - catechol could not be assigned using mass spectrometry . detection
of tetrahydroxy - o - monomethyl-6-ec - catechols in human
hepg2 cells . ( a ) extracted ion chromatogram of orbitrap full scan
at 0 h. ( b ) extracted ion chromatogram of orbitrap full scan at 24
h. ( c ) ms spectrum of the peak at 16.35 min . a bis - tetraol was detected
by identification of two bis - dehydrated - tetraols
in hepg2 cells by comparing the extracted ion chromatograms of orbitrap
full scan at 0 and 24 h in the positive ion mode ( figure s5 ) , indicating the occurrence of activation on the
two terminal benzo rings to give a bis - diol - epoxide . o - monomethyl - catechol-6-ec - ortho - quinone
was also detected in hepg2
cells by comparing the extracted ion chromatograms of orbitrap full
scan at 0 and 24 h in the positive ion mode ( figure s6 ) , indicating the occurrence of the formation of a bis - ortho - quinone . in particular , the unique biotransformation involving o - methylation
strongly indicated the formation of the catechol , thus confirming
the occurrence of the ortho - quinone pathway . further evidence for
the formation of a bis - ortho - quinone came from the detection of 6-ec - bis - ortho - quinone
in hepg2 cells when the extracted ion chromatograms of orbitrap full
scan at 0 and 24 h in the positive ion mode were compared ( figure s7 ) . 6-ec
is a representative regioisomer of c2-chrysenes present in
crude oil that may enter the food chain after oil spills . we examined
its metabolism in human hepg2 cells knowing that human exposure pathways
involve ingestion . human hepg2 cells were selected as an alternative
to human hepatocytes due to the limitations of the latter , for instance ,
scarce availability , shorter life span , phenotypic instability , and
higher individual variability . we proposed four potential pathways
of 6-ec metabolic activation :
formation of diol - epoxides , ortho - quinones , bis - electrophiles , and
ethanesulfonates . we found evidence for the first three pathways but
no evidence for activation on the ethyl side chain involving hydroxylation
and subsequent sulfonation . representative metabolites of 6-ec from
the pathways of activation were detected and identified by hplc - uv - flr
and lc - ms / ms ( table 4 ) . metabolites
4 , 5 , and 8 in figure 1 were identified as isomers of a monohydroxy - o - monomethyl - o - monosulfonated - catechol . metabolite 6 in figure 1 was identified as an o - monomethyl - o - monosulfonated - catechol . metabolite
7 in figure 1 was identified
as an o - monosulfonated - catechol or an o - monosulfonated - bis - phenol . metabolites 1 and 9 in figure 1 remain unassigned . on the basis of the peak areas of
the metabolites on uv and flr
chromatograms , the major potential metabolic activation pathways of
6-ec in human hepg2 cells involved formation of diol - epoxides and
ortho - quinones ( scheme 2 ) . we also found evidence for the potential metabolic activation
of 6-ec on both bay regions to form bis - electrophiles containing a
mono - diol - epoxide and a mono - ortho - quinone within the same structure ,
bis - diol - epoxides , and bis - ortho - quinones ( scheme 2 ) . we suspect that these metabolites were
underdetected probably due to the potential of bis - electrophiles to
form protein and dna cross - links . the number of each
metabolite
corresponds to the metabolites labeled in the uv and fluorescence
chromatograms in figure 1 . we did not find any evidence for hydroxylation
on the ethyl group
side chain at c6 followed by formation of sulfate in human hepg2 cells ,
which is consistent with the previous findings that hydroxymethylation
of 5-methylchrysene and 6-methylchrysene ( 6-mc ) is not an important
metabolic activation pathway in humans . monodehydrated
o - monosulfonated-6-ec - dihydrodiol shown in figure 2 has an identical nominal mass as that of
the sulfate conjugate of side chain monohydroxy-6-ec . however , the
fragmentation pattern in figure 2 shows the loss of the sulfate group and the subsequent
loss of the ch3 group , which rules out the possibility
of side chain hydroxylation on 6-ec . metabolic activation of
6-mc via a diol - epoxide pathway has been
previously characterized in mouse skin , human liver microsomes , and
human lung microsomes . in this pathway , 6-mc containing a methyl group located at a non - bay
region is activated by p450 1a1 to yield 6-mc-1r,2r - diol , which is further converted to 6-mc-1r,2s - diol-3s,4r - epoxide with relatively weak dna - binding and carcinogenic properties
compared with those of the diol - epoxide generated from 5-methylchrysene
containing a methyl group located at a bay region . it was also
reported that hydroxylation on the methyl group of 6-mc catalyzed
by p450 3a4 and 1a2 leads to 6-hydroxy-6-mc without carcinogenic and
tumor - initiating activities in mouse skin . however , a large amount of 6-mc was left unmetabolized after incubation
in human liver microsomes , and a few metabolites found in significant
amounts were unidentified , which may
explain why only the diol - epoxide pathway of 6-mc activation was proposed
previously . the structure of 6-ec is identical to that of 6-mc except
for the length of the side chain ; therefore , it was expected that
the diol - epoxide pathway of 6-ec would be identified in the present
study . multiple metabolites were detected which indicated that
the ortho - quinone
pathway was involved in the potential metabolic activation of 6-ec . interestingly , o - monomethyl - o - monosulfonated-6-ec - catechols and their
monohydroxy products were detected in human hepg2 cells , whereas o - monomethyl-6-ec - catechols
were not detected , which could be explained if either the o - monomethyl
catechol is rapidly sulfonated or sulfonation proceeds prior to o - methylation . we failed to observe the occurrence of o - monoglucuronidation of 6-ec - catechol ,
probably due to the low expression level of uridine 5-diphospho - glucuronosyltransferases
in human hepg2 cells . the enzymes
responsible for the potential metabolic activation
pathway of 6-ec in human hepg2 cells remain to be identified . inducible
cytochrome p450 1a1 present in human hepg2 cells is probably responsible
for the formation of diol - epoxides . several
members of the aldo - keto reductase ( akr ) superfamily , including akr1c1 ,
ak1c2 , and akr1c3 , present in human hepg2 cells would oxidize 6-ec
trans - dihydrodiols to ortho - quinones . akrs , nad(p)h : quinone oxidoreductase 1 , and carbonyl reductase can
all catalyze the redox cycling of ortho - quinones to form intermediate
catechols . formation of 6-ec - diones indicates
that subsequent conjugation of the catechols is not completely effective
in preventing redox cycling , as predicted earlier in our studies of
benzo[a]pyrene-7,8-dione . sulfotransferases ( sults ) and catechol - o - methyltransferase would
be responsible for the formation of o - monomethyl - o - monosulfonated - catechols . the sults that are expressed in human hepg2 cells and likely responsible
for o - sulfonation are sult1a1 , 1a2 , 1e1 , and 2a1 . -glutamyltranspeptidase , dipeptidase , and n - acetyl transferase would sequentially catalyze the degradation
of a gsh - ortho - quinone conjugate to generate the nac - ortho - quinone
conjugate that was detected . potential
dual metabolic activation of 6-ec on both terminal benzo rings was
consistent with the previous findings of 5-methylchrysene , which further confirmed that p450 1a1 and akrs
can work separately or together in the metabolic activation of chrysene
derivatives containing two bay regions to form bis - electrophiles . elucidation of the metabolic pathways of 6-ec in human liver cells
can be used to identify exposure biomarkers of 6-ec that could be
used for biomonitoring human urine and plasma . our earlier studies
on the metabolic activation of 5-methylchrysene indicated metabolites
in common with those found in the present study , i.e. , tetraols and
catechol conjugates . in particular , o - sulfonated - catechols observed
in both studies could be biomarkers due to the intense molecular ions
in the negative ion mode and the characteristic loss of the sulfate
group ( 80 amu ) in ms spectra . by developing a panel of
biomarkers for petrogenic pah exposure , requisite specificity and
sensitivity will likely increase as opposed to measuring a single
analyte . these biomarkers have the potential
for monitoring the consumption of oil - contaminated seafood in humans . | exposure to polycyclic
aromatic hydrocarbons ( pahs ) is the major
human health hazard associated with the deepwater horizon oil spill .
c2-chrysenes are representative pahs present in crude oil and could
contaminate the food chain .
we describe the metabolism of a c2-chrysene
regioisomer , 6-ethylchrysene ( 6-ec ) , in human hepg2 cells .
the structures
of the metabolites were identified by hplc - uv - fluorescence detection
and lc - ms / ms .
6-ec - tetraol isomers were identified as signature metabolites
of the diol - epoxide pathway .
o - monomethyl - o - monosulfonated-6-ec - catechol ,
its monohydroxy products , and n - acetyl - l - cysteine(nac)-6-ec - ortho - quinone were discovered as signature metabolites
of the ortho - quinone pathway .
potential dual metabolic activation
of 6-ec involving the formation of bis - electrophiles , i.e. , a mono - diol - epoxide
and a mono - ortho - quinone within the same structure , bis - diol - epoxides ,
and bis - ortho - quinones was observed as well .
the identification of
6-ec - tetraol , o - monomethyl - o - monosulfonated-6-ec - catechol , its monohydroxy
products , and nac-6-ec - ortho - quinone supports potential metabolic
activation of 6-ec by p450 and akr enzymes followed by metabolic detoxification
of the ortho - quinone through interception of its redox cycling capability
by catechol - o - methyltransferase and sulfotransferase enzymes . the
tetraols and catechol conjugates could be used as biomarkers of human
exposure to 6-ec resulting from oil spills . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
central venous catheters ( cvcs ) are routinely used for long intravenous access in intensive care unit ( icu ) . a 50-year - old male was admitted to icu with head and chest injury needed multiple cvc for the long - term intravenous access . he had bilateral multiple rib fractures and right - sided hemothorax , for which an intercostal drain was inserted . thoracic computed tomography ( ct ) scan with intravenous contrast was done to look for vascular injuries , which did not reveal any other injuries . right - sided internal jugular vein was cannulated uneventfully , and the tip of cvc was confirmed in the chest radiograph along the right border of the mediastinum . the patient needed long - term intravenous access , hence change of cvc was planned 15 days later . patient was placed supine with arms adducted . under aseptic precaution , left subclavian vein was cannulated by seldinger 's technique in single attempt . postprocedure chest x - ray revealed , cvc was along the left border of the mediastinum rather than along the right border [ figure 1 ] . possibility of cvc entering in one of the tributaries of left brachiocephalic vein such as internal mammary vein was thought of as possibility . to differentiate among them , a lateral chest radiograph with intravenous contrast through the cvc since the position of left subclavian cvc was not confirmed , right subclavian vein was cannulated in single attempt . chest radiograph confirmed the position of right cvc along the right border of the mediastinum [ figure 2 ] . right internal jugular central venous catheter along the right border of mediastinum and left subclavian central venous catheter along the left border of mediastinum chest x - ray anteroposterior right subclavian central venous catheter along the right border of mediastinum and left subclavian superior vena cava along the left border of mediastinum all the chest radiographs and ct scan chest were reviewed with a radiologist . an earlier performed thoracic ct scan with intravenous contrast revealed that patient had persistent left sided superior vena cava ( svc ) along with right - sided svc . thus , the patient had double svc ( persistent left svc [ plsvc ] ) , and both of them were draining into the right atrium [ figures 3 and 4 ] . contrast - enhanced thoracic computed tomography transverse view . 1 : right - sided superior vena cava , 2 : left - sided superior vena cava , 3 : aorta , 4 : pulmonary artery 1 : right - sided superior vena cava , 2 : left - sided superior vena cava , 3 : aorta , 4 : pulmonary artery the cvc that is outside normal course ( along the left border of mediastinum ) leads to suspicion of it either being intra - arterial , extravascular or in one of the tributaries of brachiocephalic vein . in our patient , the blood could be easily aspirated from all the three ports ; hence , extravascular position was unlikely . hence , the possibility of cvc being in one of the tributaries of the left brachiocephalic vein such as internal mammary vein or superior intercostal or pericardiophrenic vein was considered . in the lateral view of chest radiograph , catheters in the internal mammary , pericardiophrenic and superior intercostal veins although plsvc is a relatively rare vascular anomaly ( prevalence of 0.3%0.5% in healthy individuals ) , it is the most common congenital anomaly of the thoracic venous circulation . the thoracic embryonic venous system is composed of two large veins ( the superior cardinal veins ) which return blood from cranial aspect of embryo . the left common cardinal vein persists to form coronary sinus and oblique vein of the left atrium . during the 8 week of gestation , an anastomosis forms between right and left superior cardinal veins resulting in the brachiocephalic vein . the cephalic portion of superior cardinal veins forms the internal jugular veins and the caudal portion forms the normal right - sided svc , whereas the portion of the left superior cardinal vein caudal to the brachiocephalic vein normally regresses to become ligament of marshall . if this normal regression of the left superior cardinal vein fails to occur , a persistent left - sided vascular structure that empties into the coronary sinus results in condition plsvc . plsvc may occur with a normal right - sided svc ( 82% ) or without right svc ( as a single left - sided svc ) . when a double svc exists , the left svc descends vertically , anterior , and to the left of the aortic arch and main pulmonary artery , then swings medially to enter the coronary sinus or right atrium . there are previous reports of an incidental diagnosis of a plsvc with an abnormal left para - mediastinal course on x - ray . despite these reports the knowledge of this anomaly , its diagnosis and implications are not enough among the anesthesiologist and the intensive care specialist . this leads to many unnecessary investigations and removal of cvc for the fear of being in abnormal position . if double svc is confirmed , most of the cvc can be left in situ and used without any adverse effects , keeping in mind that left - sided svc can directly drain into the left atrium ( 8% ) ; hence , there is increased risk of systemic air or particulate emboli . second , left svc may be associated with a wide variety of other cardiac defects such as single atrium , ventricular septal defect , and patent ductus arteriosus . therefore , we conclude that when cvc is along the left border of mediastinum , the possibility of pslvc should be kept in mind , along with the possibility of it being in one of the tributaries of brachiocephalic vein . if plsvc is confirmed , it can be left in situ and used for long - term infusion . | a 50-year - old male was admitted to intensive care unit with head and chest injury needed multiple central venous catheter ( cvc ) for the long - term intravenous access .
right internal jugular vein was cannulated uneventfully , and the tip of cvc was confirmed in the chest radiograph along the right border of the mediastinum .
after few days , left subclavian vein was cannulated and the procedure was uneventful .
however , the postprocedure chest x - ray showed the cvc along the left border of mediastinum rather than the right border .
possibility of cvc in one of the tributaries of left brachiocephalic vein was thought .
due to uncertainty in position of left cvc , we inserted cvc in right - sided subclavian vein , which was in normal position along the right border of mediastinum . left subclavian cvc was removed .
reviewing the patient 's thoracic computed tomography scan revealed , patient had congenital anomaly and double superior vena cava that explained the abnormal course of left subclavian cvc along the left border of mediastinum . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the importance of hypertension in the pediatric population has not been as well appreciated as in adults . children with elevated blood pressure ( bp ) can develop target organ damage , and they are also at increased risk of cardiovascular disease in adulthood . consequently , detection and management of elevated bp at an early age may be an important means for limiting the disease burden due to hypertension . the prevalence of hbp among children in several recently conducted studies in western countries ranged from 7 to 19% [ 46 ] . a study of blood pressure levels among primary school students in kuwait found that the overall prevalence of hypertension was 5.1% , 3.6% among school children in jordan , and 4.30% among preparatory school children in alexandria . the prevalence of childhood obesity has increased markedly over the last 2 decades [ 1014 ] . this increase is associated with an increase in hypertension rates which could lead to atherosclerotic disease in adulthood [ 15 , 16 ] . primary hypertension in children has become increasingly common in association with other cardiovascular risk factors that include being overweight , insulin resistance , and dyslipidemia . many studies have shown that blood pressure is associated with being overweight in children and adolescents of western countries [ 46 , 17 ] . however , few data are available in non - western countries and in non - caucasian populations [ 1820 ] . previous studies [ 2123 ] reported ethnic variation in the relationship of overweight / obesity and change in blood pressure , and this ethnic variations could be either genetic or environmental or both . thus , the aims of the present study were ( i ) to estimate the prevalence of hbp , and ( ii ) to investigate the relationship between bp and different body composition measures in egyptian school adolescents in alexandria city , egypt . to our knowledge a cross - sectional study was conducted with a total of 1500 school adolescents from different preparatory and secondary schools in alexandria city , egypt . the minimum required sample was calculated by using epi - info software program , on assumption that the prevalence of hypertension among school children is 4.30% . the calculated sample at 95% confidence level was found to be 1475 students . thus , a total sample of 1500 school adolescents enrolled in preparatory and secondary schools was considered , to compensate for drop out . three educational zones were selected randomly of the seven zones in alexandria . of each zone , four governmental schools were allocated randomly to represent preparatory and secondary education for both sexes . of each selected school , 3 classrooms were selected at random to represent the 3 educational grades . height was measured without shoes to the nearest 0.1 cm using a portable stadiometer . weight was measured in light clothing to the nearest 0.1 kg using a digital heine portable scale . wc was measured at the narrowest area above the umbilicus or mid way between the coastal margin and the iliac crest , in a horizontal plane at the end of normal expiration , with the tape measure snugly fitted [ 24 , 25 ] . hip circumference was measured at the maximal gluteal protrusion or at the most prominent area of the buttocks at the level of symphysis pubis in a horizontal plane . the tape measure was held snugly against the body but without compression [ 24 , 25 ] . bmi was calculated as the ratio of weight ( kg ) to height ( m ) squared ( kg / m ) . the 5th , 85th , and 95th percentiles were calculated according to the international statistics standards . adolescents were classified based on these percentiles as follows : < 5th underweight , 5th85th normal weight , 85th95th overweight , and 95th + considered obese . the 90th percentile values for wc for gender and age generated in the national health and nutrition examination survey ( nhanes iii ) were used as cut - off values to identify adolescents with abdominal obesity [ 24 , 28 ] . whr was calculated by dividing waist by hip circumference , and abdominal obesity was diagnosed when the whr was > 0.80 in girls and 0.95 in boys . whtr was calculated as the ratio of waist ( cm ) and height ( cm ) . a whtr cut - off of 0.5 is used to define abdominal obesity for both 619-year - old boys and girls . bp was measured using the standardized mercury sphygmomanometer with manually inflated cuff of suitable size and a stethoscope . it was measured on the right arm after the child was sitting quietly for 5 minutes to relieve anxiety , and seated with his or her back supported , feet on the floor , right arm supported , and cubital fossa at heart level left arm . interval , and the average was recorded . the blood pressure was measured two times with zero device . sbp was determined by the onset of the tapping korotkoff sounds ( k1 ) and the fifth korotkoff sound ( k5 ) , or the disappearance of korotkoff sounds , as the definition of dbp . the mean values of blood pressure were measured and corrected for age and sex in the form of centile bands and compared with us national childhood blood pressure standards . when the bp was greater than the 90th percentile for age , gender , and height , measuring bp was repeated twice at the same visit , and average sbp and dbp were used . if bp was greater than the 95th percentile , bp was staged to stage i ( 95th percentile to the 99th percentile plus 5 mmhg ) and stage 2 ( > 99th percentile plus 5 mmhg ) . adolescents aged 18 and 19 years in the study were considered as adults and were classified as follows : hypertension is defined as average sbp > 120 and dbp > 80 mmhg . stage ii hypertension is defined as average sbp greater than or equal 160 or dbp greater than or equal 100 mmhg . in order to adjust for the many of the possible confounders for the association between obesity and high blood pressure among adolescents , all students of the study sample . personal history of chronic diseases , such as , hypertension , diabetes mellitus , renal diseases , and endocrine diseases , and taking antihypertensive drugs , corticosteroid , insulin , or penicillin . family history of chronic disease , such as hypertension , obesity , and diabetes mellitus . the far relative was considered as others such as aunt , uncle , grandfather , and grandmother . lifestyle : this includes the following : ( a ) dietary habits : intake of salty and fatty diet ( very high , high , moderate , and little consumption ) . smoking : regular smoker / nonsmoker . only daily and weekly smokers were considered as regular smokers . daily smokers were those who , at the time of the survey , smoked cigarettes every day . weekly smokers were those who smoked less than once a day but at least once a week . physically active adolescents were those who reported practicing exercise more than one hour at least 3 times per week . a pilot study was carried out on 25 students in 2 schools ( one preparatory and the other secondary ) in alexandria at the middle districts . the pilot study aimed to test the validity and clarity of the structured questionnaire for the intended school adolescents , pretest the study tools in order to reveal any modifications needed , estimate the average time needed to obtain the required information , identify any possible potential difficulties in data collection , and examine the overall survey technique to be used . statistical analysis was done with aid of the computer program spss ( statistical package for the social sciences ) . student 's t - test was used to compare between sample means for quantitative data . mantel- haenszel chi - square , and chi - square for linear trend were applied to gauge the difference between categorical data . logistic regression was used to assess the relationship between the different body composition measures and elevated bp , adjusting for age , sex , smoking behaviour , food and salt consumption , history of chronic disease , family history of hypertension , diabetes mellitus , and obesity . all participants had the right not to participate in the study or to withdraw from the measurements prior to its completion . the study protocol received ethical approval from the research committee of the high institute of public health , alexandria university , egypt . table 1 shows the distribution of study sample according to the levels of blood pressure and body composition measures . the prevalence rates of prehypertension and hypertension were 5.7% and 4.0% , respectively , with no sex difference ( = 5.86 , df = 4 , p = .12 ) . overall obesity prevalence based on bmi measure overall obesity was significantly more prevalent in male than in female adolescents ( 11.8% versus 8.7% , p = .002 ) . central obesity was prevalent in 16.1% , 4.5% , and 16.7% based on whr , wc , and whtr measures , respectively . males showed significantly higher prevalence of central obesity based on wc ( 5.9% versus 3.2% , = 6.16 , df = 1 , p = .013 ) while female adolescents showed significantly higher prevalence of central obesity based on whr ( 31.9% versus 0.3% , = 277.7 , df = 1 , p < .001 ) . figure 1 shows that there was a significant gradual increase in the prevalence of elevated blood pressure with the advancement of age of the adolescents . this prevalence increased from 4.8% in adolescents aged 13 to 14 years to 15.7% in adolescents 17 years of age . ( l = 18.603 , p < .001 ) .
table 2 and figure 2 show the association between body composition measures and high blood pressure among school adolescents . compared with those of normal weight , obese adolescents had significantly greater odds of having high blood , whether defined by bmi ( or = 3.47 , 95% ci = 2.44 4.93 ) , wc ( or = 4.27 , 95% ci = 2.97 6.15 ) , whr ( or = 1.83 , 95% ci = 1.22 2.75 ) , or whtr ( or = 7.27 , 95% ci = 4.32 12.23 ) . when all body composition measures were adjusted for all the potential confounders in a logistic analysis ( age , history of chronic diseases , family history of hypertension , and family history of diabetes mellitus ) , both bmi and the wc measures were the only significant predictors of high blood pressure in school adolescents . blood pressure was significantly high among adolescents who are obese by bmi ( or = 2.18 , 95% ci = 1.38 3.44 ) and those who are obese by wc ( or = 3.14 , 95% ci = 1.67 5.94 ) . table 3 shows that high blood pressure was significantly more prevalent among school adolescents with positive history of chronic diseases ( or = 2.46 , 95% ci = 1.46 - 4.15 ) , those with positive family history of high blood pressure ( or = 1.61 , 95% ci = 1.14 2.27 ) , and those with positive family history of diabetes mellitus ( or = 1.53 , 95% ci = 1.08 2.15 ) . however , there was no significant association between high blood pressure and family history of obesity . with regard to the lifestyles and health - related behavior of adolescents , high blood pressure showed no significant association with any of these behaviours . in the present study , these figures were lower than those among iranian adolescent girls ( 13.9% , and 19.4% ) , and figures among us adolescents ( 15.7% , and 3.2% ) . there was a gradual and progressive significant increase in the prevalence of elevated blood pressure from 4.8% in young adolescents aged 13 to 14 years up to 15.7% in those aged 17 years . however , comparison of the prevalence of elevated bp with other studies is limited due to differences in the procedures used for bp measurement across studies . few population - based data on the relation between bp and bmi are available in children and adolescents in developing countries . in a school - based survey of a representative sample of youth aged 9 , 13 , and 16 years in canada , body mass index was consistently associated with sbp and dbp in all age - gender groups . the present study revealed that adolescents with high blood pressure were 3.5 times more likely to be overweight or obese as compared to adolescents with normal blood pressure . bmi was a significant predictor of high blood pressure among adolescents of the present study , even after adjustment for all potential predictors . this finding was in agreement with a previous followup study of adolescents for 31.5 years , where a bmi above the 95th percentile in adolescence predicted adult mortality rates , and a 10-kg higher body weight was associated with a 3.0-mmhg higher systolic and a 2.3-mmhg higher diastolic blood pressure . these increases translate into an estimated 12% increased risk for chd and 24% increased risk for stroke . wc was more powerful than bmi for predicting the incidence of hypertension in a previous study . in obese adolescents , the accumulation of excess fat is known to occur predominantly in the upper body rather than in the peripheral region . the increased waist circumference is unlikely to be due to visceral adipose tissue alone ; it probably reflects both visceral and subcutaneous fat , and , hence , total fatness . in contrast , the body mass index measures the sum of fat mass and fat - free mass , and it is impossible to know the relative contributions of each . the present study showed that adolescents with high blood pressure were more than 7 times more likely to be obese with central obesity ( wc percentiles > 90th for gender and age ) as compared to those with normal blood pressure . in a study of british youth aged 1116 years , waist circumference , representing central fatness , has increased much faster than body mass index over 1020 years . it has been reported in a previous study that combination of bmi and wc is more strongly related to cvd risk factors than bmi or wc alone . in the present study , after adjustment for all other body composition measures , both wc and bmi were the only significant predictors of high blood pressure in adolescents . few studies have shown that waist circumference may be a better predictor of cardiovascular disease than bmi [ 42 , 43 ] and waist - to - hip ratio . adjustments of wc by stature ( whtr ) or hip circumference ( whr ) were found to improve its association with the incidence of hypertension . whtr was recommended , being more highly correlated with visceral fat mass and clustering of cardiovascular risk factors in children and adults . it may be a more accurate tracking indicator of fat distribution and accumulation by age , because it accounts for the growth in both wc and height over age , particularly in children and adolescents . in the present study , adolescents with high blood pressure were 2 times and 7 times to be obese according to the whr and whtr , respectively . however , after adjustment with all other body composition measures , this association was not significant . this finding is in accordance with what was reported in a previous study that the use of ratios such as whr to assess obesity in children and adolescents may not be appropriate because they are highly age dependent and may obscure stronger relations that may be present with separate circumference measurements . first , bp was measured on one single visit whereas hypertension should be based on readings taken on several visits . however , most other epidemiological studies of bp in children also relied on single - visit readings [ 4649 ] . . however , these us reference data have the advantage of being adjusted for sex , age , and height , which are the main known physiological determinants of bp in children . this might affect the estimates of elevated bp , yet it is unlikely to have biased our findings on the relationship between bp and body composition measures . third , the study relied upon a written questionnaire for information on lifestyles and health behaviours , such as exercising , food and salt consumption , and smoking behaviour . if the accuracy of self - reported lifestyles differed by obesity , then our effect estimate might have been biased . fourth , we can not be certain of the causal direction of the associations observed between obesity and high blood pressure , due to the study 's cross - sectional design . a longitudinal study with repeated measures of body composition and blood pressure would be desirable in the future . in conclusion , this study provides important evidence of the association between both overall obesity , based on bmi percentiles and central obesity , based on wc percentiles and high blood pressure among egyptian adolescents . followup and regular blood pressure measurement should be an important step in school health programs . it could be used in a health promotion program to identify individuals who should seek , and be offered , weight management and those at risk of developing hypertension . prevention of cardiovascular risk factors as early as childhood may be an important strategy to prevent noncommunicable diseases in a life course perspective , particularly in settings with scarce resources and limited health care capacity . programs and policies to limit sedentary behaviours and promote physical activity and healthy nutrition among children are recommended . | aim . to investigate the relationship between high blood pressure ( hbp ) and obesity in egyptian adolescents . methods . a cross - sectional study of 1500 adolescents ( 1119 years ) in alexandria , egypt , was conducted .
resting bp was measured and measurements were categorized using the 2004 fourth report on blood pressure screening recommendations .
additional measures included height , weight , and waist and hip circumferences .
obesity was determined based on bmi , waist circumference ( wc ) and waist - to - hip ratio ( whr ) , and waist - to - height ratio ( whtr ) indicators .
crude and adjusted odds ratios were used as measures of association between bp and obesity .
results .
prevalence rates of prehypertension and hypertension were 5.7% and 4.0% , respectively .
obesity was seen in 34.6% , 16.1% , 4.5% , and 16.7% according to bmi , whr , wc , and whtr , respectively . adjusting for confounders ,
hbp was significantly associated with overall obesity based on bmi ( or = 2.18 , 95% , ci = 1.38 - 3.44 ) and central obesity based on wc ( or = 3.14 , 95% , ci = 1.67 - 5.94 ) .
conclusion .
both overall obesity and central obesity were significant predictors of hbp in egyptian adolescents . |
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we report a case of mature teratoma of the undescended left testis diagnosed on the basis of prenatal ultrasonography . a 26-year - old gravida 1 woman with no medial or surgical illness was referred for third trimester growth profile ultrasonography to our center at 36 weeks of gestation as a routine procedure showed 2.0 1.8 cm sized , well - circumscribed lesion in pelvis . the lesion was round in shape , cystic with a solid component , located on the left lateral side of urinary bladder [ figure 1 ] . left scrotal sac was empty , while the right scrotal sac showed normal echogenicity gonad of size 1.4 0.9 cm , and there were no other structural abnormalities identified . the provisional diagnosis of cryptorchid testicular teratoma was made and follow - up ultrasonography was suggested on day 1 of postnatal life . the neonate was born via the vaginal route . after delivery , the neonate was found to have left undescended testis with normal positioned right testis . postnatal ultrasonographic examination revealed a well - defined round to oval mass ( 2.0 1.9 cm ) with foci of subcentimeter - sized calcifications located on the left lateral side of urinary bladder [ figure 2 postnatal ] . intraoperatively , a 2.0 1.5 cm sized left testicular mass was found on the left lateral side of urinary bladder [ figure 3 of surgical photos ] . histology examination showed testis with a tumor composed of elements of all germ cell layers , viz . skin with adnexal structures , wall of small intestine with mucosal and muscular layers , fibrovascular tissue , brown fat , cartilage , bone with bone marrow , lymphoid tissue , glial tissue , and few areas of immature mesenchyme . prenatal usg shows well - defined round lesion on left side of urinary bladder with foci of calcifications ( multiple white markers along margin of lesion ) . prenatal diagnosis of cryptorchid testicular teratoma was made ( images of normal testis were not included due to limitations pertaining to pcpndt ) postnatal usg shows cystic lesion with calcifications on left side of urinary bladder with no internal vascularity on doppler study . these findings were similar on prenatal usg and diagnosis of cryptorchid testicular teratoma was assured intra - operative image shows lesion excised during surgery ( excised lesion held with forceps ) the diagnosis of mature ( pure ) testicular teratoma was thus confirmed . there is 38 times higher risk of neoplasia in cryptorchid testis relative to general population . the incidence of testicular neoplasm in prepubertal age group is 0.5 per 100,000 , while that in postpubertal age group is 10 times higher , i.e. 5.4 per 100,000 . intratubular germ cell neoplasia is considered a precursor to testicular germ cell tumors and is found in 2%-8% of adults with a history of cryptorchidism , but it has historically not been found in association with prepubertal germ cell tumors . intratubular germ cell neoplasia has been reported adjacent to mature teratoma in a prepubertal patient as an exceptionally rare finding . the differential diagnosis for a prenatally detected cystic abdominal mass includes lymphangioma , neuroblastoma , gonadal teratoma , multicystic dysplastic kidney , meconium cyst , and intestinal duplication cyst . the organ of origin for most cystic abdominal masses in the neonate is kidney ; however , the pelvic location of the mass in our case suggested a gonadal origin . the appearance of the mass as mixed cystic with calcifications helps to further narrow the differential diagnosis in favor of a testicular teratoma . till date , five cases of prenatally detected testicular teratoma have been reported in literature . detected a 2.5 2.3 cm cystic and solid mass on the left side of the bladder on prenatal ultrasonography at 31 weeks gestation . shih et al . reported a 5 4 3 cm semi - solid mass with central echogenicity in front of the right fetal kidney and anterior to the bladder at 36 weeks gestation . siu et al . reported a 3 cm , well - circumscribed , round , mixed cystic and solid intra - abdominal lesion located below the liver , anterior to the right kidney , and superior to the bladder at 30 weeks gestation . evaluated an infant at 6 months of age who was previously found to have a 2.1 1.9 cm mass in the right iliac fossa at 27 weeks gestation . the mass was stable on ultrasound at 30 and 35 weeks gestation and postnatally at 3 months of age . janda et al . detected 1.2 1.0 cm sized cystic mass on the left side of urinary bladder at 22 weeks of gestation , which on postnatal post - surgery histopathology showed features of teratoma . the incidence of torsion of the testis or its appendages by 25 years was 1 in 160 men ; however , cryptorchidism was associated with a 10-fold higher risk of torsion . torsion of the intra - abdominal testicle is a rare event , and a tumor within the testicle appears to predispose the gonad to torsion . duncan et al . reported that 59.5% of torsed intra - abdominal testicles were associated with testicular malignancy . most of the intra - abdominal testicular tumors present in adulthood . in the prepubertal population , brown et al . reported that six of seven patients ( 86% ) ranging in age from 2 months to 11 years with intra - abdominal testicular torsion had testicular tumors . | testis follows a predictable course during its stepwise descent from its site of origin in the lumbar region to its final destination in ipsilateral hemi - scrotum .
undesended ( cryptorchid ) testis is more prone for neoplastic transformation .
testicular teratoma is rare relative to ovarian teratoma and occurs rarely prenatally than postnatally .
teratoma is composed of derivatives of three germ layers .
ultrasound with color doppler is the most commonly used modality for assessment of prenatal status .
prenatal diagnosis of cryptorchid testicular teratoma involves identification of well - defined , complex solid - cystic lesion with calcifications along the path of testicular descent and absent testis in ipsilateral hemi - scrotum at 36 weeks of gestation .
complications associated with cryptorchid testicular teratoma include torsion , hemorrhage , or malignant transformation .
early diagnosis is important to avoid complications , and hence , ultrasound imaging features play an important role in diagnosis . |
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measles is a febrile disease that typically occurs in small children ; the incubation period is 1014 days , after which clinical symptoms such as fever , coughing , and a characteristic rash appears . since measles is accompanied by immunosuppression , it has a high frequency of complication with secondary bacterial infections , such as otitis media or pneumonia . although developed countries are eradicating measles by promoting effective vaccination , measles remains an important issue , especially in developing countries ( griffin , 2007 ) . measles virus ( mv ) , belonging to the genus morbillivirus of the family paramyxoviridae , is an enveloped virus with a non - segmented negative strand rna genome . the mv genome has six genes that encode the nucleocapsid ( n ) , phospho ( p ) , matrix ( m ) , fusion ( f ) , hemagglutinin ( h ) , and large ( l ) proteins ( figure 1a ) . mv contains two envelope glycoproteins : the h protein , which is responsible for receptor binding and is important for determining cell tropism of mv ; and the f protein , which mediates membrane fusion ( navaratnarajah et al . , 2009 ) . the p gene encodes the p protein and the non - structural v and c proteins . the v and c proteins are important for antagonizing the host interferon ( ifn ) response ( gerlier and valentin , 2009 ) . the p gene encodes the p protein and the non - structural v and c proteins . the v protein is translated from an edited rna with a non - templated g residue . the c protein is translated from an alternative open reading frame ( purple ) initiated by the second aug . pathogenic wild - type mv strains can be isolated by inoculating throat swabs from measles patients in b95a cells . signaling lymphocyte activation molecule ( slam , also called cd150 ) , expressed in certain immune system cells including activated b and t lymphocytes , mature dendritic cells , and macrophages , is a receptor for wild - type mv and vaccine and laboratory - adapted strains of mv ( tatsuo et al . , 2000 ) . cd46 ( also called membrane cofactor protein ) , expressed in all human and monkey nucleated cells , is a receptor for vaccine and laboratory - adapted strains of mv ( dorig et al . recently , nectin-4 [ also called poliovirus - receptor - like-4 ( pvrl4 ) ] has been identified as the epithelial receptor for wild - type mv ( muhlebach et al . , 2011 ; several animal models have been used for studying the pathogenesis of mv ( griffin , 2007 ) . cotton rats , rats , hamsters , mice , and ferrets can be infected with mv and are commonly used as small animal models for mv pathogenesis . after identification of cd46 and slam as mv receptors , numerous transgenic and knock - in mice expressing human cd46 and/or slam were established and intensively used to study different aspects of mv infection ( sellin and horvat , 2009 ) . however , non - human primates are the only animals that exhibit acute disease similar to that seen in humans . in this review , we discuss recent findings regarding tropism and pathogenesis of mv ; these findings were obtained by infecting monkeys with recombinant wild - type mv . this was reported as early as 1911 , after inoculating monkeys with blood from measles patients ( anderson and goldberger , 1911 ) . in 1921 , it was reported that measles could be transmitted from humans to monkeys by placing a filtered throat swab from a measles patient into the tracheae of monkeys ( blake and trask , 1921a ) . these authors also performed histological analysis of infected monkeys ( blake and trask , 1921b ) . mv was first isolated in 1954 from a specimen obtained from a measles patient ( enders and peebles , 1954 ) . enders and peebles ( 1954 ) inoculated human and monkey cell cultures with a throat swab taken from a young boy named david edmonston and isolated mv from these cultures . after this , it was discovered that mv isolated from normal human renal cells caused clinical signs similar to those of human measles in monkeys ( peebles et al . , 1957 ) . since then , numerous studies have been carried out by infecting monkeys with mv , measles vaccines , or specimens from measles patients ( griffin , 2007 ) . in such experiments , two species of monkeys , cynomolgus monkey ( macaca fascicularis ) and rhesus monkey ( macaca mulatta ) , serve as good animal models . new world monkeys are more susceptible to mv than old world monkeys , and infection of marmoset ( saguinus mystax ) with mv results in a fulminant disease ( levy and mirkovic , 1971 ; albrecht et al . , 1980 ) . in the past , it was well known that infection of monkeys with materials from measles patients induced clinical signs similar to those of human measles ( nii et al . , 1964 ; yamanouchi et al . , 1970 however , curiously enough , infection of monkeys with mv isolated and propagated in cultured cells did not always induce these clinical signs ( enders et al . this riddle was solved by the introduction of b95a cells ( a marmoset b - lymphoid cell line ) for isolation and propagation of mv ( kobune et al . , ( 1990 , 1996 ) found that mv strains could be efficiently isolated in b95a cells using materials from measles patients . more importantly , mv strains isolated from b95a cells retained their original pathogenicity in monkeys . these studies indicated that vaccine and laboratory - adapted strains of mv previously isolated from non - lymphoid cells such as vero cells were not true mv ( figure 1b ) . a decade later , it was found that the mv receptor slam is highly expressed on b95a cells ( tatsuo et al . , 2000 ) , which accounts for the efficient isolation of pathogenic mv from patient samples . similar to mv strains isolated from b95a cells , mv strains isolated and propagated in monkey mononuclear cells , human cord blood cells , human b lymphoblastoid cell lines , and vero cells expressing slam replicated well in monkeys and induced clinical signs of measles ( van binnendijk et al . , 1994 ; mcchesney et al . these results suggest that expression of slam on cells used for isolation is important for isolation of pathogenic mv . reverse genetics refers to the methods used for recovering infectious viruses from the cdna that encodes the viral genome . by using this method , mutations or extra transcription units reverse genetics of mv was first established based on the edmonston vaccine strain ( radecke et al . , 1995 ) . however , as previously mentioned , viruses derived from the edmonston vaccine strain do not induce clinical symptoms of measles in monkeys . therefore , reverse genetics of pathogenic wild - type mv was needed for the study of mv pathogenesis in monkeys . to this end , we first determined the complete nucleotide sequence of the genome of the pathogenic wild - type ic - b strain ( nc_001498/ab016162 ; takeuchi et al . , 2000 ) , which was isolated in tokyo in 1984 by using b95a cells ( kobune et al . , 1990 ) . then , we constructed a complete cdna plasmid of the ic - b strain named p(+)mv323 , and successfully recovered infectious mv ( ic323 strain ) from the p(+)mv323 plasmid ( takeda et al . , 2000 ) . importantly , the ic323 strain induced clinical signs such as rash , koplik s spots , and lymphopenia similar to human measles in infected monkeys , indicating that the ic323 strain retains the original pathogenicity of the ic - b strain . now , infectious mv strains can be easily recovered from cdna plasmids by using an improved protocol ( takeda et al . , 2005 ) . reverse genetics of other wild - type mv strains has been reported for the hl strain isolated in japan ( terao - muro et al . , 2008 ) and the ks strain isolated in sudan ( lemon et al . , 2011 ) . for vaccine strains of mv , reverse genetics for the schwarz / moraten vaccine strain ( combredet et al , 2003 ; del valle et al . , 2007 ) and the aik - c vaccine strain ( nakayama et al . , 2001 ) have also been reported . reverse genetics for vaccine strains are being used as a platform to generate new multivalent vaccines expressing antigens of other pathogens ( billeter et al . , 2009 ) and oncolytic viruses for cancer therapy ( russell and peng , 2009 ) . the p gene of mv encodes two non - structural proteins , namely the c and v proteins . however , the function of the c and v proteins in vivo was not well understood . the c protein is a small ( 186 amino acid ) , highly positively charged protein . to study the function of the c protein in the context of the natural course of mv pathogenesis , we generated an ic323 strain deficient in the expression of the c protein , wtmv(c ) , by using the reverse genetics of wild - type mv ( takeuchi et al . notably , the growth of wtmv(c ) in cynomolgus monkeys was dramatically reduced when compared to the ic323 strain . a similar growth defect of the ic323 strain deficient in the expression of the c protein , c , in vivo was observed in rhesus monkeys ( devaux et al . , 2008 ) . interestingly , c induced more inflammatory cytokines such as tumor necrosis factor alpha ( tnf)- and interleukin ( il)-6 and interferon ( ifn)- and - in infected monkeys . the v protein is translated from an edited mrna of the p gene ( griffin , 2007 ) . thus , the amino - terminal domain of the v protein has the same amino acid sequence as the p protein , and the carboxyl - terminal domain of the v protein has a highly conserved amino acid sequence forming a zinc - binding domain , which is important for function as an interferon antagonist ( gerlier and valentin , 2009 ) . an ic323 strain deficient in the expression of the v protein , v , was generated by introducing nucleotide mutations in the rna - editing signal in the p gene ( devaux et al . , 2008 ) . the growth of v in infected rhesus monkeys was lower than that of the parental ic323 strain . v induced more inflammatory cytokines ( tnf- and il-6 ) and ifn- and -. an ic323 strain unable to antagonize stat1 function , stat1-blind virus , was generated by introducing three amino acid substitutions in the shared domain of the p and v proteins ( devaux et al . , 2011 ) . the stat1-blind virus induced short - lived viremia and no clinical signs in infected rhesus monkeys . this virus induced more inflammatory cytokines ( tnf- and il-6 ) and a th1/th2 balance cytokine ( il-12 ) in infected monkeys . taken together , these findings indicate that the c and v proteins are not non - essential gene products as previously thought , but are stringently required for antagonizing host innate immune and inflammatory responses in vivo . in contrast , in vitro studies indicated that the v protein blocks ifn-/ signal transductions in infected cells , inhibits tlr7-mediated ifn- production in human plasmacytoid dendritic cells , and inhibits ifn induction in infected cells by interacting with mda5 ( gerlier and valentin , 2009 ) . furthermore , the c protein appears to inhibit ifn induction in infected cells by regulating viral rna synthesis ( nakatsu et al . , thus , it is necessary to elucidate whether the in vivo phenotypes of c- and v - deficient viruses are similar to the in vitro phenotypes . recent advances in the study of virus tropism have included the introduction of enhanced green fluorescent protein ( egfp)-expressing viruses . in vivo tropism of morbillivirus can be visualized with high sensitivity in living animals as well as tissue samples by using egfp - expressing recombinant canine distemper viruses ( von messling et al . , 2004 ) . similarly , mv target tissues or organs can be visualized with high sensitivity by infecting cynomolgus monkeys with an egfp - expressing ic323 strain ( figure 2 ) . ( 2007 ) infected rhesus and cynomolgus monkeys with an ic323 strain expressing egfp ( hashimoto et al . , 2002 ) and examined the tropism of wild - type mv in vivo . they indicated that the major target cells of wild - type mv were b and t lymphocytes and cd11c - positive , major histocompatibility complex ( mhc ) class - ii - positive dendritic cells . this result is consistent with the fact that slam is a receptor for wild - type mv . infection of ciliated epithelial cells in the trachea and lungs was also detected , suggesting the presence of another receptor for mv in epithelial cells . schematic diagram of reverse genetics of wild - type mv expressing egfp and infection of monkeys . a plasmid carrying the full - genome cdna of the ic - b strain and the egfp gene under the control of the t7 promoter is introduced into cho cells expressing human slam ( cho / hslam ) , along with three supporting plasmids expressing n , p , and l proteins , respectively , under the control of the t7 promoter . after infection with vaccinia virus expressing the t7 rna polymerase , wild - type mv expressing egfp can be recovered by mixing with b95a cells . egfp fluorescence in tissues and organs of infected monkeys can be detected using a fluorescence microscope . regarding the early target cells of wild - type mv , classical textbooks describe that the primary targets of mv are the epithelial cells of the respiratory tract . however , slam is not expressed in these epithelial cells . to examine the early target cells of wild - type and vaccine strains of mv in the lung , de vries et al . ( 2010 ) infected cynomolgus monkeys with egfp - expressing ic323 or vaccine strains of mv via the intratracheal or aerosol route . they found that cd11c - positive cells , which include alveolar macrophages and dendritic cells , were the major targets of both viruses . interestingly , although viral replication and cellular tropism in the lungs were similar for the two viruses , only wild - type mv caused significant viremia , suggesting a growth defect of the vaccine strain in lymphocyte cells . similarly , to examine the early target cells of wild - type mv , lemon et al . ( 2011 ) infected cynomolgus monkeys with an egfp - expressing wild - type mv based on the ks strain by aerosol infection and found that the early target cells of wild - type mv in monkeys are macrophages and dendritic cells . these studies indicated that alveolar macrophages and dendritic cells but not the epithelial cells of the respiratory tract are the early target cells of wild - type mv . nectin-4 is a newly identified epithelial cell receptor ( epr ) for mv . to examine the effect of nectin-4-using activity of mv on disease outcomes in monkeys , an ic323 strain recognizing slam but not nectin-4 was generated by introducing amino acid mutations in the h protein ( leonard et al . , 2008 ) . at that time , nectin-4 had not been identified as a mv receptor , and this strain was called epr - blind virus . when rhesus monkeys were infected with epr - blind virus via the conjunctiva and nares , this virus induced viremia and clinical signs in infected monkeys but did not propagate in the lungs . this result indicates the importance of nectin-4 for the propagation of mv in the lungs , which is required for the subsequent exit of mv from the host . inversely , to examine the impact of the recognition of slam by mv , an ic323 strain recognizing nectin-4 but not slam , slam - blind , was generated ( leonard et al . , 2010 ) . when rhesus monkeys were infected with the slam - blind virus , it elicited no clinical symptoms . vaccine and laboratory - adapted strains of mv can utilize both cd46 and slam as cellular receptors . however , surprisingly , the impact of the cd46-using activity of vaccine and laboratory - adapted strains of mv on their tissue and organ tropism and attenuation is not well understood . as cd46 is ubiquitously expressed on all nucleated human and monkey cells , vaccine , and laboratory - adapted strains of mv may infect all tissues and organs of humans and monkeys . ( 2010 ) indicated that only cd11c - positive cells were infected with the egfp - expressing vaccine strain via the aerosol route , suggesting that vaccine strains do not use cd46 in vivo . however , when the replication of vaccine and laboratory - adapted strains of mv in monkeys is limited , it will be difficult to identify infected cells in tissues . furthermore , the infection of vaccine and laboratory - adapted strains of mv may be restricted because of mutations in the p / c / v genes , which are important for antagonizing the host ifn response ( gerlier and valentin , 2009 ) . therefore , the tropism shift that solely occurs via the h protein should be evaluated using the wild - type mv expressing egfp , which bears the h protein of a vaccine strain , such as the ic / edh strain we developed previously ( takeuchi et al . , 2002 ) . we recently infected cynomolgus monkeys with egfp - expressing wild - type or ic / edh strains and found that slam - expressing lymphocytes were the main targets of both strains , indicating that cd46 does not act as a receptor for vaccine and laboratory - adapted strains of mv in vivo ( figure 3 ; takeuchi et al . , 2012 ) . one possible explanation for the limited expansion of the egfp - expressing ic / edh strain in vivo is the activation status of lymphocytes . it is known that stimulated lymphocytes are efficiently infected with mv and that stimulated lymphocytes express slam ( tatsuo et al . , 2000 ) . the egfp - expressing ic / edh strain that entered into quiescent lymphocytes via the cd46 may not grow well in those cells . alternatively , the expression level of cd46 in vivo may be too low to allow mv dissemination by cell cell fusion , as it was reported that high cd46 density is required for mv - induced cell cell fusion ( anderson et al . , 2004 ) . for whatever the reason , the growth of the egfp - expressing ic / edh strain was less efficient than that of the egfp - expressing wild - type strain ( takeuchi et al . , 2012 ) , suggesting that the cd46-recognition ability of vaccine and laboratory - adapted mv strains plays a role in the mv attenuation . receptor usage of the wild - type and vaccine and laboratory - adapted strains of mv . as described in this review , our ic323 strain , recovered from a plasmid carrying the full - genome cdna of the ic - b strain , is now used as a standard wild - type mv strain in mv research . furthermore , egfp - expressing ic323 strains are ideal tools for the study of tissue and organ tropism of wild - type mv in vivo . although the use of monkeys has several limitations , monkey models still provide the only reliable animal model for the study of the pathogenesis of mv in humans . the combination of wild - type and vaccine mv strains generated by reverse genetics and monkey models will provide new insights into the relationship between viral gene functions or individual mutation(s ) and the pathogenesis of mv . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | measles virus ( mv ) is an enveloped negative strand rna virus belonging to the family of paramyxoviridae , genus morbillivirus , and causes one of the most contagious diseases in humans .
experimentally infected non - human primates are used as animal models for studies of the pathogenesis of human measles .
we established a reverse genetics system based on a highly pathogenic wild - type mv .
infection of monkeys with recombinant mv strains generated by reverse genetics enabled analysis of the molecular basis of mv pathogenesis .
the essential in vivo function of accessory genes was indicated by infecting monkeys with recombinant mv strains deficient in the expression of accessory genes . furthermore , recombinant wild - type mv strains expressing enhanced green fluorescent protein enabled visual tracking of mv - infected cells in vitro and in vivo . to date , three different molecules have been identified as receptors for mv .
signaling lymphocyte activation molecule ( slam , also called cd150 ) , expressed on immune cells , is a major receptor for mv .
cd46 , ubiquitously expressed in all nucleated cells in humans and monkeys , is a receptor for vaccine and laboratory - adapted strains of mv .
the newly identified nectin-4 ( also called poliovirus - receptor - like-4 ) is an epithelial cell receptor for mv .
however , recent findings have indicated that cd46 acts as an mv receptor in vitro but not in vivo .
the impact of the receptor usage of mv in vivo on the disease outcome is now under investigation . |
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psoriasis is a common multifactorial skin dermatitis with a strong genetic background whose prevalence is estimated to be around 2 - 2.8% . the most common type is the chronic en plaques psoriasis involving scalp , elbows and knees , but psoriatic lesions may be observed in any part of the body , including genitals . in this area , lesions may occur within a generalized form or as a localized form , in complete absence of any stage of the disease . the onset of genital psoriasis can be induced by local trauma and injury , but infections acting as a koebner phenomenon ( kph ) should also be considered . the diagnosis is not always easy to formulate since the clinical presentation is characterized by asymptomatic or light irritating thin red plaques with a well - defined edge , lacking the typical scale because of maceration . the treatment of genital lesions deserves special care due to the high sensitivity of the thin genital skin and the increased absorption of topical agents , especially steroids . in this report we describe a case of localized glans psoriasis induced as kph by a herpes simplex virus ( hsv ) infection presenting as a pseudobalanitis circinata , and we discuss the diagnostic process , the pathogenesis of kph and the therapeutic management of psoriasis of the genital area . a 45-year - old man , at that time not engaged in a steady relationship , came to our outpatient service for the sudden onset of painful grouped vesicles of the glans ( fig . 1 ) . the patient did not refer any remarkable past disease , but he reported a granduncle affected by psoriasis with regard to the family history . the clinical diagnosis of a hsv skin infection was confirmed by the serologic positivity for hsv2 igm and igg antibodies . we tested the patient for other sexually transmitted diseases : syphilis , hiv and hepatitis b and c , and the outcome was negative . the patient was given a one - week topical treatment with acyclovir three times daily and the lesions resolved completely . one month later , the patient returned to our clinic reporting the outbreak of new only slightly itching lesions in the same area , 10 days after having finished the topical treatment . the new lesions presented now as pseudo - anular circinate whitish flat papules ( fig . 2 ) . the patient did not report any symptoms of urethritis , gastrointestinal disorders or arthritis , no oral aphthae were observed ; laboratory tests allowed to rule out any ongoing infection . a biopsy was performed and histology showed acanthosis and papillomatosis of the epidermis with parakeratosis , microabscesses of neutrophils but no spongiosis ; enlarged capillaries were present at the apex of dermal papillae , and the diagnosis of psoriasis could be made ( fig . 3 ) . following a treatment with topical tacrolimus 0.1% ointment applied once a day for 3 weeks the lesions resolved completely . in absence of any other lesions on the body surface , the diagnosis of localized genital psoriasis is not easy to formulate , and many other disorders should be considered in the diagnostic process such as reiter 's syndrome or candida balanitis , seborrheic dermatitis , lichen planus , syphilis or contact dermatitis . when the clinical history and presentation can not clearly orientate , histology may be needed , as in our case . the onset of psoriatic lesions induced de novo after a hsv infection can be regarded as a kph . the proliferative and the immune response patterns in psoriasis have been recognized for their similarity to the wound response : the development of inflammatory psoriasis plaques is triggered by mechanical skin trauma , as well as infections . when the kph takes place , psoriatic lesions usually appear 714 days after the triggering event . currently , there are several lines of evidence suggesting dysregulation of the innate immune system in psoriasis . activated dendritic cells are increased in early psoriatic lesions through complexes of the antimicrobial peptide ll-37 cathelicidin and dna in a toll - like receptor 9-dependent manner . host dna is then turned in a proinflammatory stimulus breaking immunologic tolerance and followed by migration of dendritic cells to lymph nodes , activation of t cells and affluence of predominantly th17 cells to dermis and epidermis . a complex cytokine network , in which il-17 , il-22 and tnf - alpha are the most important mediators , is then induced and it maintains keratinocyte proliferation . the available data support the notion that psoriasis is probably an autoimmune - related disease characterized by chronic inflammation in the absence of currently known infectious agents or antigens . specifically , the relationship between psoriasis and viral agents is still far to be clarified . in patients with aids in whom circulating cd4 + t cells are depleted , the paradoxical exacerbation of psoriasis could be explained by the virus itself or by one or more of the associated opportunistic infections . a potential role as autoantigen or opportunistic agent has been suggested for hpv 5 detected in scrapings of lesional skin in approximately 90% of a large series of psoriatic patients . recent studies have demonstrated that hsv can also act through the expression of molecules called prins ( psoriasis - susceptibility - related rna gene induced by stress ) in the epidermis . prins belongs to the group of recently described mrna - like noncoding rna genes which are spliced , polyadenylated mrna molecules containing a high density of stop codons and lacking an extensive open reading frame . also induced by uvb and trauma , they function as a regulatory rna , playing a protective role in cells exposed to stress and contributing to psoriasis susceptibility . finally , raychaudhuri et al . have demonstrated that in stressed basal keratinocytes , upregulation of nerve growth factor , which has a wide array of biological actions on inflammation , immune system and cell proliferation , is an early event and precedes epidermotropism of t lymphocytes in the kph . for its promotion of cutaneous innate host defences against virus through toll - like receptor expression and the block of inflammatory cytokine release , topical tacrolimus , a calcineurin inhibitor , a further mode of action shared with other calcineurin inhibitiors may run over the peroxisome proliferator - activated receptors ( ppars ) : it has been recently shown that ppar/ causes a psoriasis - like skin disease in mice and that ppard enhances keratinocyte proliferation in psoriasis . on the basis of our and other observations , one may hypothesize that tacrolimus may prove an effective and well tolerable , albeit yet expensive alternative to topical steroids for the treatment of psoriasis and that the use of this substance might soon be proven the treatment of first choice in some critical body areas , such as the face or genitals . potential side effects , such as skin atrophy , and consequently pain during sexual intercourse , may be avoided by choosing this drug , and a rapid resolution of the psoriatic genital lesions with good tolerability may be observed as it was in our case . | in the absence of any other lesions on the body , the diagnosis of localized genital psoriasis can be difficult , requiring further examinations including a biopsy .
we report a case of psoriatic pseudobalanitis circinata triggered by a herpes virus infection , and we discuss the koebner phenomenon and the therapeutic management of psoriasis of the genital area . |
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although not a diagnostic symptom of autism spectrum disorder ( asd ) , deficits in face processing represent a model domain in which to understand some of the core behavioral and neural features of autism . for example , many components of face processing ( e.g. , identity recognition , expression recognition ) are developing at the very time that behavioral symptoms of autism are emerging and changing developmentally ( infancy through young adulthood ) , allowing researchers to track aberrant developmental trajectories , and thus identify vulnerable developmental periods . in addition , many of the individual neural regions comprising the broadly distributed circuitry that subserves face recognition abilities ( gobbini and haxby , 2007 ) are located within anatomical regions that show pathological structural growth patterns during infancy , toddlerhood , and adolescence in autism . these regions include the temporal and frontal lobes as well as the amygdala ( schumann et al . , 2010 ) , suggesting that they may be particularly vulnerable throughout the developmental course of the disorder . finally , given that faces are the pre - eminent social stimulus from which we extract multiple kinds of social information that guide behavior , they provide a useful index of atypical neural organization of social - information processing across a spectrum of social emotional disorders ( e.g. , evans et al . , 2008 ; kucharska - pietura et al . , therefore , understanding the profile of atypical neural activation during face processing in autism , particularly during vulnerable developmental periods , is a fruitful approach to studying a core feature of autism ; that is , disruption of the social brain and social information processing more generally . the central goal of the current project was to evaluate the nature and extent of disruption in the social brain during face processing in autism , particularly during adolescence . we focus specifically on adolescence ( i.e. , the second decade of life ) as this is a developmental period of emerging vulnerability for individuals with autism in terms of face processing behavior ( o'hearn et al . , 2010 ) and neural circuitry ( dalton et al . , 2005 ; 2004 ) . also , an estimated one - third of children with autism experience deterioration in functioning during adolescence , which is associated with concomitant neurological complications ( gillberg and steffenburg , 1987 ; kanne et al . , 2011 ) , a substantial increase in social withdrawal ( anderson et al . , 2011 ) , and a potential heightened risk for developing comorbid depression and anxiety ( brereton et al . , 2006 ; kuusikko et al . , , we include a particular focus on the functional profile of activation within the fusiform face area ( ffa ; kanwisher et al . , 1997 ) of the temporal lobe and the amygdala , two critical regions supporting multiple aspects of face processing ( i.e. , identity recognition , affective processing , trait attribution ) . our focus on atypical activation within the ffa and amygdala in autism stems from contradictions within the existing literature that have made it difficult to ascertain a profile of atypical functional activation and organization among these regions even in adulthood autism . importantly , while the amygdala is central for processing affective information about faces , it is only one of several other critical regions that make up the extended face network ( gobbini and haxby , 2007 ) . surprisingly , little is known about the neural profile of these extended regions in autism , which might be especially disrupted given the known social and affective impairments in autism . the ffa in the fusiform gyrus ( fg ) together with a lateral region in the inferior occipital cortex [ occipital face area , 2000 ] and the posterior superior temporal sulcus ( sts ; hoffman and haxby , 2000 ) comprise the core regions in the broadly distributed neural circuitry supporting face processing ( gobbini and haxby , 2007 ; haxby et al . , 2000 ) . although these core regions are strongly implicated in supporting the visuoperceptual and cognitive analysis of faces , they also receive strong inputs from the extended regions , which are implicated in the more social and emotional aspects of face processing ( said et al . , 2010 , 2011 ) . the extended face processing regions include the amygdala , insula , and medial prefrontal cortex , regions in the anterior paracingulate cortex , and the anterior temporal lobe ( gobbini and haxby , 2007 ) . these extended regions process more changeable aspects of faces , such as facial expressions and associating person knowledge with faces , including personal traits , attitudes , mental states , and intentions . the overwhelming majority of studies investigating the neural basis of face processing in autism have focused on understanding whether face - related activation in the ffa and the amygdala is atypical . many studies report hypo - activation in the ffa in individuals with autism during unfamiliar face processing ( dalton et al . , 2005 ; domes et al . , 2013 ; grelotti et al . , 2005 ; humphreys et al . , 2008 ; kleinhans et al . , 2011 ; sato et al . , 2012 ; schultz et al . , 2000 ; wang et al . , for example , we previously reported that during passive viewing of movies of faces , hypo - activation is evident in the ffa as well as other core ( i.e. , perceptual ) regions of the face - processing network in adults ( humphreys et al . , 2008 ) and adolescents ( scherf et al . , 2010 ) with high - functioning autism ( hfa ) . however , there are several studies that fail to find atypical activation within the fusiform gyrus ( bird et al . , 2006 ; , 2004 , 2007 ; kleinshans et al . , 2008 ) in autism . for example , who used a passive viewing task of static face photographs but asked participants to fixate a red fixation cross positioned on the bridge of the nose of the face images , failed to find differences in face - related activation in the fg of adults with autism ( hadjikhani et al . , 2007 ) . it would seem that encouraging participants with autism to fixate the face improves signal in the ffa ; however , a similar a study of adults with autism using the same procedure reported face - related hypo - activation in the fg ( humphreys et al . one important difference between these two studies is that the participants in the studies varied in the magnitude of their symptom severity with the participants in the study by hadjikhani and colleagues consisting of almost an equal distribution of autism , and asperger's / pdd participants whereas the study by humphrey and colleagues only included participants with autism . a review of this literature suggests that the pattern of mixed findings of face - related activation in the fusiform gyrus is not likely to be related to differences in task demands ( e.g. , passive viewing versus face matching ) or the specific contrast used to define the face activation ( e.g. , affective faces versus neutral faces , faces versus objects , faces versus shapes ) . patterns of both hypo- and comparable face - related activation in the ffa have been observed under the full range of these conditions . the pattern of mixed findings is also not likely to be related to the familiarity of the face stimuli since findings of both hypo- and comparable face - related activation have been observed when the face stimuli are familiar to participants ( hypo - active , dalton et al . , 2004 ; pierce and redcay , 2008 ) . instead , the studies appear to differ in terms of the relative severity of the autism participants . specifically , all the studies reporting comparable face - related activation in people with autism , particularly in the ffa , have included a large proportion of participants with asperger 's syndrome and pdd - nos , who are less severely impacted symptomatically than those with an autism diagnosis . in contrast , the studies reporting hypo - activation in the ffa have largely included participants with a diagnosis of autism who are more severely affected by the disorder . based on these findings , we suggest that the discrepancies in the existing literature , particularly with respect to face - related activation in the fusiform gyrus , may actually reflect a systematic relation between the magnitude of activation and the severity of autism symptoms and/or variation in face recognition behavior . understanding the potential relation between symptom severity , face recognition behavior , and ffa activation in response to faces may provide a critical step in reconciling the notable discrepancies about the development of the social brain in autism . findings about atypical amygdala activation during face processing in autism are equally discrepant . given the social impairments of autism and the reported difficulties in processing emotional expressions ( adolphs et al . , 2001 ;
dawson et al . , 2005 ) , amygdala activation is likely to be atypical , particularly in response to affective faces . however , the nature of this atypicality is controversial and the existing results conflict , with many reporting hypo - activation ( ashwin et al . , 2007 ; , 2008 ; corbett et al . , 2009 ; critchley et al . , 2000 ; grelotti et al . , 2005 ; hadjikhani et al . , 2007 ; pierce et al . , 2001 ) , some reporting hyper - activation ( dalton et al . , 2005 ; monk et al . , 2010 ; swartz et al . , 2013 ; tottenham et al . , 2014 ; weng et al . , 2011 ) , and still others reporting comparable activation ( pierce et al . , 2004 ; wang et al . , our review of this literature suggests that , instead of symptom severity , the discrepancy in findings about amygdala activation in autism may be related to methodological differences in the way neural activation is defined , particularly with respect to the comparison baseline condition . for example , studies reporting amygdala hyper - activation in autism generally contrast affective faces ( e.g. , sad , happy ) with fixation ( e.g. , dalton et al . , 2005 ; tottenham et al . , 2014 ; , hyper - activation compared to controls could result from either higher magnitude responses to the faces and/or lower responses to the fixation , which could both contribute to a larger difference score ( i.e. , hyper - activation ) across these two conditions . in contrast , studies reporting amygdala hypo - activation in autism have employed a variety of contrasts in which affective or neural faces are compared with other visual objects , shapes , or scrambled images ( e.g. , bookheimer et al . , 2008 ; corbett et al . , 2009 ; in this case , the reduced responsivity of the amygdala in autism compared to controls could result from either lower magnitude responses to faces and/or higher magnitude responses to the other visual categories , resulting in a lower difference score ( i.e. , hypo - activation ) across these two conditions . given this pattern of findings , it is difficult to assess whether aberrant activation in the amygdala in autism is largely indicative of atypical processing of faces specifically ( as might be concluded from the work contrasting faces with fixation baseline ) , or whether there is a broader atypicality in amygdala function that affects the processing of a wide array of visual objects ( as might be concluded by the work contrasting faces with more complex comparison images ) . careful investigation of the profile of amygdala activation in response to faces ( both affective and neutral ) as well as to a wide range of other visual stimuli will help address this question . in this study , we aimed to identify disruptions in neural activation through the core and extended regions supporting face processing ( and social - information processing more generally ) in adolescents with autism and to explore individual differences as reflected in the relationship between variations in behavior and/or symptom severity and face - related activation within these regions . we studied high functioning adolescents ( hfa ) with autism ( ages 1017 years ) and age - matched typically developing ( td ) adolescents . we measured brain activation using fmri while participants performed a recognition task with both affective and neutral faces as well as a range of other visual stimuli , including common objects , houses , and scrambled images . this enabled us to map and compare face - related activation in both core and extended face processing regions across the groups to determine the extent to which atypical activation exists in the full network of regions . we also interrogated the profile of amygdala activation across the entire range of stimuli in order to evaluate the claim that faces , and not other visual objects , specifically elicit atypical activation in the amygdala . we assayed the behavioral profile of face recognition abilities for upright and inverted faces outside the scanner . the face inversion effect ( i.e. , more accurate recognition for upright compared to inverted faces , yin , 1969 ) is a hallmark of typical face perception and the magnitude of the face inversion effect has been used as a measure of individual difference in face processing studies previously ( russell et al . , 2009 ) . finally , we correlated the magnitude of face - related activation throughout the brain , and separately within our a priori regions of interest , with autism symptom severity , levels of adaptive social functioning , and face recognition behavior . because of our sensitivity to the developmental course of the disorder and age as a proxy measure of that continuum , we also included age as an independent factor in all the regression analyses between neural activation and behavior / symptom severity measures . the participants included 20 male hfa adolescents ( range 1017 years ) and 12 age - matched td adolescents ( range 1117 years ) . the mean age did not differ across groups , f(1,30 ) = 0.07 , the mean iq was in the average range for both groups ( see table 1 for demographic and iq information , as determined using the wechsler abbreviated scale of intelligence ) . the td group had higher verbal iq scores , f(1,30 ) = 5.3 , p < .025 , which contributed to slightly higher full scale iqs , f(1,30 ) = 3.6 , p = .07 . the diagnosis of autism was established using the autism diagnostic interview - revised ( adi - r ) ( lord et al . , 1994 ) , the autism diagnostic observation schedule - g ( ados ) ( lord et al . , 2001 ) , and expert clinical diagnosis ( minshew , 1996 ) . the hfa adolescents were medically healthy ; had no identifiable genetic , metabolic , or infectious etiology for their disorder ; and , were free of birth or traumatic brain injury , seizures , attention deficit disorder , and depression . td participants were included if they were medically healthy , free of regular medication usage , and had good peer relationships as determined by parent , self - report , and staff observations during the screening procedures . td participants were excluded if they or their first - degree relatives had a history of autism , neurological or psychiatric illness , acquired brain injury , learning disabilities , developmental delay , school problems , substance abuse , or medical disorders with central nervous system implications . a single episode of depression in a parent during a stressful episode was not considered grounds for exclusion providing no other family members reported depressive episodes . both hfa and td adolescents were recruited to be part of a longitudinal study investigating the effects of visuoperceptual training . in this ongoing study , pairs of participants with autism are yoked to a single td participant with each triad of participants systematically matched on age , sex , and fsiq . this explains the relatively smaller sample size of the td compared to hfa adolescents described in this project . the data reported here are from the pre - training assessment . written informed consent was obtained from participants ' guardians , and written assent from the participants themselves , using procedures approved by the internal review boards of the university of pittsburgh and carnegie mellon university . parents completed two scales of social functioning about their adolescent , the social responsiveness scale ( srs : constantino et al . , 2003 ) and the vineland adaptive behavioral scales , second edition ( vabs - ii : sparrow et al . , 2005 ) . the srs is a questionnaire that measures the severity of autism spectrum symptoms as they occur in natural social settings ; higher scores reflect more severe symptoms . the vbas - ii is a standardized caregiver interview that measures communication , social , daily living and motor skills ; higher skills reflect more adaptive functioning . the cfmt ( duchaine and nakayama , 2006 ) was used to measure face recognition behavior outside the scanner . this task has been used previously with td children and with adolescents with autism ( o'hearn et al . , 2010 ) . as in our previous work ( scherf et al . , 2008 ) , participants always performed the upright version first to maximize the possibility that participants with autism would initially approach the task in an ecologically valid way prior to having to confront the less naturally occurring inverted faces . all participants were placed in a mock mr scanner for approximately 20 min and practiced versions of the tasks that were administered in the full scan . high - resolution structural images and functional images were then acquired in a single session . participants were scanned using a siemens 3 t verio mri scanner , equipped with a 32-channel adult head coil , at carnegie mellon . anatomical images were acquired using a 3d - mprage pulse sequence with 176 t1-weighted ac - pc aligned sagittal slices ( tr / te / ti = 1700 , 2.48 , 900 ms ; voxel size = 1 mm , fov = 256 256 , ipat = 2 ) . pc aligned slices , covering most of the brain and all the occipital and temporal lobes ( tr / te = 2000 , 25 ms , fov = 192 , matrix 64 64 , flip angle = 79 , voxel size = 3 mm , this task was designed to elicit activation in response to several visual categories and to actively engage recognition behavior . functional images were acquired across two runs of a 1-back localizer task , which included blocks of neutral faces , fearful faces , common objects , vehicles , houses , novel objects ( i.e. , greebles : gauthier and tarr , 1997 ) , and scrambled images ( fig . 1 ) . scrambled images were created in adobe photoshop by scrambling pixels in the images of the common objects . each run lasted a total of 9 min and 12 s and began with a 20-s block of fixation and a 12-s block of patterns . thereafter , blocks of stimuli were presented in a randomized order followed by intervening blocks of fixation ( 6 s ) . within a block , 12 stimuli were each presented for 800 ms , followed by a 200 ms fixation . participants were required to indicate , by button press , when they detected a repeated image . there were two repeats in each of the stimulus blocks , the position of which was counterbalanced across blocks . in each run , there were four blocks of each stimulus category such that in the final analysis when the two runs were combined , each participant observed 8 blocks of each stimulus category . the neuroimaging data were analyzed using brain voyager qx v2.3 ( brain innovation , masstricht , the netherlands ) . preprocessing of functional images included 3d - motion correction , slice scan time correction , filtering low frequencies , and re - sampling the voxels to 1 mm . runs in which participants exhibited spikes in motion of more than 2.9 mm in any of the six motion directions on any image were excluded from the analyses . a single run was excluded for each of two hfa and one td participant . the average motion ( between each time point ) in each group on both runs of the task was less than 1 mm in all six dimensions and did not differ between groups ( p > .10 ) . for each participant , the time series images for each brain volume were analyzed for category differences in a fixed - factor glm . each category was defined as a separate predictor and modeled with a box - car function adjusted for the delay in hemodynamic response . following the recommendations of weiner and grill - spector ( 2012 ) , the functional data were not spatially smoothed . the time series images were then spatially normalized into talairach space , which is common practice in autism neuroimaging research , particularly in the study of adolescents and adults when brain volumes are comparable to those of td adolescents and adults ( redcay and courchesne , 2005 ) . although participants viewed multiple visual categories in the localizer task , here we focus on differences in the topography of face- , common - object , and house - related activation with respect to activation elicited by scrambled images . functional rois were defined for each individual subject for the region of interest analyses . for each participant , the time series images were submitted to a fixed - effects glm in which category was a fixed factor . as in our previous work , we defined the measures of category - selectivity with respect to all other categories ( scherf et al . , 2007 , 2010 , 2012 ) . note that these definitions are extremely conservative in that they identify many fewer voxels as compared to a contrast that would define each visual category against a fixation ( or scrambled image ) baseline . critically , these contrasts identify non - overlapping sets of voxels in all participants , indicating that they identify the most selective of voxels for each visual category . for example , face selectivity was defined with the following balanced contrast : { [ 3 * ( neutral faces ) + 3 * ( fearful faces ) ] [ 2 * ( common objects ) + 2 * ( houses ) + 2 * ( scrambled images)]}.1 similarly , object selectivity was defined as { [ 4 * ( common objects ) ] [ ( houses ) + ( neutral faces ) + ( fearful faces ) + ( scrambled images ) ] } ; and house selectivity as { [ 4 * ( houses ) ] [ ( common objects ) + ( neutral faces ) + ( fearful faces ) + ( scrambled images)]}. the resulting individual maps were corrected for false positive activation using the false discovery rate procedure ( genovese et al . , 2002 ) with a q < .01 , which is appropriate for identifying individual - level regions of interest ( roi ) . the right and left ffa were defined as the most anterior cluster of contiguous significant voxels in the fusiform gyrus generated from each participant 's face - activation map . unfortunately , the amygdalae were not definable as functional rois consistently across the individual participant face - activation maps . as a result , given our a priori hypotheses about group differences in activation in the amygdala , we defined right and left hemisphere amygdala rois by creating a 6 mm sphere around functionally defined talairach coordinates from previous work ( blasi et al . , the left amygdala roi was centered at ( 19 , 5 , 17 ) and the right centered at ( 22 , 1 , 17 ) . within each of these rois , we conducted an roi - based glm on the time series data for each individual participant to generate the resulting beta weights for each visual category . the beta weights were submitted to repeated - measures anovas with the factors of visual category ( 5 ) and group ( 2 ) separately for the right and left rois . estimates of face - selectivity were also determined for each roi by computing a balanced difference score in the beta weights ( e.g. , faces objects ) . in addition , the ffa rois were quantified in terms of the size ( number of significantly active voxels ) . category selectivity was determined separately for each group ( hfa , td ) by submitting the time - series images from each participant within the group to a random - effects glm with category as a fixed factor and participant as a random factor . the contrasts used to define face- , object- , and house - related activation at the group level were the same as those for the individual level rois ( e.g. , faces vs houses , objects , scrambled ) . however , given the addition of between - subjects variance in these maps , we used a monte carlo simulation to correct the group maps for multiple comparisons ( p < .05 ) separately for the td ( 16 contiguous voxels at a t - value 2.7 ) and hfa ( 12 contiguous voxels at a t - value 2.5 ) participants , given the different number of participants in the two groups . to compare group differences in category - selectivity , the full set of time series data from all participants was submitted to a mixed - model anova including group and category as fixed factors and subject as a random factor.2 we specifically evaluated group category interactions in each voxel in a whole brain analysis based on the contrasts of interest . for example , to compare group differences in face - selective activation , we coded the following interaction : td ( faces > other ) > hfa ( faces > other ) . to correct the resulting interaction maps for false positive activations , we used a monte carlo simulation ( p < .05 required a minimum of 33 contiguous voxels at a t - value 2.0 ) . to examine associations between patterns of brain activation and participant characteristics , we evaluated correlations between cfmt accuracy , raw srs scores , and vbas - social scores with the individually defined roi metrics ( e.g. , magnitude of activation , size of roi ) as well as in whole brain analyses . the various roi metrics were submitted to separate step - wise regressions with age as the first factor and the relevant measure of interest ( e.g. , raw srs score ) as the second factor . this enabled us to determine whether age and the relevant measure of interest independently accounted for variation in each of these roi metrics . whole brain ancovas were computed in the hfa individuals to identify voxels in which there was significant co - variance between category - selective activation and age , raw srs scores , vbas - social scores , and cfmt accuracy . these analyses generated separate whole - brain correlational maps that were thresholded at a corrected r - value of p < .01 using a monte carlo simulation to determine the number of contiguous voxels ( 8 with r > .56 ) . rois that survived this threshold were defined . to illustrate the nature of the relation between the scores and activation in each of these rois , we generated beta weights for all visual categories ( faces , objects , houses , scrambled images ) by computing a separate glm within each roi for each participant . using these beta weights , a difference score was computed that reflected the original balanced category - selective contrast ( e.g. , faces > other ) , which was then plotted against the specific measure of interest . as described for the roi - based correlations , we submitted these difference scores to a step - wise regression with age as the first factor and the relevant measure of interest as the second factor in order to determine the independent contributions of these factors to variation in the profile of face selectivity in each region . the srs and vabs - ii social scores for the two groups are plotted in fig . 2 . for both measures , there was unequal variance across the groups ( p < .005 ) . hfa adolescents had significantly higher srs scores , t(26.8 ) = 13.4 , p < .001 , indicating more severe autism - like symptoms , as well as significantly lower vabs - ii social scores , t(20.6 ) = 6.2 , p < .001 , reflecting lower adaptive functioning than the td participants . separate regressions of age on the srs and vabs - ii scores failed to reveal age - related changes in these measures in either group . the hfa adolescents were less accurate and failed to show an inversion effect in the cfmt ( fig . a repeated - measures anova including the within - subject factor of orientation and the between - subject factor of group , revealed a main effect of group , f(1,29 ) = 6.1 , p < .025 , indicating that the hfa adolescents ( m = 41.6% ) performed worse than the td adolescents ( m = 48.0% ) across both the upright and inverted versions of the task . the low performance in both groups is still above the chance rate of 33% and is comparable to the performance reported of similarly aged td and asd participants on this same task ( o'hearn et al . , 2010).3 there was also a main effect of orientation , f(1,29 ) = 4.7 , p < .05 ( upright m = 46.9% ; inverted m = 42.5% ) , but this was qualified by an orientation group interaction , f(1,29 ) = 6.2 , p < .025 . paired - samples t - tests conducted separately for each group revealed an orientation effect ( i.e. , upright > inverted ) in the td group , t(11 ) = 2.5 , p < .05 , but not in the hfa group , t(18 ) = 0.3 , p = ns . separate regressions of age on the upright cfmt scores failed to reveal age - related changes in this measure in either group . as evident from fig . 3b , there were no group differences in accuracy or reaction time ( rt ) when participants performed the 1-back memory task in the scanner . a repeated - measures anova with visual category as the within - subjects factor and group as the between - subjects factor revealed neither a main effect of group , f(1,30 ) = 0.6 , p = ns , nor a group category interaction , f(1,30 ) = .985 , p = ns . there was , however , a main effect of visual category , f(1,30 ) = 8.1 , p < .005 , with reduced accuracy for neutral faces compared to fearful faces , common objects , and houses ( all bonferroni corrected p < .01 ) , but not scrambled images ( p = ns ) . there were no significant effects in the analysis of the rt data . therefore , group differences in the bold response to these different categories of visual objects can not be attributed to performance differences in the 1-back memory task during scanning . 4a b shows the category - selective activation for each group for faces ( red ) , places ( green ) and common objects ( blue ) . td adolescents exhibited extensive activation in both core ( i.e. , right ffa , bilateral occipital face area ( ofa ) , right sts ) and extended ( i.e. , bilateral amygdala , pcc , and vmpfc ) regions ( table 2 ) . although hfa adolescents exhibited some activation in a subset of the core face - processing regions ( i.e. , bilateral ffa ) , they did not exhibit face - related activation in the ofa or sts core regions , or in the anterior temporal lobe , pcc , or vmpfc ( table 2 ) . statistical comparison of the hfa and td face - related group maps revealed significant hypo - activation in multiple core regions in the hfa adolescents , including the bilateral ofa , right sts , and right ( but not left ) ffa , as well as in extended regions , including the right atl , pcc and vmpfc ( fig . in addition , there were several other regions that were hypoactive in the hfa adolescents during face processing , including parietal , medial temporal , as well as prefrontal regions ( table 3 ) . both the hfa and td groups exhibited extensive and comparable activation of the ventral visual processing stream bilaterally during common object processing ( see figs . 4b and 5b ) except that the hfa adolescents exhibited stronger object - related activation bilaterally in the precuneus than the td adolescents ( fig . additional comparisons using a more lenient contrast for determining object - related activation ( objects versus scrambled images ) revealed no group differences in object - related activation . however , the hfa adolescents exhibited weaker activation in the left ppa than td adolescents during house blocks ( fig . a one - tailed independent - samples t - test failed to reveal a significant difference between groups in the number of voxels within the individually defined right ffa roi , t(30 ) = 0.7 , p = ns . however , the groups tended to differ with respect to the magnitude of face selectivity of the activation in these individually defined rffa rois , t(30 ) = 1.5 , p = .07 , one - tailed . this finding replicates our previous findings in a new sample of hfa and td adolescents ( scherf et al . , 2010 ) . the magnitude of activation ( i.e. , beta weights from roi - based glms ) for each visual category for each group is illustrated separately for the right and left amygdala rois in fig . , there was no main effect of group , f(1 , 30 ) = 0.0 , p = ns ; however , there was a main effect of visual category , f(4 , 120 ) = 11.7 , p < .000 , as well as a group category interaction , f(4 , 120 ) = 4.1 , p < .005 . bonferroni corrected post - hoc tests revealed that , for both groups , fearful faces elicited greater activation than houses and scrambled images ( p < .001 ) and tended to elicit more activation than neutral faces ( p = .051 ) . additionally , neutral faces and objects also elicited more activation in the right amygdala than scrambled images ( p < .05 ) . separate repeated - measures anovas within each group revealed that there were main effects of visual category in both the td , f(4 , 44 ) = 13.4 , p < .001 , and the hfa , f(4 , 76 ) = 3.9 , p < .01 , adolescents . however , the bonferroni corrected post - hoc tests revealed that the groups differed with respect to the categories that elicited the strongest activation in the right amygdala . specifically , in the td adolescents , fearful and neutral faces as well as objects elicited stronger activation than scrambled images ( p < .05 ) ; however , these categories were not different from one another . in contrast , in the hfa adolescents , the only significant difference was between fearful faces and houses ( p < .05 ) ; none of the visual categories was different from the scrambled images as was evident in the td adolescents . interestingly , the negative response to scrambled images among the td adolescents in the right amygdala was 12 times larger ( m = .201 ) than the modestly positive activation to scrambled images ( m = .016 ) in the hfa adolescents . independent samples t - tests comparing activation on each visual category between groups confirmed that the scrambled images condition was the only one for which the hfa and td adolescents differed , t(30 ) = 2.0 , p < .05 . this pattern of differences in response to the baseline condition is highly relevant for considering discrepancies in the current literature about the hyper- or hypo - active signal in the amygdala in autism . in the left amygdala , there was no main effect of group , f(1 , 30 ) = 1.7 , p = ns , and no interaction between group and visual category , f(4 , 120 ) = 1.9 , p = ns . however , there was a main effect of visual category , f(4 , 120 ) = 4.5 , p < .005 . across both groups , fearful faces elicited stronger activation than scrambled images ( p < .001 ) , and tended to elicit stronger activation than neutral faces ( p = .081 ) , but no other categories were different from one another . independent samples t - tests failed to reveal group differences in the magnitude of activation to any of the visual categories in the left amygdala . there were no regions in either the hfa or td adolescents in which there was a significant correlation between age and face- , object- , or house - related activation . the whole - brain correlation analyses between srs score and face - related activation in the hfa adolescents revealed that the right ffa was negatively correlated with srs scores ( fig . 7a ) : participants with higher srs scores had consistently lower magnitude face - related activation in the right ffa . the stepwise regression including the predictors of age and raw srs score on the beta weight difference scores generated for each participant in this roi was significant , f(2 , 17 ) = 9.4 , p < .005 , r = .53 ; however , only raw srs score was a significant independent predictor of face - related activation within this roi ( p < . the locus of the right ffa identified in this analysis ( 31 , 42 , 14 ) overlapped with the same right ffa region that was identified in the group level contrasts of face - related activation ( 37 , 47 , 20 ) during face processing . similarly , the magnitude of the face - related activation in the individually defined right ffa was significantly negatively related to the raw srs score among the hfa participants ( fig . the stepwise regression including the predictors of age and raw srs score on the beta weight difference scores generated for each participant in their individually defined right ffa was significant , f(2 , 15 ) = 6.0 , p < .025 , r = .44 ; however , only raw srs score was a significant independent predictor of face - related activation within this roi ( p < participants with higher srs scores had lower face - selective activation in their individually defined right ffa roi . however , the size of these individually defined rois was not related to srs scores , f(1 , 18 ) = 2.2 , p = ns , r = .11 , nor was the age of the participants ( p = ns ) . in contrast , the level of adaptive function in the hfa group was not significantly related to the level of face - related activation anywhere in the brain . there were no regions in the td adolescents in which either srs or vineland scores correlated with face - related activation . the whole - brain correlational analyses between cfmt performance and face - related activation among the hfa adolescents revealed that activation in the right atl ( 31 , 3 , 23 ) was positively correlated with performance . importantly , the stepwise regression analyses of the beta weights extracted from the individual participant glms in this roi with the predictors of age and upright face recognition accuracy was significant , f(2,17 ) = 13.4 , p < .001 , r = .61 . however , only upright cfmt performance was an independent predictor of face - related activation in this roi ( p < .001 ) , age was not ( p = ns ) . the stepwise regression with age and inverted face accuracy was not significant , f(2,16 ) = 1.0 , p = ns , r = .12 ( fig . there were no regions in which face - related activation was related to performance on the cfmt in the td adolescents . the central goals of this investigation were to evaluate face - related activation in adolescents with hfa in both core and extended regions of the broader face - processing network , with particular focus on the fusiform gyrus and the amygdala , and to explore a potential relation between the magnitude of this face - related activation and autism symptom severity , levels of adaptive social functioning , and variations in behavioral face recognition performance . using a classic task of unfamiliar face recognition , we replicated and extended previous findings that adolescents with autism are impaired in upright face recognition abilities compared to age- and iq - matched td adolescents . in addition , to our knowledge , we are the first to use the cfmt to evaluate the magnitude of the face inversion effect ( fie : yin , 1969 ) in adolescents with autism . the fie is often taken as a marker of typical face perception ; however , findings of the presence and magnitude of an fie in autism are mixed . a recent review suggests that people with asd do not demonstrate qualitative differences in the fie ( weigelt et al . , 2012 ) . here , we report that adolescents with autism do not exhibit an fie when tested with the cfmt , which is in contrast to our own previous findings ( scherf et al . , 2008 ) . we suggest that these findings can be explained by the relative difficulty of the cfmt . this is a much harder task than has been used to test the fie in the vast majority of previous studies . there is empirical evidence of a developmental progression in performance on the upright version of the task that continues into early adulthood in td individuals , but this progression plateaus in hfa individuals in adolescence ( o'hearn et al . , 2010 ) . the td adolescents outperformed the hfa adolescents in the upright condition of this task , but the groups were indistinguishable in their performance on the inverted condition . therefore , we suggest that the fie may only be observable in autism under conditions when upright face recognition is optimized . using a paradigm that was designed to elicit activation in both core ( i.e. , visuoperceptual and cognitive ) and extended ( i.e. , motivational and affective ) regions of the face processing system , we determined that hfa adolescents exhibit hypo - activation in the majority , but not all , regions compared to td controls . specifically , although hfa adolescents , as a group , exhibited face - related activation in the pre - eminent ffa in both hemispheres ; activation in the right , but not the left , ffa was significantly hypo - active compared to the td adolescents ( fig . also , face - related activation in the right and left ofa and in the right posterior sts were hypoactive in the hfa group as well . hfa adolescents exhibited comparable activation to tds bilaterally in the loc and hyper - activation in the precuneus during object - recognition , and comparable activation in the ppa during house - recognition . these findings were evident even though the sample size of hfa adolescents was nearly twice the size of the td adolescents . it is important to note that the smaller td sample size compared to the hfa group size is not ideal , but does not likely challenge the pattern of results reported here given that the group comparison is at most risk for type ii error ( false negative ) . in spite of the fact that we have a smaller number of control participants , we still had enough power to observe strong group differences in favor of the controls . in other words , there is more power and consistency in the face - related activation of 12 td controls than among 20 hfa adolescents . this is due , in part , to the powerful signal - to - noise ratio that is generated from the blocked fmri design and the fact that we collected two independent runs of the experiment from each participant to boost signal even more . these findings largely replicate our own and other previous findings in adolescents with autism ( dalton et al . , 2005 ; grelotti et al . , 2005 ; pierce and redcay , 2008 ; scherf et al . , 2010 ) with one exception . here , we find that hfa adolescents exhibited strong , consistent face - related activation in the left ffa that was not present among the td adolescents ( table 2 ) . our finding that adolescents with hfa recruit the left ffa during face recognition task is particularly useful for understanding that some parts of the face - processing network are preserved and even highly functional in autism . one possible explanation for the left ffa activation in the hfa adolescents relates to findings of hemispheric asymmetries in the kinds of information encoded by the fusiform gyri . there is growing consensus that the right fusiform is more specialized for holistic processing , while the left fusiform is more implicated for part - based processing ( meng et al . thus , the reliance on the left ffa during face processing in the hfa adolescents may reflect the use of a more part - based representation to process face identity . this interpretation is consistent with findings that individuals with autism have biased visuoperceptual systems that emphasize feature - based processing of local details in visual scenes ( behrmann et al . , with respect to extended regions , as a group , the hfa adolescents only activated the left amygdala . they did not exhibit activation in the right amygdala , pcc , anterior sts , right or left atl , or vmpfc . in contrast , td adolescents exhibited activation in the right amygdala , pcc , and vmpfc . note that the td adolescents did not show group level activation in the atl in either hemisphere , suggesting that these regions may continue to develop through adolescence . however , when pitted against each other directly , the hfa adolescents exhibited hypo - activation in the right atl , the right anterior sts , and the pcc , as well as in several other regions compared to the td adolescents ( table 3 ) . there were no regions in which the hfa adolescents exhibited greater activation than the td adolescents during face processing . importantly , there were no group differences in the profile of activation of the left amygdala for any of the stimulus categories . both groups exhibited the strongest magnitude response to fearful faces and a negative response to scrambled images . in contrast , in the right amygdala , there were differences between the groups in the profile of activation , but these differences were not specific to faces . in the right hemisphere , the only reliably different response in amygdala activation was to scrambled images . the td adolescents exhibited a strong negative response to scrambled images , whereas there was no such negative response in the hfa adolescents . there were no other group differences in response to either fearful or neutral faces , houses , or common objects . these findings show how a contrast between fearful or neutral faces and scrambled images would lead to a conclusion that hfa adolescents exhibited hypo - activation in the right amygdala , as has been reported in previous studies that used scrambled images as a contrast to face stimuli ( ashwin et al . , 2007 ; hadjikhani et al . , 2007 ; kleinshans et al . , 2008 ) however , a contrast between fearful and neutral faces or between fearful or neutral faces and objects would lead to a conclusion of comparable amygdala activation across the groups , which is consistent with findings from one previous study ( weng et al . , , there was no contrast that reflected hyper - activation to faces in the amygdala among hfa adolescents . this finding stands in contrast with several previous findings of relative hyper - activation in the amygdala during affective face processing in autism . there are multiple potential explanations for the absence of hyper - activation of the amygdala during face processing . importantly , many of our autism participants have been in several previous research studies , including those employing functional neuroimaging . as a result , most of our hfa adolescents were experienced and especially comfortable being in the fmri scanner , which may have significantly reduced anxiety and thus amygdala activation . we suggest that this is an important consideration for other studies reporting hyper - activation in the amygdala in individuals with autism ; it may reflect more generalized anxiety about the scanner environment compared to typically developing individuals . alternatively , one might suggest that our participants were avoiding looking at the eye region of the faces , thereby reducing amygdala activation . a recent study reported hyper - activation in amygdala responses from an autism group viewing neutral faces , particularly when they were directed to look at the eye region of the face ( swartz et al . , 2013 ) . this prediction would be consistent with the hypothesis that there is decreased motivation to attend to ( i.e. , look at ) social stimuli , like faces ( dawson et al . 2002 ) , which leads to hypo - activation in the fusiform gyrus ( dalton et al . , 2005 ) . together , these findings might suggest that the adolescents in our sample were not looking at the eye region of the faces to the same extent as were the td adolescents and that this aversion to the eye region led to the hypo - activation throughout the core and extended regions of the face processing network . we did not collect eye - tracking data , which limits our ability to investigate this possibility . however , both groups performed comparably on the 1-back recognition task for faces , and all other visual objects , in the scanner . this suggests that the adolescents with autism attended to the faces sufficiently to support near ceiling performance on the recognition task while the hypo - active bold signal was being acquired . also , it should be noted that the relation between purported atypicalities in the locus of fixations during face processing and cortical activation patterns in children with autism is controversial ( see boraston and blakemore , 2007 ) . for example , one study of young adolescents with autism found no differences from td controls in fixation patterns when observing facial expressions , despite finding impressive differences in the patterns of neural activation under these same conditions ( dapretto et al . , 2006 ) . also , at least one study in adults with autism found similar patterns of face - related hypoactivation in the fg when participants were required to fixate a central dot overlaid on the center of each stimulus and under free viewing conditions ( humphreys et al . , 2008 ) . we also report novel evidence that the magnitude of hypo - activation in the right ffa among the hfa adolescents is selectively related to the severity of autism symptoms . specifically , individuals with more severe autism symptoms ( i.e. , higher srs scores ) exhibited less face - related activation in the right ffa and no other region . in other words , there was a negative relation between the magnitude of srs scores and face - activation . the illustrative plot of the beta weights from these analyses suggest that the most severely affected adolescents with autism exhibited more object- than face - related activation in the right fusiform gyrus . this finding is consistent with the notion that individuals with autism may treat faces more like common objects with respect to the visuoperceptual strategy that they employ for recognition ( mottron et al . , 2006 ) . it is also consistent with several other studies , which report that typical face - processing regions are actually object - selective in autism ( humphreys et al . , 2008 ; scherf et al . , 2010 ; schultz et al . , 2000 ) . importantly , there were no regions in which object- or house - related activation correlated with symptom severity or levels of social functioning . these highly selective results suggest that the right ffa is particularly vulnerable in autism and that activation in this region may be related to the success with which individuals with autism interact with the social world . although these findings do not indicate a causal direction of the effect ( i.e. , impaired ffa activation leads to social deficits or vice versa ) , there may be a bidirectional influence between face - processing and symptom severity and/or social functioning in autism . the individual differences approach that we employed in this work to understand brain - behavior correspondences in autism may help reconcile discrepancies in the literature concerning hypo - activation in the ffa and suggest that studies failing to report such hypo - activation are likely to have a sample of individuals with less severe symptoms . we did not find a similar relation between face - related activation in the ffa ( or any other region ) in the td adolescents and either their autism - like behaviors or their levels of adaptive functioning . this null result may be related to the limited range of individual differences on these measures among the td adolescents and the small number of participants . it is possible that td adolescents with higher numbers of autism - like traits ( as measured by the autism quotient ; baron - cohen et al . , 2001 ) might show a similar relation between face - related activation and the severity of these traits . this kind of finding would help determine whether the relation between the neural profile of activation for faces and autism symptoms / traits is specifically vulnerable in and characteristic of autism or whether it reflects a broader relation between social information processing of human faces and levels of social functioning in the population more broadly . in spite of the association between face - activation and symptom severity in the right ffa , we did not find a relation between variation in face - recognition behavioral performance and the magnitude of face - selective activation in the fusiform gyrus among the hfa adolescents . this null result is consistent with recent findings of adults with asd ( jiang et al . , these same authors also reported that , using a novel analysis of voxelwise correlations and an fmri - adaptation paradigm to probe the sparseness of face - related representations within the ffa , adults with autism who exhibit particularly poor face recognition skills have less sparse ( and therefore less selective ) neural representations for faces in the ffa ( jiang et al . , in other words , the whole - brain correlational analysis using a category - selective definition of face - related activation ( as determined by the faces other visual categories contrast ) may not have been sensitive enough to detect the brain however , in spite of this limitation within the ffa , we did find a brain behavior relation in the right atl , a region implicated in supporting face individuation ( kriegeskorte et al . , specifically , hfa adolescents who scored higher on the upright version of the cfmt outside the scanner exhibited stronger face - related activation in the right atl during the face - recognition task in the scanner . this finding suggests that there may be substantial heterogeneity in activation patterns that might be used to predict and/or identify which individuals could benefit the most from targeted cognitive remediation ( e.g. , face training ) . given that the atl is also associated with linking biographic information about faces to perceptual representations ( haxby et al . , 2000 ) , hfa adolescents who showed stronger activation in this region might benefit from strategies such as linking names to faces or encoding a semantic detail about the face ( e.g. , looks like my teacher ) . we did not observe a similar relation between face - recognition behavior and face - related activation in the right atl ( or any other region ) among the td adolescents . the small number of td participants ( n = 12 ) likely underpowered the whole - brain correlational analyses of individual differences in this group.4 at the same time , reports of brain behavior correlations within the face - processing system are actually quite limited , with some reporting positive correlations between the volume of the right ffa with recognition behavior ( golarai et al . , 2007 , 2010 ) and others reporting positive correlations between the magnitude of behavioral and neural responses to face inversion within the right ffa ( alyward et al . , 2005 ; passarotti et al . , 2007 ) in samples that combine adolescents and adults . these correlations could be driven by developmental changes in both face recognition behavior and neural organization within the ffa and/or by individual differences in these characteristics across the age range . future work investigating the developmental emergence of these brain behavior relations separate from individual differences in these relations among typically developing individuals will be critical for interpreting our findings of individual differences among hfa adolescents . in conclusion , our findings identify the right ffa as a particularly vulnerable node in the broadly distributed face - processing network in autism , particularly during adolescence when this region is maturing among typically developing adolescents . importantly , we show that it is not the only atypical node , indicating that the extent of impairment in the functional organization of neural regions supporting face processing in autism is much broader than previously reported . interestingly , conclusions about the relative hyper- or hypo - activation of the amygdala depended on the nature of the contrast that was used to define the activation . we suggest that our findings reflect a systematic relation between the magnitude of neural dysfunction , severity of autism symptoms , and variation in face recognition behavior , which provides new insight about reconciling discrepancies in the existing literature . by elucidating brain behavior relations that underlie one of the most prominent social deficits in autism , this research helps resolve discrepancies in the literature concerning hypo - activation of the social brain in autism , and points to a specific vulnerability in the development of the fusiform gyrus . | despite the impressive literature describing atypical neural activation in visuoperceptual face processing regions in autism , almost nothing is known about whether these perturbations extend to more affective regions in the circuitry and whether they bear any relationship to symptom severity or atypical behavior .
using fmri , we compared face- , object- , and house - related activation in adolescent males with high - functioning autism ( hfa ) and typically developing ( td ) matched controls .
hfa adolescents exhibited hypo - activation throughout the core visuoperceptual regions , particularly in the right hemisphere , as well as in some of the affective / motivational face - processing regions , including the posterior cingulate cortex and right anterior temporal lobe .
conclusions about the relative hyper- or hypo - activation of the amygdala depended on the nature of the contrast that was used to define the activation .
individual differences in symptom severity predicted the magnitude of face activation , particularly in the right fusiform gyrus .
also , among the hfa adolescents , face recognition performance predicted the magnitude of face activation in the right anterior temporal lobe , a region that supports face individuation in td adults .
our findings reveal a systematic relation between the magnitude of neural dysfunction , severity of autism symptoms , and variation in face recognition behavior in adolescents with autism . in so doing ,
we uncover brain behavior relations that underlie one of the most prominent social deficits in autism and help resolve discrepancies in the literature . |
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first - line treatment of cushing s disease ( cd ) is pituitary microsurgery ; medical treatment has been limited to patients with persistent hypercortisolism after unsuccessful surgery , while awaiting the beneficial effects of radiation therapy or , preoperatively , to control symptoms due to extremely severe hypercortisolism in order to reduce the surgical risk 1 . drugs so far available target adrenal cortisol production via steroidogenesis inhibition ( ketoconazole , metyrapone , etomidate ) or act by blocking glucocorticoid action at the glucocorticoid receptor ( mifepristone ) or combine antisteroidogenic and adrenolitic action ( mitotane ) 2,3 until short time ago , only cabergoline was an available pituitary targeted therapy 4 . overall , there is no substantial evidence in support of many of the drugs currently used in treatment of hypercortisolism ; many are used off - label , are unavailable in some countries and no reliable predictors of efficacy are identified . pasireotide is a novel somatostatin analog that acts on specific receptor isoforms with increased affinity toward sstr 5 in comparison with previous analogs 5 . in this case report , we show that low doses of pasireotide used as first - line treatment in a patient with cd not eligible for surgery were effective in normalizing cortisol secretion and improving the clinical condition and also in determining substantial tumor volume reduction . a 63-years - old male patient was referred to emergency room after 2 days of acute onset headache , diplopia , visual impairment , nausea , and vomiting ; when admitted to the hospital only diplopia persisted . the past medical history was unremarkable except for depression since 20 years . at brain ct and ct - angiography performed in the emergency department a pituitary mass was detected ; at subsequent gadolinium - enhanced mri , a pituitary macroadenoma with suprasellar and right parasellar extension dislocating right internal carotid was detected ( 21 mm 14.5 mm 15.7 mm , respectively , postero - anterior , cranio - caudal , and latero - lateral diameters , volume 2.53 cm ) , isointense with brain on t1 and hyperintense on t2 weighted images , compressing the normal gland and deviating to the left the pituitary stalk ( fig.1 ) . full - blown cushingoid features were present with newly diagnosed hypertension and impaired glucose regulation ( hba1c 44 mmol / mol ) treated , respectively , with hypotensive drug and diet . gadolinium enhanced mri studies performed at baseline ( left ) and after 12 months ( right ) of treatment with pasireotide 300 g subcutaneously ( s.c . ) two times a day ( b.i.d ) . high - normal plasma acth levels ( 88 and 43.2 pg / ml , n.v . 952 ) , elevated 24 h urinary free cortisol excretion ( ufc ) ( 459 g/24 h , normal range 36137 ) were detected as well as normal thyroid function and normogonadotropic hypogonadism . hypercortisolism was confirmed by repeated ufc collections ( ufc 2601 , 1075 , 6222 g/24 h ) , unsuppressibility of cortisol after low - dose dexamethasone ( 320 g / l ) and by supranormal midnight plasma cortisol ( 172 g / l , n.v . plasma acth increased by 35% after desmopressin and by 18% after crh ( basal 60 and peak 71 pg / ml at 60 min ) , whereas plasma cortisol was not responsive ( basal 393 and peak 405 g / l at 15 min ) ; plasma and urinary cortisol decreased by 75.3% and 66.3% after high - dose dexamethasone suppression test . biochemical and hormonal data at baseline and during treatment with pasireotide clinical signs and symptoms of overt cushing s syndrome were worsening since the first evaluation 2 months before : blood pressure levels had increased , cholesterol levels were higher than before , glycemic control worsened and hba1c value consistent with overt diabetes was found ( hba1c 49 mmol / mol ) ( table1 ) . pituitary surgery was recommended : as the patient refused surgery , primary medical treatment with subcutaneous pasireotide ( som230 ; novartis , basel , switzerland ) was offered at the initial dose of 0.6 g , bid . acth and cortisol variations after 100 g octreotide were compared with those recorded after the first dose of pasireotide . octreotide did not induce changes , whereas a progressive decline in cortisol concentration was recorded with maximal inhibition of 67% at 10 h after administration of pasireotide ( table2 , fig.2 ) . left : plasma acth and cortisol levels before and hourly for 6 h after a single dose of 100 g s.c . right : plasma acth and cortisol levels before and every 2 h for 10 h after a single dose of 600 g s.c . pasireotide left : plasma acth and cortisol levels before and hourly for 6 h after a single dose of 100 g subcutaneous octreotide right : plasma acth and cortisol levels before and every 2 h for 10 h after a single dose of 600 g subcutaneous pasireotide . two weeks later , plasma acth normalized ( 47 pg / ml ) and ufc decreased ( 564 g/24 h ) ( fig.3 ) . a worsening of glycemic control required an increase of metformin dose and addition of sitagliptin . urinary - free cortisol ( ufc ) excretion ( normal range : 37136 g/24 h ) and plasma acth ( normal range 952 pg / ml ) values before and during the entire course of pasireotide treatment . one month later , the patient complained of fatigue , dizziness , abdominal pain , and diarrhea , consistent with hypoadrenalism ; ufc had decreased to subnormal values ( 27 g/24 h ) . on the basis of clinical and biochemical data , pasireotide was reduced to 0.3 g twice daily . starting from the third month of treatment , the improvement of the clinical condition persisted steadily for 18 months ( at the present time ) in line with normalization of ufc concentrations ( fig.3 ) . fasting , postprandial glucose , and hba1c values were satisfactory on long - acting insulin , metformin , and sitaglyptin ( fasting glycemia values < 130 mg / dl and postprandial values < 180 mg / dl ) ; cholesterol profile improved . blood pressure normalized without treatment , weight decreased by more than 10% and waist circumference from 105 to 96 cm . although no specific quality of life measure was utilized , the patient reported improvements in the areas of self confidence , physical appearance , social activities , sleep quality , physical performances , and everyday activities . at mri , the size of the pituitary tumor decreased by 44% at 6 months and up to 52% at 18 months ( 18.9 mm 9.8 mm 11 mm , volume 1.21 cm ) ( fig.1 ) . from a clinical standpoint , a remarkable finding of this case is the effectiveness of low doses of pasireotide in obtaining a stable control of disease in a patient with clinical and biochemical features of severe hypercortisolism , a finding rarely reported previously in single patients 6,7 . as reported in the registrative trial 8 , normalization of ufc was more likely achieved in patients with mild disease , although the drug was effective also in severe hypercortisolism . if confirmed in other cases , the effectiveness of pasireotide at doses lower than previously suggested has practical implications such as the reduced risk of adverse events and the potential reduction of the costs for managing the disease . diabetes worsened during treatment , however , surrogate markers of cardiovascular risk improved as a consequence of the remission of hypercortisolism . treatment induced hypocortisolism is an adverse event to be considered in any patient on medical treatment for hypercortisolism : alerting the patient on warning symptoms might help in avoiding severe adverse consequences . the tumor volume reduction here observed is concordant with the antiproliferative effect already seen in primary cell cultures of human corticotroph adenomas 9,10 . the molecular mechanism of suppression of cell growth and the role of specific sstrs in transducing growth inhibitory signals in corticotroph tumors need further study . data in patients with a measurable tumor support the antiproliferative effect of pasireotide at high doses ( 9001200 g , bid ) 8,11 ; the present case shows that even a lower dose of pasireotide retains an antiproliferative effect . this case illustrates the long - term effectiveness of low doses of pasireotide , a tumor - directed therapy , in the biochemical and clinical control of hypercortisolism of cd . although it is clear that the control of tumoral mass is best achieved with surgery , the surgical outcome in acth - secreting macroadenomas is less favorable than in micro . the drug may be an option for long - term treatment in some patients with the disease when surgery is not feasible for many reasons . it might be an option in patients with acth - dependent macroadenomas where control of tumor mass is an important clinical endpoint . | key clinical messagethis case shows efficacy of low - dose pasireotide in biochemical and clinical control of severe hypercortisolism and in tumor volume reduction in a patient with an acth - secreting macroadenoma .
the drug may be an option for long - term treatment in some patients where control of tumor mass is an important clinical endpoint . |
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however , in the setting of appendicitis with abscess or phlegmon , initial non - operative management has been shown to be safe and effective.[13 ] the treatment of appendicitis with phlegmon has been debated for more than 100 years . conservative treatment is an established practice , but the necessity of interval appendectomy remains controversial . advocates of interval appendectomy propose that appendectomy prevents recurrence and gives definitive treatment.[46 ] appendectomy can also provide a definitive diagnosis , and may sometimes reveal an unexpected malignancy . however , another group of surgeons who oppose this policy[79 ] point out that the rate of recurrent appendicitis is around 620% , and that the complication rate of interval appendectomy is not low ( 919% ) . the aim of this study was to determine the risk of recurrent appendicitis following initial non - operative treatment for appendicitis , and evaluate factors associated with recurrence . our hypothesis was that the risk of recurrent appendicitis is low and that interval appendectomy is not routinely indicated . patients who received conservative treatment for appendicitis with lump formation were prospectively studied from june 2006 to june 2008 . baseline investigations viz complete blood count , liver function and kidney function was done in every patient . computed tomography ( ct ) was done in every patient for confirmation of diagnosis . every patient was treated with intravenous fluids , antibiotics ( third generation cephalosporin , amikacin and metronidazole ) . patients were followed up in outpatient department monthly for three months , bimonthly for another six months , and six monthly thereafter . if signs of recurrent appendicitis appeared ( such as right lower quadrant pain , or tenderness with or without fever ) , ct was repeated and appendectomy was performed if recurrent appendicitis was confirmed . data was collected and analyzed statistically using spss software . a total of 763 patients with acute appendicitis were treated from june 2006 to june 2008 . out of them 220 ( 28.8% ) patients presented with lump formation . high rate of lump formation was because of late referral and due to poor health care facilities available in our state . seven patients in this group ( 3.2% ) were operated in the index admission as they did not respond to conservative treatment . mean follow up was 32 months . out of the total ; 115 of them were men and 98 were women . most of the patients had classical history of pain , starting at the umbilicus then radiating to the right iliac region . most of the patients ( 79.8% ) had history of anorexia but nausea and vomiting was found in only 23.4% of patients . all the patients had a lump palpable in the right iliac region on clinical examination . there was no clinical difference in the primary clinical presentation between the cases that later developed recurrence and the patients who did not had recurrence [ table 2 ] . clinical presentation of patients of appendicitis with lump formation clinical presentation of patients who later had recurrence among 213 patients , 28 ( 13.1% ) had recurrent appendicitis on follow - up and they were operated for the same . all these patients had recurrences within six months of the primary treatment [ figure 1 ] . lump formation after acute appendicitis is as a result of walled off perforation of appendix . lump can be an inflammatory mass consisting of inflamed appendix , adjacent viscera , and greater omentum or a pus containing appendiceal mass . although management of acute appendicitis is primarily surgical , management of appendicitis with lump is controversial for more than a century now . mcpherson and kinmonth reported that non - operative management of appendicitis with tumor formation achieved a 76% success rate and 0.8% mortality rate . currently , conservative treatment of appendicitis with lump formation is the preferred one . the success rate of conservative treatment of appendicitis with tumor formation ranges from 76% to 97% . ct- or ultrasound - guided drainage of an appendix abscess has made surgical drainage less common . however , some surgeons still advocate early surgical intervention instead of conservative approach , although this approach has been criticized because of possibility of spread of previously localized infection , injury to surrounding structures , and overall higher complication rate . after successful conservative treatment the morbidity of interval appendectomy ranges from 3.4% to 19%[4611151921 ] which can be compared to the reported incidence of recurrent appendicitis after conservative treatment of an appendix mass that ranges from 0% to 20%.[4681012 ] the danger of recurrence is reported to be greatest during the first 6 months after the initial episode and minimal after 2 years . lai et al . , also suggested that routine elective appendectomy can be safely omitted in more than80% of patients . , in their studies also support the practice of nonsurgical treatment without interval appendectomy in patients with appendiceal abscess or phlegmon . the risk of recurrence is low , which does not motivate routine interval appendectomy after initial conservative treatment . | background : interval appendectomy after acute appendicitis with lump formation ( phlegmon ) remains controversial .
we conducted this study to determine the risk of recurrent appendicitis following initial non - operative treatment for appendicitis , and evaluate factors associated with recurrence .
secondarily , we evaluate the efficacy of interval appendectomy versus no appendectomy.materials and methods : patients who received conservative treatment for appendicitis with lump formation were prospectively studied from june 2006 to june 2008 .
these patients were followed for recurrence of appendicitis.results:of 763 patients with acute appendicitis some 220 patients had lump formation ( 28.8% ) .
median age was 28 years .
conservative treatment was successful in 213 ( 96.8% ) patients .
the rate of recurrence was 13.1% , all occurring within six months after the index admission .
mean follow - up was 2618 months.conclusion:conservative treatment of appendicitis with lump formation is efficient and the recurrence rate is low .
routine interval appendectomy after initial conservative treatment for lump formation is not a cost - effective intervention and not recommended . |
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it is well known that vascular disease , including atherosclerosis , hypertension , stroke , and pulmonary arterial hypertension , are usually caused by multifactorial pathogeneses involving both genetic and environmental factors ( fig . the conventional hypothesis - driven approaches to vascular research , however , mainly focus on individual factors that are presumably involved in the pathophysiology of vascular disease . while some of these factors have been extensively studied and become the mainstream targets of current clinical treatment of vascular disease , many other factors have been largely neglected or not studied at all [ 2 - 5 ] . a deeper understanding of the genetic basis of vascular disease may provide new insights into the mechanisms underlying the pathophysiology and facilitate development of novel diagnostic and therapeutic modalities . traditionally , the phenotype - genotype relationship of a disease is studied using strategies of either forward genetics ( top - down ) , which starts with one phenotype and considers its link with one or many genotypes ( such as genetic linkage studies ) , or reverse genetics ( bottom - up ) , which starts with one genotype and see whether its relationship with the phenotype can be confirmed ( such as functional studies of gene overexpression or knockout , fig . the emergence of many omics technologies and platforms , including genomics , proteomics , and metabolomics , has provided many unbiased approaches to unravel the etiology and pathophysiology of many vascular diseases in the fashion of global and integrated biological system rather than single gene - based individual molecular components ( fig . 3 ) . as a consequence of these omics studies the current definition of clinical phenotypes of complex disease and combination drug therapy can no longer this has created major challenges for a complete understanding of the relationship between the genomic / proteomic / metabonomic variability and the phenotypic vascular disease in individual patients the color spectrum of development of clinical phenotype of vascular disease schematically shows that in most cases the vascular disease is caused neither solely by genomic factors ( green ) nor totally by environmental ( red ) factors . the clinical phenotypes of vascular disease in an individual patient are usually a sum result of the complex of interplay between genetic and environmental contributions . although in some conditions patients with a positive genetic susceptibility to vascular disease may have a major genetic component , whether the patient will manifest the full clinical phenotypes of the vascular disease is dependent on the contributing environmental factors . conversely , patients may have different thresholds of an environmentally induced vascular disease depending on the individual s genetic background . a variety of biomarkers ( including genes , micrornas , proteins , small molecules of metabolites , and imaging ) can be deployed to delineate disease progression in vascular disease . however , it is the environmental imprints on the genetic background that are responsible for the dynamic production of abnormal unique proteins and/or small metabolites that foster disease progression in the susceptible individuals . the environmental prediction of vascular disease can be detected using protein or metabolite biomarkers . as the disease progresses further in the susceptible individual the traditional phenotype - genotype association study follows the principle of one gene , one protein , one phenotype ( disease ) and uses either forward ( top - down ) genetics or reverse ( bottom - up ) genetics strategy to identify the genetic variability responsible for the phenotype or disease . principle has guided the traditional drug research with identification of one pure drug target as the ideal drug with pure therapeutic efficacy and the least side - effect or toxicity . the post - genome approaches to phenotype - genotype association study use similar strategies to those of genetic approaches but at an omics the top - down strategy uses a set of technologies and approaches including genomics , transcriptomics , proteomics , interactomes , and metabolomics to study the association of phenotype ( a disease ) and genome , transcriptome , proteome , interactome , and metabolome . disease phenotype may be associated with multiple single - nucleotide polymorphisms ( snps ) or mutations of multiple genes , or changes in expression and/or posttranslational modification profiles of multiple proteins . the bottom - up strategy uses functional genomics , functional proteomics and functional metabolomics to test whether the findings from the top - down studies are indeed correlated to the phenotypes . this also leads to current challenges in the definition of clinical phenotypes and pure drug targets . and the translation of the knowledge of genomic / proteomic/ metabonomic research into clinical practice of combination therapy of vascular disease [ 2 , 6 , 7 ] . clearly , newer approaches are needed for the redefinition of disease using not only the traditional signs and symptoms but also the underlying genomic / proteomic causes and other factors . here , we introduce the phenomics strategy that will make the transition from the conventional focused genotype / phenotype studies to the global genome / phenome approaches to a systematical understanding of pathophysilogical mechanisms and combination therapy of vascular disease . we also discuss about the advance from the genome - wide association studies ( gwas ) to the phenome - wide association studies ( phewas ) in vascular medicine . it is believed that the application of phenomics in the study of vascular disease will identify systemically integrated biomarkers for diagnosis and prognosis and novel treatment targets for combination therapy and thus make a revolutionary paradigm shift in the clinical treatment of vascular disease . a wide variety of approaches have been used to study the association of genetic polymorphisms to phenotypes of vascular disease , evolving from genetics , to genomics and gwas with the improvement of genotyping analyses . the candidate gene approach is the earliest and simplest method using the strategy of forward genetics based on a priori hypothesis to determine whether a gene is responsible for an intermediate phenotype , such as a molecular and cellular function , or a clinical manifestation . the focus of candidate gene study is on single genes mainly located in exons with polymorphisms that have a major role in determining the gene expression and the transduced protein or changes in cellular function . they showed that homozygotes with a deletion in the angiotensin converting enzyme ( ace ) gene were at higher risk of coronary artery disease and myocardial infarction . this association was more evident in subjects lacking of some well - established non - genetic risk factors , suggesting the importance of studying both genetic and environmental factors . later , it was noted that in a gene of 30 kb there are 10100 polymorphisms that are potentially responsible for variations of the genetic effect . therefore , later studies started to genotype more than one polymorphism in the same gene . for example , iacoviello et al . found that two polymorphisms in distant regions of the gene encoding for coagulation factor vii were associated with myocardial infarction ( mi ) and suggested a higher gene effect for the presence of both risk alleles . this has increased the analysis complexity and furnished an enormous amount of information in a big - data fashion . since the completion of the human genome project in 2000 , millions of single nucleotide polymorphisms ( snps ) have been discovered in the human genome . while the snp is deliberately chosen to mark a gene hypothesized a priori to play a mechanistic role in determining the trait in a candidate gene study , snps from across the entire genome are studied agnostically , without premeditation regarding their possible function in a genome - wide association study ( gwas ) . the patterns of linkage disequilibrium ( ld ) in the human genome have also been characterized through the international hapmap project . with the availability of high - throughput genotyping platforms and significantly decreased costs of genotyping and genome sequencing , gwas has become more affordable and has gradually overwhelmed the analyses on a single candidate gene . gwas has provided a systematic approach to investigation of the impact of common genomic variations on human vascular disease , such as acute coronary artery syndrome and hypertension [ 10 - 14 ] . while some studies started to use the clustering of common clinical domain phenotypes , most of current gwas are still using the hypothesis - driven strategy for identifying genetic mechanisms and remain largely focused on clinical categories that do not provide adequate etiological information . in gwas , a single pre - defined disease or clinical phenotype ( trait ) is studied and the accrual of a large number of single gene variant phenotype associations has serendipitously identified one gene affecting or responsible for more than one phenotypic characteristic or single loci associated with multiple diseases ( pleiotropy ) . in addition to the levels of encoded protein in basal conditions , the link between genetic variants and phenotypic diversities could be resulted from different responses to an environmental stimulus ( fig . 1 ) . a final target environmental risk factor can interact with a variety of molecular pathways , which are in turn regulated by multiple genetic factors . consequently , the effects of environmental risk factors could be modified by different genetic background . as a classic example of how genetic background could impact in disease pathogenesis , it was found that patients with different genotypes showed different levels of monocyte - production of cytokines in response to stimulation with lipopolysaccharide , corresponding to a different response of monocytes to inflammatory stimulation and risk of ischemic vascular disease ( mi and stroke ) at young age . actually , a risk factor together with other risk factors such as high salt intake , hypercholesterolaemia , smoking , obesity , sedentary life - style , kidney dysfunction , and vascular inflammation , is very common in the development of hypertension [ 13 , 17 - 20 ] . these risk factors lead to early functional and structural changes of the vasculature ( e.g. endothelial dysfunction and increased vascular stiffness ) that then gradually develop into early and advanced organ damage and failure and hypertension ( fig . the genome remains largely unchanged , whereas it is clear that the expression and function of proteins , metabolisms , structure of tissues and organs undergo substantial changes . therefore , while genetic factors define an individual s potential or risk for hypertension in early life , their applications in the diagnosis and prognosis of hypertension and prediction of an individual s risk of developing overt organ damage are very limited . due to the dynamic nature of disease development , the transcriptome , proteome , metabolome , and interactome will not only change due to disease processes but also in response to therapeutic measures and can therefore be used to unravel the pathophysiological pathways responsible for vascular disease and to monitor treatment effects . in order to gain a more comprehensive and quantitative understanding of the complex interplay of multiple mechanisms in vascular disease the omics-based studies have been developed in vascular medicine in the past few years [ 21 - 24 ] . omics strategies , including genomics , transcriptomics , proteomics , and metabolomics , however , are still based on the traditional molecular biology principle that dna is transcribed to rna and translated to proteins that control an organism s metabolism . accordingly , these approaches start from a global study of a large number of molecules and then dissect the changes in the expression of individual genes and proteins under the influence of vascular disease or drug treatment . as a consequence of these omics studies the current definition of clinical phenotypes of complex disease and combination drug therapy this has created major challenges for a complete understanding of the relationship between the genomic / proteomic/ metabonomic variability and the phenotypic vascular disease in individual patients and the translation of the knowledge of genomic / proteomic / metabonomic research into the clinical practice of combination therapy of vascular disease [ 2 , 6 , 7 ] . pleiotropy of vascular diseases remain largely focused on clinical categories that do not adequately reflect the etiology of the diseases . the reason for the limitation is that the conventional concept of vascular disease , for example hypertension , is defined as a clinical phenotype , not a clinical phenome ( fig . , the current definition of vascular disease is out of date and newer approaches are needed for the redefinition of vascular disease using not only the traditional signs and symptoms but also the underlying molecular causes at the scale of genome / proteome and the interaction with environmental factors and their modifying role over a longer period of time . the newly developed phenomic approach that assembles coherent sets of phenotypic features across individual measurements and diagnostic boundaries provides a new opportunity for systematical investigations of established biological pathways and complements the traditional candidate gene strategy or gwas focused on single genotype - phenotype association [ 25 , 26 ] . this new approach provides a novel paradigm for the redefinition of vascular disease as new category of clinical phenome ( fig . a phenome is the sum of complete phenotypic characteristics ( phenomic traits ) that signify the expression of the whole genome , proteome , and metabolome under specific environmental influence [ 26 - 29 ] . phenomics is a recently developed new transdiscipline that studies phenome in order to correlate complex traits to variability not only in genome , but also in proteome , metabolome , interactome , and environmental factors [ 28 - 32 ] . it provides a suite of new technologies and platforms for the transition from focused phenotype - genotype study to a systematic phenome - genome approach , which can be used to redefine the clinical phenotypes of diseases . phenomics is a multi - phenotype approach that requires collection of a wide breadth of phenotypes with fine resolution , phenomic analysis with constructing heat maps , cluster analysis , pathway analysis , text mining and big data analysis ( fig . clinical phenomics is the systematic measurement and analysis of qualitative and quantitative clinical phenome ( traits ) , including clinical signs and symptoms , and laboratory results obtained by biochemical , genomic , proteomic , metabolomic , and imaging methods , for the refinement and characterization of a clinical phenome . what will emerge from a phenomics ( including clinical phenomics ) approach is a more valid and etiologically - based systematic definition of disease phenome ( fig . 4 ) that may be quite different from those of current disease phenotypes defined by using the clinical symptoms at an organ ( e.g. , coronary artery , heart , kidney etc . ) or system ( e.g. , cardiovascular or nervous system ) level alone , as has been the tradition in clinical practice of western medicine for the past centuries ( fig . , disease should now be defined as a clinical phenome that is the sum total of a patient s clinical characteristics or phenomic traits that signify the expression of the whole genome , proteome , and metabolome under specific environmental influence ( fig . , the identification of multiple disease genes or proteins at the genomic and proteomic levels provide a solid platform for novel definition of the systematically integrated ( or clustered ) hypertensive phenotypes ( hypertension phenome ) under the scope of the whole genome / proteome / metabolome ( fig . , the hypertension phenome will now include not only clinical symptoms of elevated blood pressure ( hypertension ) but also a series of other systematically - defined phenotypic characteristics at different levels , including vascular morphology and function , heart - kidney - neural circuits / networks , metabolites , proteins , and genes ( fig . it is interesting that in the traditional chinese medicine ( tmc ) no hypertension was defined as a single disease ( or clinical phenotype ) . the symptoms of hypertension were included in 4 to 5 different zheng - hou ( fig . 4 ) defined as liver - yang hyperactivity ( or gang - yang - shang - kang ) , liver - qi stagnancy ( or gang - qi - yu - jie ) , ying - deficiency with yang - hyperactivity ( or yin - xu - yang - sheng ) , or abundant phlegm - dampness ( or tan - shi - yong - sheng ) . in current terms , zheng - hou can be explained as a clinical phenome while hypertension is a phenotype . therefore , phenomics study of tmc may help to redefine these hypertension - associated zheng - hou at the omics level , i.e. , the hypertensive phenome of vascular disease ( fig . 4 ) . with well - defined disease phenomes and a suite of new phenomics technologies and platforms ( fig . 5 ) the phenomics approach may be used to characterize the phenomes of vascular disease and also to identify the corresponding therapeutic targets for combination therapy at the level of systems biology . therefore , phenomics will complement other omics approaches , including genomics , proteomics , and metabolomics , and provide a new paradigm for the study of vascular disease and for rationally selecting drug targets and treatments suitable for personalized medicine ( fig . it can be envisaged that phenomics will refine the definition and diagnosis of vascular disease phenome and with systematically - defined therapeutic targets and improve predictive validity for outcomes of drug treatment . hypertensive phenotypes will be systematically - integrated and defined as a new hypertensive phenome of vascular disease according to the association with other related ( clustered ) clinical phenotypes such as vascular dysfunction , vascular inflammation , lipid metabolism , diabetes , and/or kidney dysfunction , multiple disease genes , snps , or proteins identified under the scope of the whole genome / proteome/ metabolome , using either the top - down ( comparative genomics , proteomics , metabolomics , gwas , etc . ) or bottom - up ( functional genomics , proteomics , metabolomics , phewas , etc . ) or both strategies . in the traditional chinese medicine ( tmc ) , the symptoms of hypertension were included in different zheng - hou defined as liver - yang hyperactivity ( or gang - yang - shang - kang ) , liver - qi stagnancy ( or gang - qi - yu - jie ) , ying - deficiency with yang - hyperactivity ( or yin - xu - yang - sheng ) , or abundant phlegm - dampness ( or tan - shi - yong - sheng ) . therefore , the hypertensive phenome will now include not only clinical symptoms and signs of elevated blood pressure ( hypertension ) but also a series of other systematically - defined phenotypic characteristics at different levels , including vascular morphology and function , heart - kidney - neural circuits / networks , metabolites , proteins , and genes . the phenomics approach will also provide a novel paradigm for drug research and development with the identification of multiple targets corresponding to the disease genes and proteins in the disease phenome , which will be suitable for personalized combination therapy . as a mimic to the gwas , denny et al . proposed in 2010 a novel approach termed phenome - wide association phenomics is a novel transdiscipline as a complimentary approach to several other omics approaches , including genomics , transcriptomics , proteomics , and metabolomics in the study of vascular disease and drug research and development , which will ultimately lead to individualized drug therapy and personalized medicine . phewas utilizes phenomics and big - data technologies to analyze all genetic / proteomic variants and all available phenotypic information from emrs , electronic health records ( ehrs ) , or observational cohort containing all types of diagnoses of clinical phenotypes such as data from the clinical data warehouse ( cdw ) in the estimation of association between genome - phenome and detection of pleiotropy [ 25 , 26 , 28 , 31 , 36 - 38 ] . with phewas , associations between a specific genetic variant and a wide range of physiological and/or clinical outcomes and phenotypes can be explored either by using algorithms to parse the data collected from ehrs , emrs , or cdw . denny et al . used the international classification of disease ( icd9 ) billing codes available in most electronic medical records ( emrs ) to develop a code translation table for automatic definition of 776 different disease populations and used the prevalent icd9 codes derived from the emr database to define their controls . using this phewas algorithm , they genotyped 6005 european - americans in the biovu , vanderbilt 's dna biobank , accrued at five snps with previously reported disease associations : coronary artery disease , atrial fibrillation , crohn 's disease , carotid artery stenosis , multiple sclerosis , systemic lupus erythematosus and rheumatoid arthritis . the phewas software was used to generate cases and control populations across all icd9 code groups for each of these five snps and to analyze the disease - snp associations . in addition , they also identified 19 previously unknown statistical ( p < 0.01 ) snps - diseases associations . this study demonstrates the feasibility of using phewas analysis as an alternative method that complements gwas , to investigate genotype - phenotype ( snp - disease ) associations . since 2010 , phewas has been used to investigate whether snps associated with one phenotype are also associated with other phenotypes [ 35 , 39 , 40 ] . more recently , denny and colleagues expanded their phewas using phenotypic data from the emrs of 13,835 individuals to look for associations between 1,358 phenotypes and 3,144 snps that had previously been found to show association with one or more traits in gwas . in cases for which the phewas was sufficiently powered , they found 66% of associations from gwas were replicated . in addition , they also uncovered 63 cases of previously unknown associations , potentially as pleiotropic effects . emr - based phewass provide a much simpler approach to pleiotropy analysis than the current gwas - based approach , which requires complex integration of data from multiple studies . robust test of the emr / ehr - based phewas allows unbiased interrogation across all domains of disease ( heart diseases , hypertension , stroke , brain diseases , diabetes , cancers , etc . ) , and can not only replicate what is known about individual genotype - phenotype associations with various snps but also uncover novel associations with a wide range of phenotypes in the emr / ehr - based cohorts . larger emr / ehr - based phewass may reveal more pleiotropy than has been estimated from gwas and have the potential to significantly improve our understanding of the molecular etiologies of diseases . with the fast advance in big - data technology and phenomics , we believe that the application of the emr / ehr data - based phewas in vascular medicine opens important avenues to enhance systematically - integrated analysis of the genomic basis of human cardiovascular disease and responses to drug therapy and to reform our understanding and clinical treatment of these devastating diseases with a new concept of wholism . a wide variety of approaches have been used to study the association of genetic polymorphisms to phenotypes of vascular disease , evolving from genetics , to genomics and gwas with the improvement of genotyping analyses . the candidate gene approach is the earliest and simplest method using the strategy of forward genetics based on a priori hypothesis to determine whether a gene is responsible for an intermediate phenotype , such as a molecular and cellular function , or a clinical manifestation . the focus of candidate gene study is on single genes mainly located in exons with polymorphisms that have a major role in determining the gene expression and the transduced protein or changes in cellular function . they showed that homozygotes with a deletion in the angiotensin converting enzyme ( ace ) gene were at higher risk of coronary artery disease and myocardial infarction . this association was more evident in subjects lacking of some well - established non - genetic risk factors , suggesting the importance of studying both genetic and environmental factors . later , it was noted that in a gene of 30 kb there are 10100 polymorphisms that are potentially responsible for variations of the genetic effect . therefore , later studies started to genotype more than one polymorphism in the same gene . for example , iacoviello et al . found that two polymorphisms in distant regions of the gene encoding for coagulation factor vii were associated with myocardial infarction ( mi ) and suggested a higher gene effect for the presence of both risk alleles . this has increased the analysis complexity and furnished an enormous amount of information in a big - data fashion . since the completion of the human genome project in 2000 , millions of single nucleotide polymorphisms ( snps ) have been discovered in the human genome . while the snp is deliberately chosen to mark a gene hypothesized a priori to play a mechanistic role in determining the trait in a candidate gene study , snps from across the entire genome are studied agnostically , without premeditation regarding their possible function in a genome - wide association study ( gwas ) . the patterns of linkage disequilibrium ( ld ) in the human genome have also been characterized through the international hapmap project . with the availability of high - throughput genotyping platforms and significantly decreased costs of genotyping and genome sequencing , gwas has become more affordable and has gradually overwhelmed the analyses on a single candidate gene . gwas has provided a systematic approach to investigation of the impact of common genomic variations on human vascular disease , such as acute coronary artery syndrome and hypertension [ 10 - 14 ] . while some studies started to use the clustering of common clinical domain phenotypes , most of current gwas are still using the hypothesis - driven strategy for identifying genetic mechanisms and remain largely focused on clinical categories that do not provide adequate etiological information . in gwas , a single pre - defined disease or clinical phenotype ( trait ) is studied and the accrual of a large number of single gene variant phenotype associations has serendipitously identified one gene affecting or responsible for more than one phenotypic characteristic or single loci associated with multiple diseases ( pleiotropy ) . in addition to the levels of encoded protein in basal conditions , the link between genetic variants and phenotypic diversities could be resulted from different responses to an environmental stimulus ( fig . a final target environmental risk factor can interact with a variety of molecular pathways , which are in turn regulated by multiple genetic factors . consequently , the effects of environmental risk factors could be modified by different genetic background . as a classic example of how genetic background could impact in disease pathogenesis , it was found that patients with different genotypes showed different levels of monocyte - production of cytokines in response to stimulation with lipopolysaccharide , corresponding to a different response of monocytes to inflammatory stimulation and risk of ischemic vascular disease ( mi and stroke ) at young age . actually , a risk factor together with other risk factors such as high salt intake , hypercholesterolaemia , smoking , obesity , sedentary life - style , kidney dysfunction , and vascular inflammation , is very common in the development of hypertension [ 13 , 17 - 20 ] . these risk factors lead to early functional and structural changes of the vasculature ( e.g. endothelial dysfunction and increased vascular stiffness ) that then gradually develop into early and advanced organ damage and failure and hypertension ( fig . the genome remains largely unchanged , whereas it is clear that the expression and function of proteins , metabolisms , structure of tissues and organs undergo substantial changes . therefore , while genetic factors define an individual s potential or risk for hypertension in early life , their applications in the diagnosis and prognosis of hypertension and prediction of an individual s risk of developing overt organ damage are very limited . due to the dynamic nature of disease development , the transcriptome , proteome , metabolome , and interactome will not only change due to disease processes but also in response to therapeutic measures and can therefore be used to unravel the pathophysiological pathways responsible for vascular disease and to monitor treatment effects . in order to gain a more comprehensive and quantitative understanding of the complex interplay of multiple mechanisms in vascular disease the omics-based studies have been developed in vascular medicine in the past few years [ 21 - 24 ] . these omics strategies , including genomics , transcriptomics , proteomics , and metabolomics , however , are still based on the traditional molecular biology principle that dna is transcribed to rna and translated to proteins that control an organism s metabolism . accordingly , these approaches start from a global study of a large number of molecules and then dissect the changes in the expression of individual genes and proteins under the influence of vascular disease or drug treatment . as a consequence of these omics studies the current definition of clinical phenotypes of complex disease and combination drug therapy this has created major challenges for a complete understanding of the relationship between the genomic / proteomic/ metabonomic variability and the phenotypic vascular disease in individual patients and the translation of the knowledge of genomic / proteomic / metabonomic research into the clinical practice of combination therapy of vascular disease [ 2 , 6 , 7 ] . pleiotropy of vascular diseases remain largely focused on clinical categories that do not adequately reflect the etiology of the diseases . the reason for the limitation is that the conventional concept of vascular disease , for example hypertension , is defined as a clinical phenotype , not a clinical phenome ( fig . , the current definition of vascular disease is out of date and newer approaches are needed for the redefinition of vascular disease using not only the traditional signs and symptoms but also the underlying molecular causes at the scale of genome / proteome and the interaction with environmental factors and their modifying role over a longer period of time . the newly developed phenomic approach that assembles coherent sets of phenotypic features across individual measurements and diagnostic boundaries provides a new opportunity for systematical investigations of established biological pathways and complements the traditional candidate gene strategy or gwas focused on single genotype - phenotype association [ 25 , 26 ] . this new approach provides a novel paradigm for the redefinition of vascular disease as new category of clinical phenome ( fig . a phenome is the sum of complete phenotypic characteristics ( phenomic traits ) that signify the expression of the whole genome , proteome , and metabolome under specific environmental influence [ 26 - 29 ] . phenomics is a recently developed new transdiscipline that studies phenome in order to correlate complex traits to variability not only in genome , but also in proteome , metabolome , interactome , and environmental factors [ 28 - 32 ] . it provides a suite of new technologies and platforms for the transition from focused phenotype - genotype study to a systematic phenome - genome approach , which can be used to redefine the clinical phenotypes of diseases . phenomics is a multi - phenotype approach that requires collection of a wide breadth of phenotypes with fine resolution , phenomic analysis with constructing heat maps , cluster analysis , pathway analysis , text mining and big data analysis ( fig . clinical phenomics is the systematic measurement and analysis of qualitative and quantitative clinical phenome ( traits ) , including clinical signs and symptoms , and laboratory results obtained by biochemical , genomic , proteomic , metabolomic , and imaging methods , for the refinement and characterization of a clinical phenome . what will emerge from a phenomics ( including clinical phenomics ) approach is a more valid and etiologically - based systematic definition of disease phenome ( fig . 4 ) that may be quite different from those of current disease phenotypes defined by using the clinical symptoms at an organ ( e.g. , coronary artery , heart , kidney etc . ) or system ( e.g. , cardiovascular or nervous system ) level alone , as has been the tradition in clinical practice of western medicine for the past centuries ( fig . , disease should now be defined as a clinical phenome that is the sum total of a patient s clinical characteristics or phenomic traits that signify the expression of the whole genome , proteome , and metabolome under specific environmental influence ( fig . , the identification of multiple disease genes or proteins at the genomic and proteomic levels provide a solid platform for novel definition of the systematically integrated ( or clustered ) hypertensive phenotypes ( hypertension phenome ) under the scope of the whole genome / proteome / metabolome ( fig . , the hypertension phenome will now include not only clinical symptoms of elevated blood pressure ( hypertension ) but also a series of other systematically - defined phenotypic characteristics at different levels , including vascular morphology and function , heart - kidney - neural circuits / networks , metabolites , proteins , and genes ( fig . it is interesting that in the traditional chinese medicine ( tmc ) no hypertension was defined as a single disease ( or clinical phenotype ) . the symptoms of hypertension were included in 4 to 5 different zheng - hou ( fig . 4 ) defined as liver - yang hyperactivity ( or gang - yang - shang - kang ) , liver - qi stagnancy ( or gang - qi - yu - jie ) , ying - deficiency with yang - hyperactivity ( or yin - xu - yang - sheng ) , or abundant phlegm - dampness ( or tan - shi - yong - sheng ) . in current terms , zheng - hou can be explained as a clinical phenome while hypertension is a phenotype . therefore , phenomics study of tmc may help to redefine these hypertension - associated zheng - hou at the omics level , i.e. , the hypertensive phenome of vascular disease ( fig . 4 ) . with well - defined disease phenomes and a suite of new phenomics technologies and platforms ( fig . 5 ) the phenomics approach may be used to characterize the phenomes of vascular disease and also to identify the corresponding therapeutic targets for combination therapy at the level of systems biology . therefore , phenomics will complement other omics approaches , including genomics , proteomics , and metabolomics , and provide a new paradigm for the study of vascular disease and for rationally selecting drug targets and treatments suitable for personalized medicine ( fig . it can be envisaged that phenomics will refine the definition and diagnosis of vascular disease phenome and with systematically - defined therapeutic targets and improve predictive validity for outcomes of drug treatment . hypertensive phenotypes will be systematically - integrated and defined as a new hypertensive phenome of vascular disease according to the association with other related ( clustered ) clinical phenotypes such as vascular dysfunction , vascular inflammation , lipid metabolism , diabetes , and/or kidney dysfunction , multiple disease genes , snps , or proteins identified under the scope of the whole genome / proteome/ metabolome , using either the top - down ( comparative genomics , proteomics , metabolomics , gwas , etc . ) or bottom - up ( functional genomics , proteomics , metabolomics , phewas , etc . ) or both strategies . in the traditional chinese medicine ( tmc ) zheng - hou defined as liver - yang hyperactivity ( or gang - yang - shang - kang ) , liver - qi stagnancy ( or gang - qi - yu - jie ) , ying - deficiency with yang - hyperactivity ( or yin - xu - yang - sheng ) , or abundant phlegm - dampness ( or tan - shi - yong - sheng ) . therefore , the hypertensive phenome will now include not only clinical symptoms and signs of elevated blood pressure ( hypertension ) but also a series of other systematically - defined phenotypic characteristics at different levels , including vascular morphology and function , heart - kidney - neural circuits / networks , metabolites , proteins , and genes . the phenomics approach will also provide a novel paradigm for drug research and development with the identification of multiple targets corresponding to the disease genes and proteins in the disease phenome , which will be suitable for personalized combination therapy . as a mimic to the gwas , denny et al . proposed in 2010 a novel approach termed phenome - wide association phenomics is a novel transdiscipline as a complimentary approach to several other omics approaches , including genomics , transcriptomics , proteomics , and metabolomics in the study of vascular disease and drug research and development , which will ultimately lead to individualized drug therapy and personalized medicine . phewas utilizes phenomics and big - data technologies to analyze all genetic / proteomic variants and all available phenotypic information from emrs , electronic health records ( ehrs ) , or observational cohort containing all types of diagnoses of clinical phenotypes such as data from the clinical data warehouse ( cdw ) in the estimation of association between genome - phenome and detection of pleiotropy [ 25 , 26 , 28 , 31 , 36 - 38 ] . with phewas , associations between a specific genetic variant and a wide range of physiological and/or clinical outcomes and phenotypes can be explored either by using algorithms to parse the data collected from ehrs , emrs , or cdw . denny et al . used the international classification of disease ( icd9 ) billing codes available in most electronic medical records ( emrs ) to develop a code translation table for automatic definition of 776 different disease populations and used the prevalent icd9 codes derived from the emr database to define their controls . using this phewas algorithm , they genotyped 6005 european - americans in the biovu , vanderbilt 's dna biobank , accrued at five snps with previously reported disease associations : coronary artery disease , atrial fibrillation , crohn 's disease , carotid artery stenosis , multiple sclerosis , systemic lupus erythematosus and rheumatoid arthritis . the phewas software was used to generate cases and control populations across all icd9 code groups for each of these five snps and to analyze the disease - snp associations . in addition , they also identified 19 previously unknown statistical ( p < 0.01 ) snps - diseases associations . this study demonstrates the feasibility of using phewas analysis as an alternative method that complements gwas , to investigate genotype - phenotype ( snp - disease ) associations . since 2010 , phewas has been used to investigate whether snps associated with one phenotype are also associated with other phenotypes [ 35 , 39 , 40 ] . more recently , denny and colleagues expanded their phewas using phenotypic data from the emrs of 13,835 individuals to look for associations between 1,358 phenotypes and 3,144 snps that had previously been found to show association with one or more traits in gwas . in cases for which the phewas was sufficiently powered , they found 66% of associations from gwas were replicated . in addition , they also uncovered 63 cases of previously unknown associations , potentially as pleiotropic effects . emr - based phewass provide a much simpler approach to pleiotropy analysis than the current gwas - based approach , which requires complex integration of data from multiple studies . robust test of the emr / ehr - based phewas allows unbiased interrogation across all domains of disease ( heart diseases , hypertension , stroke , brain diseases , diabetes , cancers , etc . ) , and can not only replicate what is known about individual genotype - phenotype associations with various snps but also uncover novel associations with a wide range of phenotypes in the emr / ehr - based cohorts . larger emr / ehr - based phewass may reveal more pleiotropy than has been estimated from gwas and have the potential to significantly improve our understanding of the molecular etiologies of diseases . with the fast advance in big - data technology and phenomics , we believe that the application of the emr / ehr data - based phewas in vascular medicine opens important avenues to enhance systematically - integrated analysis of the genomic basis of human cardiovascular disease and responses to drug therapy and to reform our understanding and clinical treatment of these devastating diseases with a new concept of wholism . | vascular diseases are usually caused by multifactorial pathogeneses involving genetic and environmental factors .
our current understanding of vascular disease is , however , based on the focused genotype / phenotype studies driven by the one - gene / one - phenotype hypothesis . drugs with pure target at individual molecules involved in the pathophysiological pathways are the mainstream of current clinical treatments and the basis of combination therapy of vascular diseases .
recently , the combination of genomics , proteomics , and metabolomics has unraveled the etiology and pathophysiology of vascular disease in a big - data fashion and also revealed unmatched relationships between the omic variability and the much narrower definition of various clinical phenotypes of vascular disease in individual patients . here
, we introduce the phenomics strategy that will change the conventional focused phenotype / genotype / genome study to a new systematic phenome / genome / proteome approach to the understanding of pathophysiology and combination therapy of vascular disease .
a phenome is the sum total of an organism s phenotypic traits that signify the expression of genome and specific environmental influence .
phenomics is the study of phenome to quantitatively correlate complex traits to variability not only in genome , but also in transcriptome , proteome , metabolome , interactome , and environmental factors by exploring the systems biology that links the genomic and phenomic spaces .
the application of phenomics and the phenome - wide associated study ( phewas ) will not only identify a systemically - integrated set of biomarkers for diagnosis and prognosis of vascular disease but also provide novel treatment targets for combination therapy and thus make a revolutionary paradigm shift in the clinical treatment of these devastating diseases . |
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although there is a long history of attempts to implement telecytology , it still has only limited applications and acceptance . this stems largely from the challenge involved in making imaging systems of sufficient quality to consistently and accurately reproduce cytology material present on glass slides . cytology material is difficult to image , relative to histology , for a number of reasons . paraffin - embedded tissue is cut to create a single uniform plane of tissue for staining and examination . cytology material , on the other hand , frequently contains a significant element of three - dimensionality resulting from cell clustering or as an artifact of preparation techniques . furthermore , diagnostic cytology material , unlike histology , is more frequently widely and randomly distributed across glass slides , especially in smeared preparations , requiring more intensive screening of a larger area in order to find the best areas for diagnostic interpretation . the greater reliance of cytologists on subtle features of individual cells and background material is another problem , as this requires greater magnification and higher quality images . finally , the frequent need for complimentary stains , usually papanicolaou and romanowsky stains in tandem , increases the number of slides that must be examined to render a confident diagnosis . counterbalancing the problematic aspects of telecytology , however , are powerful incentives to develop imaging technology for use in cytology material . unlike histology , where a tissue block remains behind for potential recuts if a slide is lost or broken , many cytology slides are unique and irreplaceable . it is not uncommon for life - altering patient diagnoses to rest entirely on the appearance of a few cells present on a single cytology slide . for this reason , developing telecytology capabilities that could reduce the need to physically send precious glass slides over long distances , or even to create a permanent digital copy that could be stored long term , is highly desirable . along the same lines , cytology education and testing suffer because of the difficulty of acquiring a sufficient number of comparable and high - quality slides for wide distribution and easy access . telecytology offers the possibility of providing users with access to digitized teaching sets comprised of typical and unique cases . ideally , outstanding examples from glass slide teaching sets scattered in laboratories around the world could be compiled into a universally accessible database for the benefit of cytologists everywhere , akin to the effort involved in creating the online static digital bethesda system for cervical cytology . the evolution of telecytology is approaching a critical stage in which it is becoming more reasonable to expect much broader application and acceptance of this technology . increasing numbers of cytology procedures requiring immediate evaluation are being performed in settings where expert cytopathologists are a limiting factor , for instance fine needle aspiration of thyroid nodules . most telecytology studies in the literature focus on the review of static digital microphotographs or video microscopy that only allow the remote viewer to assess a tiny fraction of the total case material selected by the host reviewing the actual glass slides . advances in whole - slide imaging ( wsi ) , however , offer the possibility of creating higher quality reproductions of entire glass slides that can be stored and accessed in real time . moreover , current applications that accompany wsi viewing permit teleconferencing whereby several individuals can remotely log in to simultaneously view a case . this opens up entirely new possibilities for telecytology , including the prospect of providing final diagnoses based on virtual images alone . telecytology depends , fundamentally , on the ability to convert optical information presented in a microscope eyepiece into a digital image that can be remotely transmitted . digital imaging devices ( e.g. , digital camera , wsi scanner ) , computers , and networks ( i.e. the internet ) make this task ever - easier . a digital image is represented in a computer by a two - dimensional array of numbers , each element of which represents a pixel ( short for picture element ) . the imaging process involves capturing , saving , editing ( if necessary ) , and sharing digital images . there are multiple types of devices that can be used to acquire digital images [ figure 1 ] . many of these devices , particularly microscope - mounted cameras , are already widely installed in many pathology laboratories and used for several purposes including teaching and tumor board presentations . commercial software is available for a few thousand dollars that allows for remote live viewing of the images produced by such cameras . other systems , such as remotely controlled robotic microscopes and whole - slide scanners are being used , but to a lesser degree , largely for telepathology of surgical pathology material ( teleconsultation ) and frozen sections ( including brain smears ) . input devices for creating digital images : ( far left ) digital camera attached via adapter to a light microscope , ( middle left ) whole - slide scanners showing ( upper ) the aperio scanscope xt cs scanner , scans up to 120 slides ; and ( lower ) the omnyx vl4 whole - slide scanner that scans up to four slides at a time and ( middle right ) robotic microscopes including ( upper ) the nikon coolscope ii , one glass slide scanner and ( lower ) the trestle 5l50 , 50 slide loaders ( far right ) cambridge research and instrumentation ( cri ) nuance multispectral imaging ( msi ) camera microscopic digital images can be static ( still images ) , viewed live ( real - time video or robotic microscopy ) , or viewed after scanning of the glass slides ( wsi or virtual microscopy).[25 ] efforts are underway to standardize the process of acquiring , storing , and displaying digital images in pathology in a manner similar to radiology.[69 ] wsi is a digital imaging modality that uses computerized technology to scan and convert ( digitize ) glass slides into digital images so that they can be viewed on a computer using viewing software . this software allows a user to scan from field to field and increase or decrease the magnification , simulating panning around and zooming in or out with a conventional microscope . current wsi technology provides rapid , high - quality image capture and storage , integrated with image viewer software to allow virtual microscopy anywhere in the world over the internet . advantages of whole slide imaging in cytopathology practice disadvantages of whole - slide imaging in cytopathology practice wsi technology has started to have a significant impact on cytopathology practice in various aspects including telecytology , performing cytology quality assurance , cytology education , competency assessment , image analysis , and research [ figure 2].[1216 ] recent advanced capabilities include a decrease in the time to scan slides ( only a - few - minute range ) and the ability to handle a large number of slides with automated feeders holding up to 300 slides . the z - stack function ( so called because the planes of focus are along the z - axis ) offers manual - focusing capability of selected areas on virtual slides . an individual wsi device currently costs approximately $ 135,000 with basic viewing software , with some systems being considerably more . in addition to the devices themselves , substantial costs are incurred for labor needed to operate and troubleshoot the scanner , long - term image storage and secure transmission , and other less obvious items such as upgraded monitors for pathologists , image analysis software , and integration of the scanner software with the lis . depending on the volume of scanning and the number of sites that need to be served , wsi systems routinely cost hundreds of thousands or even millions of dollars . scan times have improved considerably but still can be quite lengthy , especially for slides that are required to be digitized at high magnification ( e.g. 40 ) , have a large area to be scanned , and need to be scanned in using multiple planes of focus , as is often the case in cytology . longer scan times correspond to larger image files , increasing the challenges involved in their storage and transmission . furthermore , not all wsi devices are capable of scanning multiple planes of focus , while others only allow z - stack functionality to be used for delimited portions of the virtual slide . traditionally , scanner manufacturers have focused on histology applications because of these issues . however , as scanning technology improves and servers become capable of handling larger files at lower cost , cytology applications are becoming ever - more feasible . in the future , decreasing barriers and increasing demand should bring about the introduction of more scanners with advanced capabilities suitable for telecytology applications . a whole - slide image ( digitized slide ) of signet - ring carcinoma in pleural effusion illustrating the viewer software provided by the vendor to allow for remote viewing and manipulation of images by the cytopathologist . ( a ) zoom slider , ( b ) thumbnail image , ( c ) magnified field , ( d ) circled area is the annotation layer information used to mark up areas of interest , ( e ) drawing tool bar telecytology has been used in niche applications as a part of clinical practices for a number of years . most of the literature reporting applications of telecytology for patient care revolves around either static images for consultation[2232 ] or streaming of live video images for remote immediate assessment.[3335 ] use of remotely controlled ( dynamic ) robotic systems for telecytology has also been reported.[3638 ] only a few applications of wsi for clinical use have been published to date , but this technology seems to be spurring renewed interest in research in the area of telepathology . studies reporting on the use of static images are typically somewhat older and often are the result of attempts at making cytopathology expertise more readily available either by enabling the transmission of cytopathology materials from areas with limited cytology resources or by speeding up more routine consult processes . all that is required is a microscope - mounted camera of the type widely used by many pathologists for other functions , and hence is readily available without the need to purchase new equipment . the static images also require only a small amount of computer memory ; hence they can be readily transmitted and viewed over existing networks . however , the images produced by these cameras can only capture a tiny fraction of the information contained in a glass slide . this makes the remote viewer entirely dependent on the judgment of the person who captures the images . static images are also subject to quality issues , with seemingly minor variations in image parameters ( e.g. , color , contrast ) potentially leading to markedly different interpretations . reported uses of static images for telecytology include gynecologic material ( pap tests ) as well as nongynecological specimen types including aspirates of breast , thyroid , and pancreas , as well as pleural fluid specimens . in general , past studies demonstrated good concordance between diagnoses made using still photomicrographs presented on a computer screen when compared to those made following review of the corresponding glass slides [ table 3 ] . most important was the inability to reliably depict the entire case material with only a few representative photographs . this drawback of static telecytology inevitably led to diagnostic difficulties , since overall cellularity is an important factor in many cases , especially for nongynecologic specimens . indeed , different studies of breast aspirates showed a tendency toward both false - positive diagnoses and excessive hedging resulting in a tendency to call true positive cases only suspicious . two separate studies of pap tests found that small cells , including hsil and carcinoma , were especially difficult to accurately classify using only a single focus plane . a system of z - stack videos showing areas of interest has been proposed as an alternate way to overcome this difficulty , but so far this has had only very limited application . another issue is that the most challenging cases , those most in need of expert consultation , tend to perform the most poorly . one study of telecytology consultation found that 9% of telecytology consults cases had a major discrepancy between the initial diagnosis and the final glass slide diagnosis , despite the fact that 11% of the telecytology consult attempts were deferred . another study found only 48% diagnostic concordance between the telecytology consultant opinion and the opinion of the referring pathologist , with a 31% discrepancy rate between the diagnosis based on static images and the final glass slide diagnosis in those cases where the slides were reviewed . a pilot study marrying static telecytology with automated pap test slide scanning has also been published . the authors demonstrated that remote interpretation of the pre - selected fields of view selected by the scanning algorithm yielded sensitivity levels comparable to those seen for the same scanning device in prospective trials that did not employ the telecytology component . the goal of the study was to demonstrate the feasibility of using such scanners to provide access to cytology screening in parts of the world that lack cytotechnologists and pathologists . studies of this kind raise the exciting possibility of exporting cytopathology expertise to the developing world . summary of studies using static images for telecytology a few studies have reported the use of remote robotic microscopy as a means of overcoming the shortcomings of telecytology based on static photographs.[3638 ] remotely controlled slides offer the possibility of real - time ( dynamic ) simulated microscopy enabling thorough examination of potentially all the material present on slides . one study found that remote robotic examination of pancreas diff -- quik adequacy slides had an interobserver kappa of 0.61 when compared with the final diagnosis , compared to a kappa of 0.79 for the same slides viewed on site with a conventional microscope . the difference was not statistically significant and most discrepancies occurred early in the study , leading the authors to conclude that with sufficient training and experience this technology could be a viable alternative to live attendance at remote sites . however , this technology is slow and awkward due to the delays involved in sending instructions to the robotic controls from the remote location , then having to wait for the image to refresh before making additional adjustments . this problem is especially acute for specimens that are thick or sparse and require extensive scanning . the equipment is also expensive , often no longer supported by vendors , and has no other uses , unlike the competing technology of wsi . another technology that has proven to be popular for telecytology is remote live video microscopy [ figure 3 ] . many standard microscope - mounted cameras have the capacity to stream images , making the technology relatively affordable . studies to date have applied this technology to remote locations within the same medical center,[3335 ] allowing cytopathologists to view cases for immediate adequacy purposes collected in suites inconveniently distant from the main laboratory . the findings support the use of the technology , with adequacy interpretation results that are comparable for video telecytology and conventional microscopy , reporting concordance rates in excess of 95%.[3335 ] the main drawback of this technology is that the quality of the interpretation depends heavily on the ability of the person who moves the slide and selects the fields of view . although feedback by telephone can help the consultant guide the remote microscope operator , the system works better if that individual has enough training to locate and recognize critical cells independently . typically , a senior pathology resident , cytopathology fellow , or cytotechnologist is employed for this purpose . surprisingly , to the best of our knowledge , there are no published reports of the use of video microscopy across institutions for remote consultations . undoubtedly the technology has been used in this way , but its utility in this setting may be limited by the need to have the requesting and consulting pathologists simultaneously involved in case review , creating cumbersome scheduling problems . newer technology using high - definition ( hd ) cameras and secure networking telecommunication solutions offers promising improvements . case of a lymph node fna that was suspicious for lymphoma requiring submission of the fresh sample for flow cytometric study wsi offers the possibility of overcoming many of the aforementioned problems associated with other methods . since the whole slide is available for review by the remote viewer , issues of field selection and lack of low - power impression are largely nullified . commercially available scanners capable of producing z - stack images can also overcome the plane of focus problem . software for manipulating the virtual image on the computer screen of the viewer eliminates the need for a skilled person on site to move the slide or for the user to struggle with a cumbersome remote robotics application [ figure 4 ] . although wsi scanners are presently more expensive relative to standard microscope - mounted digital cameras , they have numerous other potential uses to help justify their cost ( e.g. image analysis ) . the digital cameras incorporated within these wsi scanners are automated and provide consistent images , reducing concerns about image quality . there are very few publications in the literature regarding the use of wsi for clinical telecytology . one study has been published where the investigators report that wsi performed using pap tests demonstrated excellent performance of the virtual images , with diagnostic accuracy comparable to that glass slides . however , issues of speed in this study were of concern , causing users to prefer the glass slide over the wsi equivalent . a survey of cytotechnologists given wsi virtual slides to examine also found that the slowness of the system was the predominant complaint . wsi manufacturers are currently working on alternative means of displaying and manipulating the images in order to make diagnosis faster and easier , looking to create the pathologist cockpit with optimized monitors and control devices . upper : documents and images ( static or whole slide ) uploaded via the portal are available for review . lower : launching imagescope viewer allows virtual slides to be viewed online clearly , a key problem for wsi is the scanning time required for digitizing cytology slides , which at present takes approximately 10 minutes for a conventionally prepared smeared slide at 20 with one focus plane , and more than an hour for multi - plane scanning necessary to generate z - stack images . this makes immediate consultation difficult due to the delays introduced by the extra scanning step . although frozen section diagnosis using wsi has been successfully demonstrated , the application of wsi for immediate cytology adequacy interpretation on a routine basis will most likely have to wait for significant technological improvements in scanners . another barrier is the file sizes needed to adequately capture large three - dimensional cytology slides . such digital image files may be in the hundreds of megabytes to gigabyte range , making their storage and transmission challenging , although this issue seems to be less imposing with the passage of time as compression algorithms continually improve and data storage options become ever cheaper . information technology restrictions of institutional firewalls and other security issues , in addition to secure routing of private patient metadata information along with standardized dicom images , will also need to be overcome . finally , issues related to standardization and validation are extremely important for wsi because of the potential for this technology to completely replace the conventional optical microscope . this has caused the us food and drug administration to consider requiring wsi manufacturers to get approval for their devices before they can be used for clinical purposes . partly in response to this possibility , the college of american pathologists ( cap ) has developed recommendations for validating wsi systems for routine clinical diagnostic use , including cytology specimens that will require cytology - specific validation . although error is difficult to define in anatomical pathology because much of the interpretation is subjective with high interobserver variability , laboratories are still required to participate in quality assurance ( qa ) measures , such as the independent review of cases in an effort to identify significant diagnostic errors and take corrective action . for gynecologic cytology , although the cap publishes a series of checklists to guide cytopathology qa , the specific implementation of such a program is intentionally left flexible to accommodate the wide variety of pathology practices that exist . typical approaches to cytology qa include a review of cases in a variety of settings such as intradepartmental , interdepartmental , or extradepartmental review and second opinions requested by patients or clinicians . discrepancies should be noted and resolved between pathologists and , if necessary , amended reports or addenda issued . there is good reason behind extensive qa programs in anatomical pathology and cytopathology as it has been estimated that the actual error rate likely ranges from 1% to 5% , and in a study of self - reported discrepancies among 72 institutions , 6.7% of anatomical pathology diagnoses were found to be discrepant at second review . in 1% of these cases , statistics based upon second review in the same institution are likely to reflect an underrepresentation of errors because traditional case reviews are associated with a number of potential biases , including reviewer knowledge of the original diagnosis and/or the identity of the sign - out pathologist . therefore , establishing mechanisms for qa case review with truly independent outside pathologists has great appeal as a means of achieving maximal quality improvement impact . a major hindrance to establishing a multifacility qa program , however , is the expense and difficulty of moving and managing slides between facilities , especially if the qa is to be done before or soon after to the sign - out date when the detection of potential errors would be of most benefit to the patient . automated wsi , in which all the slides in a case are imaged in their entirety at high resolution and made available to cytopathologists on a network , is a modality that may prove useful in cytopathology case review . a digitized case could allow a qa system to hide the identity of the original sign - out pathologist and , if desired , the original diagnosis . more importantly , however , digital slides available on a network can mitigate the problems of glass slide logistics and , by so doing , enable routine multifacility cytopathology qa . digital imaging is beginning to replace the traditional classroom with microscopes in medical education , including cytopathology.[5456 ] digital imaging undoubtedly offers significant advantages over the traditional light microscope in education and training . also , the use of glass slides is limited by the fact the colors of stains fade over time , glass slides can be easily broken or lost , the slides can be used only by one person at a time , and a microscope is necessary for viewing . a web - based virtual slide library can be permanently stored , enabling users to review cytological educational material anytime , anywhere without microscopes or glass slides . in contrast , the traditional microscopy classroom is costly to set up and maintain , and high - quality cytology glass slides are impossible to duplicate or replace . virtual slides may be annotated and shared by participants , such as pathology residents before conferences . with digital slide conferences , advantages include improved access to large teaching sets , enhanced annotations , and instant access to linked related clinical data such as radiology images . due to the increase in usage of wsi in pathology and cytology education , demands for adequate training in digital imaging is becoming an increasingly necessary component of training in pathology residency and cytotechnology training programs . digitzed slides in cytology can even be employed to support a " virtual " rotation for trainees . wsi technology also offers the ability to introduce effective online cytology educational programs and online cytology atlases such as the uscap virtual slide box , which offers unknown cases in anatomical pathology and cytopathology . the international academy of cytology ( iac ) recently provided several digital educational materials on their web site , including cases with virtual slides and static images and online lectures , seminars , and workshops . the american society of cytology ( asc ) has also begun to incorporate wsi into its case studies for continuing education and is building a virtual atlas that will be available to members online . it is conceivable that in the near future cytopathlogy testing will be greatly altered by the use of wsi . virtual slides solve many of the thorniest issues involved in testing diagnostic proficiency . having a single digital copy of a slide that can be viewed by all test takers not only eliminates the logistical nightmare of maintaining and distributing large sets of glass slides , but also ensures that every test participant is viewing identical images . this greatly reduces the burden of validation and increases fairness and reproducibility , as long as participants are adequately trained to view such images . the american board of pathology has already been using virtual slides for a subset of its pathologist certification examination questions for a number of years . another obvious area for application of this technology would be pap test proficiency testing mandated in the usa by clia 88 . educational testing , such as the popular interlaboratory comparison program offered by the cap , would be another logical venue for wsi for the same reasons . the practice of cytology is evolving rapidly , and cytologists must prepare now for the digital tomorrow . in the coming years , several changes such as the advancement of personalized medicine , adoption of standards like dicom , and the emergence of technological advances like digital pathology will greatly impact how a cytologist performs his / her job . early efforts to use digital images and the internet to render diagnoses via telecytology have shown promise despite suboptimal older technology that initially was restricted to reproducing only a tiny fraction of the material on a glass slide . wsi offers the prospect of true virtual microscopy , and may in time even replace glass slides in routine practice . we are rapidly approaching this reality as vendors continue to build newer , faster , and cheaper scanners with sophisticated software to improve digital pathology workflow . cytologists can look forward to accessing , reviewing , sharing , and even analyzing the digital data in their digitized slides . | telecytology is the interpretation of cytology material at a distance using digital images . for more than a decade ,
pioneering efforts to introduce telecytology into clinical practice have been reported .
a medline search for telecytology and cytology reveals a voluminous literature , though much of what has been published to date is based on technologies that are rapidly becoming obsolete .
the technological limitations of previous techniques , including the transmission of static digital images and dynamic streaming images , have limited telecytology to minor niches .
the primary problem with these technologies is that the remote viewer can only see a small fraction of the material on the original slides , introducing the possibility of diagnostic error based not only on image quality but also on image selection .
remote robotic microscopy offers one possible solution to this problem , but to date has found limited acceptance , principally attributable to slow operating times .
whole slide imaging seems to be a much more promising solution , though cytology - specific literature regarding its use is still scant .
the advent of whole slide imaging opens up new possibilities for telecytology by enabling high - quality images of entire cytology specimens to be available to anyone , anywhere via the internet .
although challenges remain , especially with regard to capturing the full microscopy experience including multiple planes of focus and sharp high - powered images , rapidly advancing technology promises to overcome these limitations .
increasing application of whole slide imaging technology in surgical pathology will undoubtedly also increase its application to cytology due to the increasing affordability and practicality of the equipment as it serves a larger number of useful roles within a pathology department .
the current and expanding applications of telecytology for clinical practice , education , quality assurance , and testing will be reviewed . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
vitiligo is a common depigmenting skin disorder[13 ] characterized by acquired , idiopathic , progressive , circumscribed hypomelanosis of the skin , and hair . it is generally agreed that there is an absence of functional melanocytes in vitiligo skin and that this loss of histochemically recognizable melanocytes is the result of destruction . vitiligo is the most prevalent pigmentary disorder with an incidence rate between 0.1 - 2% showing multifactorial etiology and polygenic inheritance . the effects of vitiligo can be cosmetically and psychologically devastating , resulting in low self - esteem , poor body image , and difficulties in sexual relationships . because the disease is still not understood , there is a plethora of different treatments including topical corticosteroids , calcineurin inhibitors , vitamin - d derivatives , phototherapy ( ultraviolet [ uv ] a , narrowband uvb ) , photochemotherapy ( psoralen plus uva [ puva ] , psoralen with sunlight [ puvasol ] ) , surgical techniques , and combination of topical therapies and light treatment . on the other hand , there are few controlled trials assessing efficacy of natural health products ( e.g. , vitamins , minerals , herbal medicines , and other supplements ) for vitiligo , but those that have been published generally show weakly positive outcomes with few adverse reactions . for the first time , in one study , we showed that topical corticosteroid in combination with oral zinc is more effective than topical steroid alone . although this efficacy was not statistically significant in this study , it appears that more robust long - term randomized controlled trials with more patients , maybe with high doses of oral zinc are needed to fully establish the efficacy of oral zinc in management of vitiligo . vitiligo is a multifactorial disorder,[571011 ] related to both genetic and nongenetic factors . due to the observed variation in clinical manifestations of the disease various physiological , biochemical , histochemical , and enzymatic studies have been done to clarify the pathogenesis of vitiligo . genes certainly play a role in all aspects of vitiligo pathogenesis , even response to environmental triggers , and so genetics really should not be separated out as a distinct phenomenon . in the past few years , studies of the genetic epidemiology of generalized vitiligo have led to the recognition that vitiligo is part of a broader , genetically determined , autoimmune , and auto inflammatory diathesis . currently , there is strong support only for hla , ptpn22 , nalp1 , and perhaps ctla4 , all genes associated with autoimmune susceptibility . hla types associated with vitiligo in more than one study include a2 , dr4 , dr7 , and cw6 . jin et al . reported the linkage signals on chromosomes 1 , 7 , and 17 in caucasian families with generalized vitiligo and associated autoimmune diseases and identified the risk loci of chromosomes 17 and 1 as nlpr1 ( nalp1 ) and foxd3 , respectively . the major reasons for this hypothesis are the coexistence of other autoimmune disorders , detection of various autoantibodies including antithyroid and antimelanocyte antibodies in the serum of vitiligo patients and alteration of t - cell population showing decreased t - helper cells . the facts that vitiligo is found more frequently in patients with metastatic melanoma and is associated with an improved prognosis , that vitiligo - like depigmentation has been observed following immunotherapy of melanoma and that vitiligo has also developed in recipients of bone marrow transplant or lymphocyte infusions from patients with vitiligo , further support a crucial role for cell - mediated immunity in the pathogenesis of the disorder . histological and immunohistochemical studies in prelesional skin suggest the involvement of cellular immunity in vitiligo . focal spongiosis and epidermal mononuclear cell infiltrate was found in 48% and 80% of both marginal and vitiliginous skins , respectively , in vitiligo patients . thus , the vitiligo is an inflammatory disease and the epidermal lymphocytic infiltrate is most likely the primary immunologic event . there are some clues that point to heritable biological properties that might make the melanocyte of some people susceptible to environmental triggers or other stressors , possibly resulting in melanocyte death by necrosis , apoptosis , or pyroptosis , consequent presentation of tolerogens and loss of immune tolerance , and ultimately autoimmunity directed against melanocytes . as hypotheses , accumulation of toxic compounds , altered cellular environment , impaired melanocyte migration , and/or proliferation , infection , such as virus , psychological ( stress and personality characteristics of patients ) , neural and autocytotoxic factors can all contribute to vitiligo . the autocytotoxic theory postulates that cytotoxic precursors to melanin synthesis accumulate in melanocytes , causing cell death ( self - destruct theory of lerner ) . there may be a relationship between stress and the development of vitiligo via increasing levels of neuroendocrine hormones . increases level of neuroendocrine hormones affects the immune system and alters the level of neuropeptides , which may be the initial steps in the pathogenesis of vitiligo . the histological and some laboratory data support apoptosis rather than cell necrosis as the mechanism of melanocyte loss.[12731 ] therefore , programmed melanocyte death or destruction due to inherent sensitivity to oxidative stress arising from either toxic intermediates of melanin or other factors including immune cytokines , environmental factors or other molecular mechanisms had been proposed by many authors . showed that highly significant increase in the percentage of apoptotic peripheral blood mononuclear cells in active vitiligo patients versus stable vitiligo patients or controls . it is suggested that the imbalance in the oxidant - antioxidant system rather than oxidative stress might play such role in vitiligo . research at the molecular level has also demonstrated deficiency of antioxidant substances in vitiliginous skin . this leads to cytotoxic action of reactive oxygen species such as superoxide anion and hydroxyl radical that are generated by the ultraviolet damaged epidermis . a significant depletion of enzymatic and nonenzymatic antioxidants characterizes the epidermis of patients with active vitiligo , and represents a fingerprint of abnormal oxidative stress leading to impaired cofactor 6-tetrahydrobiopterin ( 6-bh4 ) levels , which inhibit melanogenesis interfering with the conversion of 1-phenylanine to tyrosine . melanins are colloidal pigments , known to have a high affinity for metal ions ; therefore , certain metal ions such as copper , zinc , and iron were found in high levels in pigmented tissues involved in melanin synthesis . as melanocyte degeneration was greater in active vitiligo , so there should be decreased zinc and copper in pigment tissues with their defective share in melanin synthesis reflecting their higher serum level . therefore , high serum zinc and copper levels were the result rather than the cause of the disease . zinc -2-glycoprotein ( zag ) , a plasma glycoprotein is a member of the immunoglobulin gene super family and has a three - dimensional structure that is highly homologous to major histocompatibility complex class i and ii molecules . b-16 recombinant human zag tumors have decreased levels of tyrosinase protein and minimal tyrosinase activity . for the first time , we pointed that there may be relation between the molecule of zag and vitiligo . in summary , the exact etiology and pathogenesis of this disease is not clear and the discovery of biological pathways of vitiligo pathogenesis will provide novel therapeutic and prophylactic targets for future approaches to the treatment and prevention of vitiligo . essentiality of zinc is related mainly to its function as the metal moiety of important enzymes . historically , for more than 3000 years , zinc salts such as zinc oxide or calamine were applied topically to facilitate wound healing . in the past 50 years since it was first shown to be essential for aspergillus niger by raulin in 1869 , zinc has been shown to be a cofactor of over 300 metalloenzymes and over 2000 transcription factors . in one study , we studied the efficacy of zinc in the treatment of vitiligo . in this study , we revealed that combination of zinc and topical steroid is more effective than the topical steroid alone . zinc constitutes less than 0.005% of total body weight , and present in all cells and is indispensable for the normal functions of cells , tissues and organs of the body . normal serum zinc level ranges from 70 to 180 gm/100 ml with the mean value of 120 22 gm/100 ml . no statistically significant difference in the mean values of serum zinc level was observed in relation to sex , age , race , food , habits , and diurnal variation . copper , zinc , selenium , and molybdenum are involved in many biochemical processes supporting life . the most important of these processes are cellular respiration , cellular utilization of oxygen , dna and rna reproduction , maintenance of cell membrane integrity , and sequestration of free radicals . zinc takes part in virtually all body functions from spermatogenesis to growth to abstract thought processes . zinc and vitamin zinc is integral part of a number of metalloenzymes necessary for normal protein , carbohydrate , lipid , and nucleic acid metabolism . copper , zinc , and selenium are involved in destruction of free radicals through cascading enzyme systems . the trace elements , copper and zinc , are linked together in cytosolic antioxidant- defense against reactive oxygen species , being constituents of copper , zinc - superoxide dismutase that catalyze the dismutation of reactive oxygen species to o2 or h2o2 . on the other hand , these trace elements , through this antioxidant - defense mechanism , may stimulate the protective antiapoptotic cellular stress - signaling cascades and thereby may stabilize cell proteins rendering them less prone to oxidation . decline in intracellular zinc below a critical threshold level may not only trigger pathways leadings to caspase activation but may also facilitate the process by which the caspases are activated . caspase families of enzymes are apoptotic regulators . zinc in combination with other micronutrients such as copper , cobalt , nickel , iron , manganese , and calcium plays an important role in the process of melanogenesis . they catalyze the rearrangement of dopachrome to form 5,6-dihydroxy indole-2 carboxylic acid ( dica ) and enhancement of eumelanin polymer formation from monomers . one of the importance of zinc is its involvement in gene transcription on several levels , via participation in histone deacetylation reactions and in actual transcription by factors possessing zinc - finger motifs . moreover , a high concentration of zinc is present in metallothioneins , a member of the shock proteins , expressed abundantly in the adenohypophysis . evidence exist that , in bovine pituitary glands , metallothioneins may be involved in the synthesis and release of -melanocyte stimulating hormone . hence , in a zinc - deficient state , melanogenesis may be impaired at the cellular level and the hypophyseal control mechanism on this process may be disturbed . bioavailability of dietary zinc varies widely between different sources , but is about 20% to 30% . zinc is distributed throughout the body with the highest concentrations found in muscle , bone , skin , eye , and prostatic fluids . it is primarily excreted in the feces , and regulation of fecal losses is important in zinc homoeostasis . zinc is effective in treatment of acne vulgaris,[375052 ] diaper dermatitis , seborrheic dermatitis , dandruff , psoriasis , androgenic alopecia , recalcitrant viral warts , old - world cutaneous leishmaniasis , and chronic cutaneous ulcers.[35052 ] success or failure of systemic treatment with zinc of acne vulgaris , or leg ulcer may be explained by understanding the metabolism of zinc in the organism interpreted as a feedback mechanism : treatment with zinc is only successful if zinc is not available in sufficient amount for the organism , i.e. , in case of primary zinc deficiency . several groups reported oral zinc sulfate to be effective in the treatment of severe acne and less efficient for the treatment of mild to moderate acne . when compared with systemic tetracycline treatment , zinc salts appear to be equal to or less effective than this class of antibiotics . one study reported a cure rate of over 80% of recalcitrant viral warts with oral zinc sulfate . yaghoobi et al . also showed the effectiveness of oral zinc sulfate in treatment of viral warts . contrary to popular belief , no association was found between systemic deficiency and alopecia areata . conversely , helmy et al . showed that serum zinc and copper levels were significantly higher in active vitiligo patients compared to controls . while serum zinc and copper levels insignificantly higher in active vitiligo versus stable disease and in stable patients versus controls . it appears that increased apoptosis of peripheral blood mononuclear cells in active vitiligo will lead to release of zinc and copper in serum ( as zinc is present maximally intracellular ) , resulting in increasing their serum levels significantly in active vitiligo . inamadar et al . reported appearance of vitiligo - like depigmented cutaneous lesions in two siblings with acrodermatitis entropathica who developed decreased serum zinc level due to discontinuation of zinc supplements . to compare probable pathogenesis related to vitiligo development in various studies and functional significance of zinc in humans , it appears zinc is important in preventing and treatment of vitiligo with following mechanisms :
zinc is a potential antiapoptotic factor . on the other hand , based on histological and some laboratory data , apoptosis of melanocytes has been suggested as a probable mechanism of vitiligo . hence , zinc , via preventing apoptosis of melanocytes may be able to control vitiligo.zinc and other trace elements are linked together in cytosolic antioxidant - defense system against reactive oxygen species . one of the theories regarding to pathogenesis of vitiligo is oxidative stress leading to destruction of melanocytes . hence , zinc can control vitiligo through inhibiting production of free radicals.zinc is one of trace elements that play an important role in the process of melanogenesis . hence , zinc may be effective in melanogenesis in vitiliginous lesions.some study , revealed significant increase in the percentage of apoptotic peripheral blood mononuclear cells in vitiligo . on the hand , accumulation of toxic compounds , altered cellular environment and infection zinc may affect in preventing vitiligo via destructing these probable environmental factors through prevention of these immunity - related cells.zinc deficiency is one of the many factors involved in the nonspecific suppression of cell - mediated immunity . hence , zinc may stimulate cell - mediated immunity against probable infective and other factors contributing in vitiligo development.zinc has a role in the synthesis and release of -melanocyte stimulating hormone in bovine . hence , zinc may be effective in melanogenesis in human via release of this hormone.zag regulates melanin production by normal and malignant melanocytes . we proposed that zinc via precipitating the zag in site of vitiligo patches may be effective in the treatment of vitiligo . zinc is a potential antiapoptotic factor . on the other hand , based on histological and some laboratory data , apoptosis of melanocytes has been suggested as a probable mechanism of vitiligo . hence , zinc , via preventing apoptosis of melanocytes may be able to control vitiligo . zinc and other trace elements are linked together in cytosolic antioxidant - defense system against reactive oxygen species . one of the theories regarding to pathogenesis of vitiligo is oxidative stress leading to destruction of melanocytes . zinc is one of trace elements that play an important role in the process of melanogenesis . some study , revealed significant increase in the percentage of apoptotic peripheral blood mononuclear cells in vitiligo . on the hand , accumulation of toxic compounds , altered cellular environment and infection can all contribute to vitiligo . zinc may affect in preventing vitiligo via destructing these probable environmental factors through prevention of these immunity - related cells . zinc deficiency is one of the many factors involved in the nonspecific suppression of cell - mediated immunity . hence , zinc may stimulate cell - mediated immunity against probable infective and other factors contributing in vitiligo development . zinc has a role in the synthesis and release of -melanocyte stimulating hormone in bovine . hence , zinc may be effective in melanogenesis in human via release of this hormone . we proposed that zinc via precipitating the zag in site of vitiligo patches may be effective in the treatment of vitiligo . | vitiligo is a common depigmenting skin disorder ( prevalence 0.1 - 2% ) , still represents a cause of stigmatization and quality of life impairment in a large population .
several theories on vitiligo etiopathogenesis have been suggested including in trauma , stress , and autoimmune and genetic predisposition , accumulation of toxic compounds , altered cellular environment , imbalance in the oxidant - antioxidant system , impaired melanocyte migration and/or proliferation , infection , and psychological factors .
zinc , as a trace element , has many vital functions in human .
it is antiapoptotic factor and needed as a cofactor for antioxidant defense system .
it plays an important role in the process of melanogenesis
. it may be effective in prevention and treatment of vitiligo via some mechanism .
herein , we suggested some probable protective mechanism for zinc in association with vitiligo . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
in 2008 , the government of india launched its flagship health insurance scheme for the poor . the rashtriya swasthya bima yojana ( rsby ) combines cutting edge technology with an unusual reliance on incentives to provide inpatient insurance coverage up to an annual sum of rs . presently , rsby is being implemented in 27 states across india , covering more than 27 million families . the comprehensive demand - side financing scheme offers a business plan that offers adequate incentives for all the key players of the program , i.e. the beneficiary , the insurance company , healthcare providers or hospitals , and the government . the scheme offers a front - end that starts from village - level enrollment so as to enable the beneficiary to receive a smart card almost at his doorstep , with the only eligibility criterion being his / her name has to be in the bpl list . rsby is an intervention that enables consumer choices among competing facilities through a demand - side subsidy . it aims to provide greater financial protection for poor households and foster better quality care through increased competition . the scheme allows for cashless hospitalization services at any of the empanelled hospitals - across the country for up to rs . the national interoperability feature is one of the many unique features that have been designed to be the most important asset of the scheme . additionally , the scheme is supposed to be paperless with features of online data transfer . the use of technology enables the hospitals to submit the claims online and the insurers are also supposed to make online payment to the hospitals . finally , a robust backend data management system ensures smooth flow of data from across india to both the state and central governments in real time that can ensure effective monitoring of the scheme . almost 4 years into the scheme , and despite having the potential of changing the access to healthcare scenario in the country , the scheme seems to be crippled with many problems being faced by all the stakeholders . the present paper is an attempt to get an insight to understand the bottlenecks faced by the service providers , with an overall goal to understand the issues in complete roll out of rsby and its successful implementation across country . rsby was implemented in the five pilot districts of gujarat in 2008 - 2009 . in the year 2009 - 2010 finally , in 2010 - 2011 , the entire rural below poverty line ( bpl ) populations of all the 26 districts of gujarat were covered under the rsby scheme . out of the total 29.69 lakh rural bpl families , the objective of the present study was to undertake the stakeholder analysis and understand the service providers perspective to rsby . the broader objective of the present study was to ensure effective implementation of rsby in india . the qualitative tool utilized in the present study was in - depth interview of service providers of rsby in patan district of gujarat state . patan district was the first district in gujarat where phase i of rsby was implemented . in - depth interview of practitioners associated with empanelled hospital in the district patan was conducted . a desk review of the 2010 claims in the district under rsby by the service provider was undertaken . selection of the practitioner / hospital was based on the claim rates under rsby for the district patan . it was attempted to interview service providers from different specialties like obstetrics and gynecology , surgery , and medicine . in all , 10 in - depth interviews were conducted . informed consent was obtained from the service providers and all efforts were taken to maintain the confidentiality of the respondents . the themes of the in - depth interview and open - ended interviews were based on the qualitative analysis of the published articles and studies published in journals , government website , case studies , and other available documents on rsby . audio recording of the interview was done wherever respondents permitted , or else the notebook method was followed to capture the responses . the review of the present status of rsby reflects a huge disparity in uptake of rsby at a national level ; while states like delhi and karnataka have less than 20% enrollment , himachal pradesh has more than 85% of its bpl population covered under the scheme . on one hand , the scheme is being extended to many newer states ; on the other hand , there are states like andhra pradesh who are not keen on adopting rsby as it has its own parallel scheme there are other states like karnataka , maharashtra , and tamil nadu which are offering rsby , along with piloting state - owned health insurance scheme that although in theory can complement rsby , can generate possibility of competition as well as corruption at grassroot level . the extreme example comes from rajasthan , which after initial experimentation , stopped implementing rsby altogether and has started its own scheme that covers hospitalization at government hospitals . in the states that are implementing rsby , including gujarat , the recent enrollment drive the annual mission to renew the policy by providing fresh smart cards has shown decline in the enrollment of the scheme . in gujarat , while the overall enrollment rate is 57% , the same in the nine districts where the scheme is in third year , the enrollment is only 48% . the enrollment in pioneer districts like banaskantha and patan remain abysmally low at 40% and 45% , respectively . stakeholders in rsby include members of the community , insurance company , and the service provider . while members of the community have been studied , the other two stakeholders have either never been studied or not been adequately studied . the present study reports qualitative observations from service providers perspective on rsby , which are discussed below . the scheme offers excellent business proposition to hospitals in terms of a ) untapped rural market that was not being able to access services and b ) ability to not refuse healthcare services to the needy eligible clients who otherwise could not have been catered to . the very same reasons can also make the scheme prone to excessive claims through fraudulent or unnecessary procedures . majority of the public and the private service providers of the studied district opined that even though theoretically the insurance companies abide to make efforts to enroll high percent of eligible members of the community and make the smart card available at the village level , they rarely undertake this step . hence , the awareness about its utility remains a challenge owing to a ) poor literacy , b ) ineffective information education and communication ( iec ) , and c ) mere power equation wherein hired staff of the insurance company along with government staff visits the selected portion of community and provides a card using sophisticated technology to the family . as per the views of the service providers , an ineffective iec around the utility was documented during the present study . this ineffective iec for the usefulness of smart card can originate from a ) the fact that the role of iec has been heavily relied upon by the insurance company , which possibly has wasted interest in not promoting the utility of the card to reduce claims , and b ) the added responsibility of iec on the ever - burdened peripheral health staff . one of the stakeholders at the district level narrated : the annual renewal is at times a cumbersome effort at all levels ; while insurance companies that are struggling with burning portfolios would be interested in enrolling less people , the beneficiaries may not even understand the need of renewing the card that they have got previous year . similarly , one of the providers opined , since the premium is per card issued, there is no incentive to ensure enrollment of all five members . it was also observed that the district authorities , with the only responsibility of enrollment and limited or no authority to ensure it , can have limited interest in enrollment drive vis - - vis other indicator - driven health programs . one of the respondents also believed , beneficiaries who have either experienced or have known about experience of refusal to enrollment or utilization can have lesser interest in utilization in the absence of a strong iec . while the scheme relies heavily on technology to ensure paperless cashless services , on field , it was observed in the present study that the claim settlements are done through physical documents . the physical documents like identity proof to verify age and gender at times become a necessity as the quality of smart card data is suboptimal . however , there are instances of insurance companies insisting on hard copies of every case paper to be sent to state headquarters , in addition to the online transfers . one of the empanelled service providers narrated : without submitting the hard copies , original or xerox , you can not receive your legitimate claims , rsby being a paperless scheme is only on paper . another important issue is of delay in payment , as narrated by majority of empanelled service providers , which was of inter - insurance claim settlement . although the draft mou between an insurance company and state nodal agency ( sna ) indicates that all the payments shall be made electronically within 21 days of the receipt of electronic claim documents , this can not be followed as there are more than one insurance company operating in the state . even the state government in one of the presentations admitted that more than half of the claims in rsby have been settled beyond 4 weeks time . this delay is peculiar in inter - insurance company claim settlements . as per the guidelines for inter insurance company claim settlement , in case of usage of cards in a hospital not empanelled by the issuing insurance company or in a district where the issuing insurance company is not operating the district server , it is the responsibility of the insurance company operating the district server to ensure that the transaction data reaches the nodal person of the concerned insurance company . given that the draining of patients from one district to another is very common and if a different insurer is operating in the neighboring district , the inter - insurer payment transfer becomes cumbersome . there have been instances of delays and in some cases denials altogether after a few months . with these happening , the source hospitals prefer to avoid the clients from destination districts , and on the other hand , the destination insurance companies are found to be influencing the choices of the clients by encouraging them to take services of the respective district . this kind of practice defeats the very national portability purpose of the scheme . one of the observations as narrated by the service providers was a constant threat of being suspended from the list / de - empanelment of the provider by the insurance company . one of the providers narrated : it is just one email that makes you from being empanelled to de - empanelled . the service provider further added : insurance company is always skeptical about work load , they feel the more you work , the more claim you generate , which is essentially false . the service providers opined thus : insurance companies are the happiest if you generate no claim , but if you work , you are doing malpractice as companies have to pay the claims . the implementation of rsby is heavily dependent on the smooth coordination amongst insurance company , the state and district administration , and the hospitals . as perceived by many , one of the common features of any health insurance scheme , more so in case of a scheme like rsby , is supply - side moral hazards . the hospitals theoretically have short - term incentive to undertake malpractices to generate more business out of the scheme . in the light of various complaints of such fraudulent practices , rsby issued advisory for de - empanelment of hospitals in may 2011 . according to this , there is a three step procedure for de - listing the empanelled hospital , viz . a ) putting the hospital on watchlist at any doubt on the performance of a hospital , b ) suspension of the hospital , and c ) detailed investigation and action by insurance company , which can lead to de - empanelment . power to the insurance company to suspend the empanelment of any hospital , followed by intimation to sna and a formal investigation . the process of investigation , i.e. from suspension to result of investigation , can take up to 30 days , during which the said hospital remains suspended and no rsby transaction is valid . the hospital , on the other hand , has been given the option to approach the grievance redressal committee for the redressal , which will take a final view within 30 days of the receipt of representation . the hospital continues to be de - empanelled till the time a final view is taken by the grievance redressal committee . the procedure for suspension can be initiated solely by the insurance company , and the investigation as well as de - empanelment involves interaction only between insurance company and state authorities ; however , the hospitals are left to start their redressal from the district - level agencies . this arrangement can create power imbalance as insurance company can unilaterally generate suspension that can go unchallenged for at least 30 days . while such a provision is indeed necessary to curb malpractices by certain providers , it has the potential to be used as a vehicle to ensure cream skimming by insurance providers by at least initiating 30-day suspension against hospitals that are not conducive for profit maximizing . such cases can be on the rise if the sna colludes with insurance company , which in the long run can affect the virtuous cycle of better enrollment better utilization better renewal . district patan has witnessed change in the insurance company in recent years ; many of the service providers narrated instances of negotiation of settlement of claim to a much lesser amount than actual by the outgoing insurance company and the service provider has to accept the formula of negotiation fearing a loss of entire claims , and hence settle for no profit or even low loss in the claimed amount . the scheme offers excellent business proposition to hospitals in terms of a ) untapped rural market that was not being able to access services and b ) ability to not refuse healthcare services to the needy eligible clients who otherwise could not have been catered to . the very same reasons can also make the scheme prone to excessive claims through fraudulent or unnecessary procedures . majority of the public and the private service providers of the studied district opined that even though theoretically the insurance companies abide to make efforts to enroll high percent of eligible members of the community and make the smart card available at the village level , they rarely undertake this step . hence , the awareness about its utility remains a challenge owing to a ) poor literacy , b ) ineffective information education and communication ( iec ) , and c ) mere power equation wherein hired staff of the insurance company along with government staff visits the selected portion of community and provides a card using sophisticated technology to the family . as per the views of the service providers , an ineffective iec around the utility was documented during the present study . this ineffective iec for the usefulness of smart card can originate from a ) the fact that the role of iec has been heavily relied upon by the insurance company , which possibly has wasted interest in not promoting the utility of the card to reduce claims , and b ) the added responsibility of iec on the ever - burdened peripheral health staff . one of the stakeholders at the district level narrated : the annual renewal is at times a cumbersome effort at all levels ; while insurance companies that are struggling with burning portfolios would be interested in enrolling less people , the beneficiaries may not even understand the need of renewing the card that they have got previous year . similarly , one of the providers opined , since the premium is per card issued, there is no incentive to ensure enrollment of all five members . it was also observed that the district authorities , with the only responsibility of enrollment and limited or no authority to ensure it , can have limited interest in enrollment drive vis - - vis other indicator - driven health programs . one of the respondents also believed , beneficiaries who have either experienced or have known about experience of refusal to enrollment or utilization can have lesser interest in utilization in the absence of a strong iec . while the scheme relies heavily on technology to ensure paperless cashless services , on field , it was observed in the present study that the claim settlements are done through physical documents . the physical documents like identity proof to verify age and gender at times become a necessity as the quality of smart card data is suboptimal . however , there are instances of insurance companies insisting on hard copies of every case paper to be sent to state headquarters , in addition to the online transfers . one of the empanelled service providers narrated : without submitting the hard copies , original or xerox , you can not receive your legitimate claims , rsby being a paperless scheme is only on paper . another important issue is of delay in payment , as narrated by majority of empanelled service providers , which was of inter - insurance claim settlement . although the draft mou between an insurance company and state nodal agency ( sna ) indicates that all the payments shall be made electronically within 21 days of the receipt of electronic claim documents , this can not be followed as there are more than one insurance company operating in the state . even the state government in one of the presentations admitted that more than half of the claims in rsby have been settled beyond 4 weeks time . this delay is peculiar in inter - insurance company claim settlements . as per the guidelines for inter insurance company claim settlement , in case of usage of cards in a hospital not empanelled by the issuing insurance company or in a district where the issuing insurance company is not operating the district server , it is the responsibility of the insurance company operating the district server to ensure that the transaction data reaches the nodal person of the concerned insurance company . given that the draining of patients from one district to another is very common and if a different insurer is operating in the neighboring district , the inter - insurer payment transfer becomes cumbersome . there have been instances of delays and in some cases denials altogether after a few months . with these happening , the source hospitals prefer to avoid the clients from destination districts , and on the other hand , the destination insurance companies are found to be influencing the choices of the clients by encouraging them to take services of the respective district . this kind of practice defeats the very national portability purpose of the scheme . one of the observations as narrated by the service providers was a constant threat of being suspended from the list / de - empanelment of the provider by the insurance company . one of the providers narrated : it is just one email that makes you from being empanelled to de - empanelled . the service provider further added : insurance company is always skeptical about work load , they feel the more you work , the more claim you generate , which is essentially false . the service providers opined thus : insurance companies are the happiest if you generate no claim , but if you work , you are doing malpractice as companies have to pay the claims . the implementation of rsby is heavily dependent on the smooth coordination amongst insurance company , the state and district administration , and the hospitals . as perceived by many , one of the common features of any health insurance scheme , more so in case of a scheme like rsby , is supply - side moral hazards . the hospitals theoretically have short - term incentive to undertake malpractices to generate more business out of the scheme . in the light of various complaints of such fraudulent practices , rsby issued advisory for de - empanelment of hospitals in may 2011 . according to this , there is a three step procedure for de - listing the empanelled hospital , viz . a ) putting the hospital on watchlist at any doubt on the performance of a hospital , b ) suspension of the hospital , and c ) detailed investigation and action by insurance company , which can lead to de - empanelment . power to the insurance company to suspend the empanelment of any hospital , followed by intimation to sna and a formal investigation . the process of investigation , i.e. from suspension to result of investigation , can take up to 30 days , during which the said hospital remains suspended and no rsby transaction is valid . the hospital , on the other hand , has been given the option to approach the grievance redressal committee for the redressal , which will take a final view within 30 days of the receipt of representation . the hospital continues to be de - empanelled till the time a final view is taken by the grievance redressal committee . the procedure for suspension can be initiated solely by the insurance company , and the investigation as well as de - empanelment involves interaction only between insurance company and state authorities ; however , the hospitals are left to start their redressal from the district - level agencies . this arrangement can create power imbalance as insurance company can unilaterally generate suspension that can go unchallenged for at least 30 days . while such a provision is indeed necessary to curb malpractices by certain providers , it has the potential to be used as a vehicle to ensure cream skimming by insurance providers by at least initiating 30-day suspension against hospitals that are not conducive for profit maximizing . such cases can be on the rise if the sna colludes with insurance company , which in the long run can affect the virtuous cycle of better enrollment better utilization better renewal . district patan has witnessed change in the insurance company in recent years ; many of the service providers narrated instances of negotiation of settlement of claim to a much lesser amount than actual by the outgoing insurance company and the service provider has to accept the formula of negotiation fearing a loss of entire claims , and hence settle for no profit or even low loss in the claimed amount . it is emerging as a boon for millions of poor patients who were not able to access healthcare due to financial inaccessibility . while the scheme is being expanded nationwide and with declining enrollment in recent years , there is a need to set and monitor control knobs to ensure smooth functioning of the scheme at all levels . three such knobs are already discussed in mous between various state government and insurance companies . to ensure effective enrollment , there can be a penalty for insurance company for not enrolling sufficient proportion of bpl families and also members within families ; calculation of premium rate can be directly linked to this proportion . the promotion role of insurance company needs to be critically looked at ; instead of leaving the iec job entirely to the insurance company , there can be a mechanism wherein the insurer allocates some percentage of the premium for the iec / bcc activity and the fund can be passed onto a neutral organization which can promote the scheme . similarly , instead of leaving the hospital insurance company to deal with each other , a tripartite mou should be arranged wherein in case of grievances around suspensions , genuine hospitals can have fair deal as well . there definitely remains a greater and more serious role of government , which ranges from ownership to larger issue of governance . however , like any other government - sponsored schemes , there are several limitations that the scheme is struggling with . there are also loopholes that have left ample opportunities for collusions between various players , and thus scope for corruption . it is imperative that genuine interests of all stakeholders are addressed and possibilities of corruption are minimized simultaneously . india neither need wait nor can afford another comptroller and auditor general ( cag ) report indicating irregularities in another of its nationwide flagship health scheme . | context : government of india in 2008 , launched its flagship health insurance scheme for the poor .
the rashtriya swasthya bima yojana ( rsby ) combines cutting edge technology with an unusual reliance on incentives to provide inpatient insurance coverage .
the scheme allows for cashless hospitalization services at any of the empaneled hospitals .
stakeholders in rsby include members of the community , insurance company and the service provider.aim:the study manuscript is an attempt to get an insight to understand the bottle necks in faced by the service providers with an overall goal to understand issues in complete roll out of rsby and its successful implementation across country .
it was conducted to undertake the stakeholder analysis and understand the service providers perspective to rsby.setting and design : the present study was conducted in the patan district of gujarat state .
qualitative tool mainly in - depth interview of service providers of rsby in patan district of gujarat state was utilized for the data collection.results and conclusion : service providers opined an ineffective iec around the utility of the rsby service in the community . in spite of the claim that scheme relies heavily on technology to ensure paperless cashless services , on field ,
it was observed in the present study that the claim settlements are done through physical documents .
the service providers had a perceived threat of being suspended from the list / de - empanelment of the provider by the insurance company .
there is an urgent need for improved and effective iec for the service and possibilities of an arrangement for to settle the case of grievances around suspensions ao that genuine hospitals can have fair deal as well .
there definitely remains a greater and more serious role of government , which ranges from ownership to larger issue of governance . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the prevalence rate of juvenile obesity
has broad and serious health implications , since obesity can increase the risks of type 2
diabetic mellitus ( t2 dm ) , hypertension , cardiovascular disease ( cvd ) , sleep apnea , and
cancer1 . abnormal glucose tolerance ,
disturbed glucose tolerance , and t2 dm have been identified as increasing the risk of
cognitive impairment , and are been known to induce cognitive damage , both of which are
early - stage symptoms of alzheimer s disease and dementia2 . diabetes mellitus is also known to have adverse effects on the
peripheral nervous system leading to detrimental changes in brain structure and other
neurophysiological traits3 . brain - derived neurotrophic factor ( bdnf ) is generated and distributed from platelets in the
peripheral and central nervous systems , endotheliocytes , smooth muscle , immunocytes , and
skeletal muscle . it is a nerve growth and positively contributes to neurogenesis ,
neurodegeneration , and hippocampal neural plasticity , and enhances memory and learning4 . a lower than normal bdnf level has been
observed among individuals with obesity and t2dm5 , and this has been reported to bring about neuropsychosis , severe
depression , and alzheimer s disease6 , and
has also recently been found to affect metabolism in the skeletal muscles6 . inflammatory factors are highly prevalent among individuals with obesity , t2 dm , and
metabolic syndrome , and such inflammatory factors are associated with obesity and
inactivity . high levels of inflammatory factors have also been identified as an independent
factor in the progress of t2 dm and cvd7 . research on regular exercise and related neurotrophic factors has determined that exercise
can be divided between that which greatly increases the resting bdnf level8,9,10 and that which does not11,12,13 , but the findings are not conclusive due
to variations in exercise mode and duration . studied the effects of regular exercise on inflammatory factors by
observing middle - aged men and women over the course of 12 months during which they performed
60 minutes of exercise once a week . there results show that as body mass index ( bmi )
decreased with progression of the exercise training , serum il-6 and c - reactive protein ( crp )
levels at rest decreased correspondingly . christiansen et al.7 observed men and women over 12 weeks while they performed 6075
minutes of endurance training 3 times a week , accompanied by diet control . they also
reported a decrease in resting levels of il-6 and monocyte chemoattractant protein-1 ( mcp-1 )
after training . in addition , crp levels increased after one intense bout of exercise15 , and a regular exercise training group had
lower levels of crp than a control group16 . although , the relationship between neurotrophic and inflammatory factors and regular
exercise has been investigated in recent research , the debate on whether regular exercise
can alter the resting bdnf level and inflammatory factor levels continues , and existing
research on the subject mainly concerns adults and the elderly . insufficient research has
been conducted on juvenile obesity and t2 dm , which are rapidly increasing . as adolescence
marks an important period of learning and development , the investigation of the effects of
regular exercise on juvenile obesity and t2 dm is indispensable . in this study , we examined the effects of exercise by measuring the resting levels of
neurotrophic factors ( bdnf , ngf , and tropomyosin receptor kinase b [ trkb ] ) , and inflammatory
factors ( il-6 , tumor necrosis factor [ tnf- ] , and crp ) of juveniles assigned to an obesity
group ( og , n = 8) , t2 dm group ( tg , n = 7 ) , and control group ( cg , n = 11 ) following 12 weeks
of aerobic exercise 3 times a week . we hypothesized that juveniles with obesity and t2 dm would have lower resting bdnf levels ,
and that 12 weeks of regular exercise would result in increased levels of resting
neurotrophic factors and decreased levels of inflammatory factors . this experiment initially recruited originally began with 45 teenagers who were assigned to
the og ( n = 15 ) , tg ( n = 15 ) , and cg ( n = 15 ) . however , due to withdrawals , only 26
teenagers in the og ( n = 8 , boys = 4 , girls = 4 , age ; 16.3 0.91 ) , tg ( n = 7 , boys = 2 ,
girls = 5 , age ; 15.5 2.14 ) , and cg ( n = 11 , boys = 11 , age ; 16.4 1.36 ) completed the 12
weeks program . all participants were between the ages of 13 and 19 years , and there were 17
boys and 9 girls . the og teenagers were selected based on the 1998 standards for children
and juvenile growth of the korean pediatrics society : bmi > 95th percentile , or obesity
index > 120% . the tg teenagers were selected from among d university hospital out - patients
whose 2-hour glucose tolerance test was 140 mg / dl blood sugar 200 mg / dl , and who had no
other complicating diseases . the cg was created by age - matching with the tg and og
juveniles . the
study was approved by the d university hospital institutional review board ( irb ) and the
subjects were included after medical examination and diagnosis by medical specialists .
additionally , signed consent forms were obtained from the guardians of all participants
prior to the commencement of the study . height , weight , and bmi were measured as the physical characteristics , and body fat
percentage was measured using an impedance analyzer ( venus 5.5 , jawon medical , gyeongsan ,
korea ) . changes in baseline characteristics following 12 weeks of aerobic exercise
trainingvariablegroupbaseline12 weeksage(yrs)cg16.451.36og16.370.91tg15.572.14height(cm)cg171.115.17171.34*5.07og171.226.31172.255.93tg161.157.70161.277.86weight(kg)cg65.9214.5866.1014.84og80.207.5073.06*10.91tg61.8413.6862.6114.23bmi(kg / m)cg22.353.9422.374.07og27.472.5124.70*3.24tg23.724.4724.074.52body fat(%)cg16.566.9616.847.18og31.775.1925.52*5.52tg28.204.7727.187.73vo2max(ml kg
min)cg33.283.7633.973.44og29.205.4533.91*4.60tg28.604.8832.86*4.56means sd . cg , control group ; og , obesity group ; tg , t2 dm group . a , significant difference
( p<0.05 ) cg vs. og ; b , significant difference og vs. tg ; c , significant difference
cg vs. tg . a submaximal oxygen consumption exercise test ( 85% vo2submax test )
was conducted before the start of the exercise training . all participants were tested on a
quinton q65 treadmill ( quinton co. , usa ) in a laboratory with 5055% humidity and a
temperature 2425 c . the subjects wore a polar system ( polar electro , kempele , finland ) ,
and were measured using a breath - by - breath test . each respiration was automatically measured
for ventilatory volume , oxygen consumption , carbon dioxide emission , respiratory exchange
ratio ( rer ) , and heart rate ( hr ) . cg , control group ; og , obesity group ; tg , t2 dm group . a , significant difference
( p<0.05 ) cg vs. og ; b , significant difference og vs. tg ; c , significant difference
cg vs. tg the exercise test was conducted using a treadmill exercise test and the bruce protocol17 . this method involves steadily increasing
the exercise level until the target heart rate ( thr ) reaches 85% of the maximum oxygen
consumption ( vo2max ) . no subjects exhibited cardiac abnormalities or high blood
pressure before reaching the thr . to prevent accidents , participants were asked to
subjectively identify the intensity of exercise on the borg scale of rate of perceived
exertion18 . aerobic exercise was conducted for a total of 4060 minutes per session , 3 sessions a week ,
for 12 weeks . participants attained 50% of their oxygen consumption as measured by the
vo2max test . between week 1 and week 4 , they engaged in 3040 minutes
walking / running aerobic exercise on a school field under the instruction of a professional
trainer while wearing a polar system at a calculated heart rate recovery ( hrr ) of 50%
vo2max . between week 5 and week 12 , the participants exercised in a similar
manner but for 4050 minutes at a hrr equivalent to 60% of vo2max . each session
was preceded and followed by a 5-minute warm up and cool down , respectively . samples were
centrifuged at 3,000 rpm for 10 minutes , stored at 80 c , and directly analyzed . the biochemical analyses of serum il-6 , tnf- , and ngf were performed using enzyme - linked
immunosorbent assays for quantitative detection of human il-6 , tnf- ( ebioscience , vienna ,
austria ) and ngf ( abcam , ma , usa ) . all factors were quantified using polyclonal antibodies
that recognized native human il-6 , tnf- , or ngf and a series of plates containing wells
coated with predetermined amounts of recombinant human il-6 , tnf- , or ngf . serum bdnf levels were analyzed with an r & d system ( minneapolis , minn . , usa ) kit and
serum trkb levels were analyzed with a sino biological kit ( sino biological inc , beijing ,
china ) . then , substrate solution and
streptavidin - hrp were added , the reaction was terminated with a stop solution , and the
results obtained by measuring the optical density at 450 nm . serum crp was analyzed with an automatic analyzer ( adipogen , seoul , korea ) using latex
cohesion turbidimetry with a crp(ii ) x2 latex reagent . for all data , the mean and standard deviation were calculated using spss for windows ver . repeated measures two - way anova was used to control for
differences between groups , test periods , physical characteristics , and biochemical blood
component changes caused by exercise , using scheffe s method for the post hoc test . subjects physical characteristics were recorded at baseline and after the 12 weeks of
regular aerobic exercise . there were no significant differences in weight among the groups , but
there was a difference between the test periods . there was an interaction effect between
group and time ( f = 12.779 , p<0.05 ) , and further analysis of the interaction effect
revealed a significant weight reduction ( p<0.05 ) in the og after the 12 weeks of regular
exercise . there were no significant differences in bmi among the groups , but there was a significant
difference between the test periods ( f = 8.011 , p<0.05 ) . there was an interaction effect
between group and time ( f = 12.685 , p<0.05 ) , and further analysis of the interaction
effect revealed a significant bmi reduction ( p<0.05 ) in the og after the 12 weeks of
regular exercise . there were significant differences in body fat percentage among the groups ( f = 10.726 ,
p<0.05 ) and test periods ( f = 16.820 , p<0.05 ) . there was an interaction effect between
group and time ( f = 12.898 , p<0.05 ) , and further analysis of the interaction effect
revealed a significant body fat percentage reduction ( p<0.05 ) in the og after the 12
weeks of regular exercise . there were no significant differences in the vo2max among groups , but the
vo2max between the test periods differed significantly ( f = 19.348 , p<0.05 ) . there was an interaction effect between group and time ( f = 3.441 , p<0.05 ) , and further
analysis revealed a significant vo2max increase ( p<0.05 ) in the og and tg
after the 12 weeks of regular exercise . the serum bdnf , ngf , and trkb levels were recorded at baseline and after the 12 weeks of
regular aerobic exercise . changes of the serum neurotrophic factors and inflammatory factors following 12
weeks of aerobic exercise training.variablegroupbaseline12-weekbdnf(pg/ml)cg21,178.33 4,306.1420,670.405,021.47og8,003.796,559.1019,617.53*8,098.60tg25,136.143,814.1625,104.585,389.95ngf(pg / ml)cg5.142.356.211.78og8.838.6630.5544.04tg5.583.337.808.86trkb(pg / ml)cg182.7132.07187.1165.44og123.2050.80146.7729.98tg112.0240.30143.8556.52il-6(pg / ml)cg1.422.851.472.84og0.900.421.521.42tg0.920.720.610.40tnf-(pg / ml)cg3.351.748.1213.33og4.271.733.711.89tg3.181.772.930.84crp(mg / dl)cg0.100.060.220.45og0.150.130.130.19tg0.060.050.080.09means sd . cg , control group ; og , obesity group ; tg , t2 dm group . a , significant difference
( p<0.05 ) cg vs. og ; b , significant difference og vs. tg ; c , significant difference
cg vs. tg . there was a significant difference in bdnf levels among the groups ( f =
13.673 , p<0.05 ) and test periods ( f = 6.338 , p<0.05 ) . there was an interaction effect
between group and test period , and further analysis of the interaction effect revealed a
significant bdnf increase ( p<0.05 ) in the og after the 12 weeks of regular exercise . cg , control group ; og , obesity group ; tg , t2 dm group . a , significant difference
( p<0.05 ) cg vs. og ; b , significant difference og vs. tg ; c , significant difference
cg vs. tg there were no significant difference in ngf levels among the groups or test periods , and
there was no interaction effect between group and test period . there was a significant difference in trkb levels among the groups ( f = 6.400 , p<0.05 ) ,
but there was no interaction effect between group and test period . the 12 weeks regular aerobic exercise resulted in little changes in the inflammatory
factors ( serum il-6 , tnf- , crp ) . there were no significant differences or interaction
effects of il-6 , tnf- , and crp among groups , or between test periods , or group and test
period . we aimed to test whether 12 weeks of regular aerobic exercise would have a positive effect
on neurotrophic and inflammatory factors . as described earlier , bdnf is mainly responsible for nerve survival , growth , maintenance ,
learning , and memory . patients with alzheimer s disease exhibit low levels of bdnf
expression in the hippocampus19 and low
serum bdnf20 . similarly , patients with
parkinson s disease and huntington s disease have low levels of hippocampal bdnf21 , and patients with depression have low
levels of serum bdnf22 . this experiment
revealed that the resting bdnf level at baseline before the 12 weeks of aerobic exercise was
significantly lower ( p<0.05 ) in the og than in the cg . this result is in agreement with
previous research5 , which has reported low
levels of bdnf among people with obesity and t2 dm . it also agrees with the findings of
previous research conducted on adults , which revealed that bdnf plays a very important role
in controlling energy homeostasis and weight23 , 24 , and has an inverse relation with weight
and age25 . it is also was interesting to note that the tg and cg had similar resting bdnf levels ,
i.e. , the resting bdnf levels of the tg teenagers of normal weight were not greatly
depressed . this raises the question as to whether the resting bdnf levels of juveniles with
t2 dm have a greater effect on body composition , such as weight , than the disease itself . another interesting point is that after 12 weeks of regular aerobic exercise , the resting
bdnf level of the og increased to 19,617 pg / ml , a level close to the adult normal range of
22,600 pg / ml25 . this was a significant
increase ( p<0.05 ) and it was only observed in the og ( table 2 ) . we suggest that there was no significant increase in the
resting bdnf level after the 12 weeks of exercise in the tg , because the exercise did not
result in significant changes in body composition ( weight , bmi , body fat percentage ) . trkb is a bdnf receptor that is expressed abundantly and widely in the brain26 , and ngf plays an important role in the
sustenance of sympathetic and sensory neurons as well as biological activities , including
cell growth27 . schulz et al.13 studied the effects of 8 weeks of aerobic
ergometry exercise on patients with multiple sclerosis ( ms ) , and reported no significant
difference in the ngf level of the exercise group . bansi et al.28 conducted 3 weeks of regular exercise ( aquatic vs.
overland ) for middle - aged patients with multiple sclerosis and also failed to significantly
alter resting ngf levels . while our present findings did not reveal a significant difference
in resting ngf levels in the og , compared to the cg and tg a
previous study of 146 adults29 , also
reported the ngf levels of obese subjects were higher than those of normal weight subjects . although trkb levels increased in the og and tg following the 12 weeks of aerobic exercise ,
there was no significant difference between the two groups . however , comparison of the
resting trkb levels of the cg with og and tg at baseline revealed that og and tg had
significantly lower resting trkb levels than cg , and we consider this proof of them having
decreased bdnf receptor levels , as trkb is a bdnf receptor ( table 2 ) . one of the limitations of this experiment was that while there have been many studies of
neurotrophic factor bdnf , few human studies of ngf and trkb have been conducted . this
renders comparative analysis difficult , and much more research concerning ngf and trkb
levels will need to be conducted in the future . high levels of inflammatory cytokines are observed in patients with obesity , t2 dm , and ms ,
and such high levels of inflammatory factors are an independent factor in the development of
cvd7 . additionally , arteriosclerosis and
coronary artery disease are associated with increased levels of il-6 , crp , and tnf-30 , 31 , and higher il-6 and crp have been observed in metabolic disabilities
such as t2 dm , ms , and obesity31 , 32 . il-6 , a typical inflammatory factor , is a multifunctional cytokine responsible for
pleiotropism in immunomodulation33 , and
was recently reported to be a myokine , involved in energy metabolism in skeletal muscle34 . crp also serves as an indicator of
systemic inflammatory response . an increased crp level in obese patients is explained by
il-6 originating from adipose tissue , and crp levels are significantly decreased by aerobic
exercise and diet35 . christiansen et
al.7 conducted a study of men and women
engaged in aerobic exercise lasting 6075 minutes per session , 3 sessions a week for 12
weeks . they noted that there were no significant reductions in il-6 , il-15 , il-18 , or mcp-1
without dieting or significant reductions in weight . kohut et al.36 studied patients with metabolic disease who showed no
reduction in weight over the course of 10 months by comparing a group that did aerobic
exercise for 45 minutes per session , 3 sessions a week , with a resistance / flexibility
training group . they found that the aerobic exercise group showed significant reductions in
crp , l-6 , and il-18 . additionally , a study of middle - aged men and women performing endurance
exercise , consisting of one 60-minute training session per week for 12 months , reported a
significant decrease in serum crp levels following a reduction in bmi14 . to date , reducing circulating inflammatory factors has been proposed to be associated with
weight reduction37 and adipose tissue
reduction36 , 38 , 39 , but our 12 weeks of
exercise failed to result in a significant reduction in inflammatory factors ( especially
il-6 ) in the og , despite a significant reduction in weight , body fat percentage , and bmi . we
believe that , compared to the 1012-month study periods of other experiments14 , 36
that saw reductions in il-6 through regular training , the exercise period of this study was
too short to reduce resting il-6 levels . another interesting point is that this study
recorded very low levels of il-6 ( 0.91.4 pg / ml ) compared to the average resting il-6 level
( 3.45.0 pg / ml ) reported by previous studies of adults ( healthy people in their 30s and
middle - aged patients with t2 dm ) and the elderly over 64 years of age7 , 14 , 36 , 40 . as the immune
and inflammatory systems progressively change with age41 , perhaps the subjects youth may have been a factor influencing
their lower il-6 levels , despite their obesity and t2 dm . the resting il-6 levels of the
teenagers were also close to the 1.11.6 pg / ml of young adults aged between 20 and 30 years
with simple obesity42 . previous research related to exercise has reported significant reductions in inflammatory
factors ( il-6 , tnf- , hs - crp ) following long - term training lasting 1012 months14 , 36
rather than a 12-week period42 , 43 . the only exception was an 8-week aerobic training and
diet restriction study of a group of children with obesity . that study reported a
significant reduction in inflammatory markers ( il-6 , tnf- , hs - crp ) , and it argued that the
significant changes in body composition ( body weight , bmi , body fat percentage ) , induced by
exercise and diet restriction , had a strong effect on the inflammatory factors44 . we also observed slight changes in tnf- and crp levels after the 12 weeks of aerobic
exercise , but did not find significant differences , in agreement with previous studies40 , 42 , 43 . taken together , our present results and
those of other studies40 , 45 indicate that exercise has limited or no effect on
inflammatory markers . while many studies of exercise effects have been conducted using adults and the elderly ,
comparisons with our results were limited by the lack of researches involving juveniles
between the ages of 13 and 17 years , and differences in training period , exercise intensity ,
exercise frequency , and sample size used in the other studies of bdnf and inflammatory
markers . the juveniles with obesity and t2 dm exhibited reduced levels of resting neurotrophic
factors ( bdnf , trkb ) at baseline , but after the 12 weeks aerobic exercise , there was a
significant increase in the bdnf level of the og . however , these findings are limited , and
the effects of regular exercise on neurotrophic factors and inflammatory factors in juvenile
obesity and t2 dm were not conclusively proven . | [ purpose ] the purpose of this study was to investigate the effects of 12 weeks regular
aerobic exercise on brain - derived neurotrophic factor ( bdnf ) and inflammatory factors in
juvenile obesity and type 2 diabetes mellitus ( t2 dm ) .
obesity and t2 dm , typically common
among adults , have recently become more prevalent in the korean juvenile population ,
affecting not only their lipid profiles and oxidant stress levels , but also their bdnf and
inflammatory factor levels .
[ subjects ] this study enrolled 26 juveniles ( boys = 15 , girls
= 9 ) who were assigned to a control group ( cg , n = 11 ) , obesity group ( og , n = 8) , or t2 dm
group ( tg , n = 7 ) .
[ methods ] the outcome of a 4060-minute aerobic exercise session that
took place three times per week for 12 weeks at a maximum oxygen intake
( vo2max ) of 50~60% was investigated .
[ results ] the exercise resulted in a
significant reduction in the resting serum bdnf and trkb levels ( baseline ) among juveniles
in the og and tg as compared to those in the cg .
additionally , the 12 weeks of regular
aerobic exercise led to significant reductions in body weight , body fat percentage , and
body mass index in the og and a significant increase of vo2max in the og and
tg . however , no significant differences in serum ngf or inflammatory factors were found
among the three groups . there was a significant increase in resting serum bdnf levels
following the 12 weeks regular exercise only in the og .
[ conclusion ] while 12 weeks of
regular aerobic exercise had a positive effect on body composition , and increased bdnf
levels of juveniles in the og , it did not affect the inflammatory factor levels and had no
effect on the tg . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
hepatic arterial pseudoaneurysms ( hap ) are uncommon and usually occur secondary to accidental or iatrogenic trauma . the iatrogenic causes include percutaneous procedures ( liver biopsy , biliary drainage ) , laparoscopic cholecystectomy , and open surgeries ( liver transplantation ) . pseudoaneurysm may also result from adjacent inflammation and infection ( as in cholecystitis , pancreatitis , and abscess ) or from primary vascular causes such as vasculitis . the clinical presentation is usually in the form of upper gastrointestinal tract ( git ) bleeding ( as a result of pseudoaneurysm rupturing into the biliary tree or gall bladder ) . the bleeding may also be intraperitoneal ( leading to hemoperitoneum ) or into the adjacent liver abscess ( leading to blood within drain content ) . hap as a complication of liver abscess are rare , and multiple pseudoaneurysms associated with cholangitic abscesses have not been reported in the literature . we present a case of severe acute pancreatitis with stent block cholangitis and multiple cholangitic abscesses that developed multiple hap . the role of computed tomography angiography ( cta ) and successful radiological interventional management is highlighted in the article . a 33-year - old male patient presented to the emergency department with epigastric pain , fever , and vomiting . investigations revealed neutrophilic leukocytosis ( white blood cell count of 28,400/mm and 90% neutrophils ) , raised serum alkaline phosphatase ( 2833 iu ) , and elevated total and conjugated bilirubin ( 11.5 and 9.2 mg / dl , respectively ) . endoscopic retrograde cholangiopancreatography ( ercp ) and common bile duct ( cbd ) stenting were done for cholangitis 2 months before . laparoscopic pancreatic necrosectomy , abscess drainage , and cholecystectomy were done 1 month prior to his present admission . the patient continued to have high - grade spiking fever and raised neutrophil counts in the post - operative period , and ultrasonography ( usg ) showed persistent peripancreatic fluid collections . the patient was managed with multiple pigtail drainages , but there was no symptomatic relief ; therefore , the patient was referred to our institute . usg revealed multiple hypoechoic lesions in both lobes of liver , few showing internal debris , with the largest in the left lobe ( measuring 9 8.5 cm ) , suggestive of abscesses . usg - guided 10f pigtail catheter was inserted into the large left lobe abscess , which drained foul - smelling purulent material . on the third day of admission , altered blood was seen draining from the pigtail catheter , which persisted for next 48 h. even though the hemoglobin level of patient did not show any significant fall ( 8.4 g / dl on the day of admission and 8.2 g / dl subsequently ) and the patient was hemodynamically stable , cta was planned in view of persistent blood in the catheter drain . cta revealed three contrast - filled outpouchings from hepatic arteries , which followed the attenuation of aorta in venous phase as well , suggestive of pseudoaneurysms [ figure 1 ] . one of these pseudoaneurysms was arising from the left hepatic artery ( lateral branch ) and was located just medial to the pigtail catheter . two other pseudoaneurysms were seen to arise from the right hepatic artery ( from the anterior and posterior branches ) . the abscesses adjacent to these pseudoaneurysms showed hyperdensity on the non - contrast scan , indicating presence of acute hemorrhage . the venous phase demonstrated multiple liver abscesses , mild central intrahepatic biliary radical dilatation , along with post - necrosectomy changes in the pancreatic bed and peripancreatic collections . ( a ) axial maximal intensity projection ( mip ) image of the arterial phase acquisition shows three pseudoaneurysms , arising from the anterior and posterior branches of right hepatic artery and the lateral branch of left hepatic artery ( black arrows ) ( b ) volume - rendered image gives three - dimensional overview of the pseudoaneurysms ( short white arrows ) and arterial anatomy , depicting replaced right hepatic artery ( long white arrow ) ( c ) axial post - contrast venous phase ct image demonstrates multiple cholangitic abscesses in both lobes of liver . black arrow depicts the contrast - filled pseudoaneurysm arising from the right anterior hepatic artery , paralleling the attenuation of aorta with these imaging findings , the patient was planned for angiographic embolization [ figure 2 ] . digital subtraction angiography ( dsa ) done through transfemoral route using 5f rc-1 catheter ( cook , bloomington , in , usa ) and 2.7f microcatheter ( progreat ; terumo , tokyo , japan ) confirmed the hap . all three hap were embolized using four microcoils ( size 18 - 4 - 6 ; cook ) . follow - up usg done 24 h later showed recurrence of one of the hap ( arising from the anterior branch of right hepatic artery ) [ figure 3 ] . it was subsequently embolized under usg guidance using 22 g chiba needle , by injecting 2 ml of 100 u / ml concentration human - derived lyophilized thrombin ( floseal ; baxter healthcare corporation , hayward , ca , usa ) [ figure 3 ] . a follow - up ct done after 3 months did not show any recanalization of the pseudoaneurysms . ( a ) selective replaced right hepatic artery dsa shows two pseudoaneurysms , one from a branch of anterior division and the other from a branch of posterior division ( black arrows ) ( b ) dsa after coil embolization shows non - filling of the pseudoaneurysms ( c ) dsa after selective catheterization of the lateral branch of left hepatic artery shows a pseudoaneurysm ( black arrow ) without any active contrast extravasation ( d ) dsa of hepatic artery after coil embolization shows non - filling of the pseudoaneurysm usg - guided thrombin injection . ( a ) gray - scale image shows anechoic pseudoaneurysm arising from the anterior branch of right hepatic artery , which shows yin - yang pattern of color flow within ( b ) indicating recanalization ( c ) shows usg - guided placement of 22 g needle tip within the pseudoaneurysm ( d ) increased echogenicity within the pseudoaneurysm is observed following thrombin injection and there is absence of color flow , suggestive of complete thrombosis hap are uncommon , though they are the second most common site of acquired visceral pseudoaneurysms . these may rupture into biliary tree , peritoneal cavity , duodenum , adjacent focal lesion , or may develop fistulous communication with portal vein branches . there are only few case reports of hap associated with liver abscess , etiologies being amebic and pyogenic . the occurrence of hap with cholangitic abscesses and cholangitis has not been reported in the literature . moreover , in our case , there were multiple pseudoaneurysms , associated with multiple abscesses . because of the risk of rupture due to surrounding inflammation , such pseudoaneurysms need to be tackled urgently . the conventional management of such cases has been surgery with hepatic artery ligation or segmental resection . but currently , radiological interventions are the treatment of choice because of lower morbidity and mortality , and better patient comfort . also , these procedures can be repeated in case of rebleed which may occur because of background inflammation . the endovascular techniques include embolization with coils ( most commonly used ) , glue , and use of covered stents to exclude the pseudoaneurysm from the circulation . coils need to be placed across the neck of the pseudoaneurysm , as proximal embolization may lead to recurrence due to the presence of arterio - arterial collaterals . percutaneous thrombin injection is a simple technique for pseudoaneurysms within solid organs , can be done under ct or usg guidance , and is especially useful to manage recurrence after failure of coil embolization . the importance of early detection of hap in case of cholangitic abscess can not be overemphasized , given its catastrophic complications and relatively easy management . cta is instrumental in detecting these lesions and also demonstrates the precise location and identifies the affected artery . the wealth of information obtained on ct enables optimal planning of radiological interventions and also serves to reduce radiation and contrast dose . but recanalization of one pseudoaneurysm occurred , which was promptly treated by percutaneous technique , without any complications . the case is unique in that it addresses diagnosis of an unusual entity , its proper evaluation using ct , and two methods of minimally invasive radiological management for treating this potentially life - threatening entity . | hepatic artery pseudoaneurysms ( hap ) are uncommon , occurring mostly as a complication of trauma ( accidental or iatrogenic ) .
liver abscess rarely causes hap and multiple hap associated with cholangitic abscesses have not been reported in the literature .
we present a patient of acute necrotizing pancreatitis with stent block cholangitis and multiple cholangitic abscesses who developed hemorrhagic output through drainage catheter in the liver abscess .
a multiphasic ct angiography demonstrated three hap , which were treated with a combination of endovascular coil embolization and percutaneous thrombin injection .
the fact that cholangitic abscesses may be associated with pseudoaneurysms should not be neglected , considering the potentially catastrophic complication and relatively easy radiological management .
ct angiography permits accurate diagnosis and lays down the roadmap for endovascular procedures . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
carotid stenosis is a late complication of radiotherapy ( rt ) , and has a significant impact on long - term mortality and morbidity . in a previous study , we documented that neurological symptomatology is under - reported during routine oncology follow - up . the detection of this complication therefore relies on routine screening by duplex ultrasound . with limited resources , serial duplex ultrasound for we attempt to identify the crucial factors for development of this complication , with the aim of highlighting high - risk patients . in the period of june 1999november 2001 , 189 nasopharyngeal carcinoma ( npc ) patients completing curative - intent rt for at least 1 year were recruited to this study . seven patients were followed - up in our neurology clinic because of their co - existing neurological problems . the rt ports for treatment of npc covered the nasopharynx , adjacent tissues at risk , and both sides of the neck . the whole neck was treated by an anterior 6 mv photon beam with a 11.5 cm wide midline shield to protect the spinal cord . the study was approved by the ethics committee of the authors affiliated institution and informed written consent was obtained from all subjects . all patients had the duplex ultrasound performed with an atl hdi 5000us scanner ( bothell , wa , usa ) with a linear array ultrasound probe ( l12 - 5 ) . the carotid arteries were classified into internal carotid arteries , carotid bulbs , common carotid arteries and external carotid arteries . the diameters of the residual lumen and the whole artery were taken by electronic calliper . when true transverse images were difficult to obtain or when the boundaries of the arteries were ill defined , degree of stenosis was predicted by the ratio of peak systolic velocity of the internal carotid artery to that of the ipsilateral common carotid artery . severe stenosis ( > 70% diameter reduction ) was diagnosed when the ratio was equal to or more than 1.5 . all the arteries were classified into four grades : no stenosis , mild stenosis ( with < 30% diameter reduction ) , moderate stenosis ( more than 30% but < 70% diameter reduction ) and severe stenosis ( < 70% diameter reduction ) . a patient with fasting blood sugar > 6.1 mmol / l was diagnosed to be diabetic . a patient with fasting cholesterol higher than 6.2 mmol / l was diagnosed to be hyperlipidaemic . for each of the risk factors , fisher s exact test or t - test was used to explore the relationship with outcome ( classified as no / mild / moderate and severe ) . the statistical analyses were performed with the outcome ( classified as no / mild / moderate and severe ) , using the package spss installed on a personal computer . the classification - tree method was then used to develop a rule for the prediction of severe outcome . the analysis was carried out by s - plus 2000 ( seattle , wa ) . the sensitivity , specificity , positive predictive value and negative predictive value of the rules were calculated . one hundred and eighty - nine npc patients ( 53 female , 136 male , with age range 3276 years with a mean of 51.94 9.72 years old , time lapsed after rt ranged from 1 to 22 years with a mean of 6.87 3.61 years ) were recruited to the study . out of 378 common carotid arteries , 204 arteries showed no degree of stenosis , 34 arteries showed mild stenosis , 131 arteries showed moderate stenosis and 9 arteries showed severe stenosis . out of 378 internal carotid arteries , 314 arteries showed no stenosis , 17 arteries showed mild stenosis , 34 arteries showed moderate stenosis and 13 arteries showed severe stenosis . out of 378 carotid bulbs , 320 segments showed no stenosis , 23 segments showed mild stenosis , 35 segments showed moderate stenosis and no segment showed severe stenosis . out of 378 external carotid arteries , 306 arteries showed no stenosis , 11 arteries showed mild stenosis , 55 arteries showed moderate stenosis and 6 arteries showed severe stenosis . fifteen ( 7.9% ) patients developed more than 70% stenosis of either internal carotid artery or common carotid artery ; 67 ( 35.4% ) patients developed moderate stenosis of either internal or common carotid artery . sex , hyperlipidaemia , diabetes mellitus , smoking history , history of cerebrovascular disease , history of hypertension were found to have no association with the development of severe carotid stenosis . only 178 out of 189 patients had blood results available , either because of the patients refusal for blood to be taken or technical error . only the time lapsed after rt and the age of the patient were found to be significant factors ( table 1 ) . with the classification - tree analysis , patients older than 57 years and with rt more than 12 years ago were at high risk of development of severe carotid stenosis ( fig . when these rules were applied , the sensitivity was 46.7% , specificity 98.2% , positive predictive value 77.8% and negative predictive value 95.6% . figure 1
classification - tree for development of severe radiation - induced carotid stenosis . the uppermost number shown inside each square indicates the total number of patients at each node . the first number inside the parentheses represents the number of patients with no / mild / moderate outcome , the second shows the number with severe outcome . age 57.table 1
association of individual risk factor with development of severe radiation - induced carotid stenosis (
f
refers to fisher s exact test , t
refers to t - test )
no / mild / moderatesevere
p valuesexfemale503male124120.564 ( f)cholesterolnormal12412high3931.000 ( f)glucosenormal14912high1431.000 ( f)smokingno12611yes4840.939 ( f)cerebrovascularno17113symptomsyes321.000 ( f)hypertensionno16713yes721.000 ( f)agemean sd ( years)51.14 9.5261.13 7.13<0.0005 ( t)rt durationmean sd ( years)6.47 3.1511.53 5.17<0.0005 ( t )
classification - tree for development of severe radiation - induced carotid stenosis . the uppermost number shown inside each square indicates the total number of patients at each node . the first number inside the parentheses represents the number of patients with no / mild / moderate outcome , the second shows the number with severe outcome . association of individual risk factor with development of severe radiation - induced carotid stenosis (
f
refers to fisher s exact test , t
refers to t - test ) radiation - induced carotid stenosis is not an uncommon event among patients with head and neck tumour receiving rt[24 ] . in our institution s treatment protocol and in most centers , the neck is always included in the radiation post even for n0 stage npc . we have used duplex ultrasound in this study as it is accurate , safe and easily accessible . according to the european carotid trial and the north american symptomatic carotid endarterectomy trial , patients with more than 70% stenosis benefit from surgical intervention . for patients with severe stenosis , we have therefore adopted cheng s classification of patients and attempted to identify the crucial factors for patients at risk of developing carotid stenosis of more than 70% . in our study , 15 out of 189 patients ( 7.9% ) developed severe ( > 70% ) carotid stenosis . the internal carotid artery was the most common artery segment with severe stenosis . this group of patients might benefit from surgical intervention and should be evaluated fully for the planning of management . preoperative digital subtraction angiography ( dsa ) for accurate assessment of extent of involvement would be necessary . a significant number of our patients ( 67 patients ) developed moderate stenosis of either the common carotid artery or internal carotid artery . follow - up with duplex ultrasound to monitor the temporal progress of the carotid stenosis might be a more practical approach . dubec recommends routine surveillance of radiation - induced carotid artery stenosis by duplex ultrasound in patients with head and neck malignancy treated with rt . we agree that this is the best way to detect carotid stenosis and identify candidates for more invasive evaluation and intervention . resources are unfortunately always limited , and routine duplex ultrasound for all post - rt patients might not be feasible . presence or absence of neurological symptomatology is not a reliable predictor for severe carotid stenosis . most post - rt patients under - report their symptomatology during routine oncology follow - up . we have therefore performed the classification - tree analysis in order to establish rules for the identification of high - risk patients . the chi - squared test or t - test was used to explore the association of each of the risk factors with the outcome . the classification - tree method was used to develop a rule for the prediction of severe carotid stenosis . the rule was represented in the form of a binary tree . in our analysis , only time lapsed after completion of rt and the age of the patient were associated with the development of severe stenosis . out of these two factors , age of the patient had a higher association with the development of severe stenosis . it was therefore chosen as the first selection criterion ; the time lapsed after rt was used as the second selection criterion . when these rules were applied , we had a high specificity ( 98.2% ) and a high negative predictive value ( 95.6% ) in exclusion / identification of patients who might benefit from surgical intervention or stenting . it was interesting to note that diabetes mellitus , smoking history , hyperlipidaemia , hypertension , neurological symptoms were not found to be associated with development of severe stenosis in these postradiation patients . we postulated that the impact of age and radiation were so significant that the impact of other risk factors were out - weighted . as a conclusion , we have found that up to 43.3% patients develop moderate to severe carotid stenosis post - rt . this is a long - term radiation - induced complication that should not be overlooked . when routine screening is not feasible practically and financially , patients more than 57 years old and with radiation completed more than 12 years ago are useful guidelines for the identification of patients at high risk of developing severe carotid stenosis . | over a period of 3 years , we have collected the carotid ultrasound findings of 189 post - radiotherapy ( rt ) nasopharyngeal carcinoma patients .
the patients had duplex ultrasound examination for assessment of extracranial carotid stenosis .
factors including age , sex , hyperlipidaemia , diabetes mellitus , smoking history , time lapsed after rt , symptoms of cerebrovascular disease were recorded .
association of individual factor with development of severe stenosis was assessed.eighty-two ( 43% ) patients developed moderate to severe stenosis of the common carotid and/or internal carotid artery .
age and time lapsed after rt were the only factors found to be associated with development of severe stenosis .
when age > 57 years and > 12 years post - rt were used as rules to identify patients with severe stenosis , the sensitivity was 46.7% , specificity 98.2% , ppv 77.8% and npv 95.6%.radiation - induced carotid stenosis is a common event . if available resources do not allow routine surveillance , age and time lapsed after rt help to identify high - risk patients . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
alpha - tocopherol ( -t ) , the main form of vitamin e in mammal organisms , is a potent fat - soluble antioxidant and scavenger of hydroperoxyl radicals formed during the lipid peroxidation chain reaction . however , -t also possess alternative , non - antioxidant , functions that occur through the regulation of signaling molecules and the transcriptional control of groups of genes involved in key cellular events such as cell cycle progression and apoptosis , mitochondrial function , xenobiotic and lipid metabolism , etc . protein kinase c ( pkc ) appears to be central to vitamin e signaling ; in fact , -t can potently inhibit pkc activity , as documented in various in vitro model systems and in vivo in both the developing and adult brain . -t is regarded as an important molecule during fetal and early postnatal life , playing fundamental roles in protecting the developing organism against oxidative stress . in fact , free radicals have been indicated as causative agents of pregnancy - related disorders , such as preeclampsia and maternal diabetes , inducing serious complications in both the mother and fetus . blood -t concentrations are reduced in abnormal pregnancies ; moreover , preclinical in vitro evidence pointed to a potential therapeutic role of vitamin e in reducing placental oxidative stress induced by nicotine . on the bases of this rationale , an increase of vitamin e intake is recommended in complicated pregnancy to prevent free radical damage to the fetus . however , beneficial effects of vitamin e in risky pregnancy are currently debated , and the anti - pkc and antimitotic activity of -t raises important caveat against an indiscriminate use of vitamin e in gestation because of its potential effects on brain development . in fact , pkc plays a pivotal role in brain maturation , and alterations in its activity and/or expression levels have been proposed as the mechanism underlying the neuroteratogenicity of several chemical or physical agents . in previous works , we found that maternal supranutritional dietary intake of -t through pregnancy and lactation in rats markedly depresses pkc activity also reducing synaptic long - term potentiation ( ltp ) in developing hippocampus . importantly , a deficit in hippocampal synaptic plasticity was produced that persisted in adulthood , accompanied by alterations in hippocampus - dependent learning performances . in particular , adult rats born to tocopherol - supplemented dams performed less efficiently in spatial learning tasks , but displayed improved contextual fear conditioning , thus showing that two kinds of hippocampus - dependent learning abilities display differential sensitivity to the same manipulation of early nutritional environment .. collectively , these findings indicated that exposing rats to an excess of -t during development can lead to long - term electrophysiological and neurobehavioral consequences in adulthood , i.e. , when tocopherol intake and -t brain accumulation , as well as pkc activity , have returned to normal levels . the possibility that adult offspring of tocopherol - supplemented dams may carry brain structural changes that could help explaining functional and behavioral alterations in adulthood , , using the very same rat model of maternal dietary -t supplementation , anatomical studies were carried out at the electron microscopy level in order to investigate , in adult offspring , possible changes in density and morphological features of asymmetric , thus excitatory and presumably glutamatergic , axo - spinous synapses within hippocampal ca1 stratum radiatum , i.e. , the brain region where we have previously found a marked reduction of ltp induction and the area involved in mediating hippocampus - related learning abilities . sprague - dawley rats ( charles river , calco , lc , italy ) were used in this study . animal care and experimentations were performed in compliance with the italian laws on animal experimentation and with the guidelines of the local ethical committee of the university of urbino carlo bo . virgin female rats ( 200 - 250 g ) were housed individually with free access to food and water and maintained at a room temperature of 21c with 12-h light / dark cycle ( light on at 6 a.m. and off at 6 p.m. ) . dams were randomly divided into two groups : a control group ( ctrl ; n=4 ) , fed a commercial standard diet , and a supplemented group ( treat ; n=4 ) , fed a diet highly enriched in the natural rrr--tocopherol form ( 15 g / kg pellet ; laboratori dottori piccioni , gessate , mi , italy ) , as previously described . briefly , -t - enriched diet was administered constantly from 2 weeks before mating throughout pregnancy and lactation . after weaning , supplementation protocol did not affect course of pregnancy , newborn survival rate , weight of pups , and litter size , and no obvious teratogenic effects were observed in treat newborn . no significant intergroup difference in ponderal growth as well as in the timing of major maturational steps was found , neither obvious abnormalities were observed in older pups . for the present experimental purposes , ctrl and treat offspring ( 6 rats for each group , from 3 different litters ) were sacrified at adult age ( postnatal day 60 , p60 ) . rats were deeply anesthetized with sodium pentothal ( 45 mg / kg ) and perfused through the ascending aorta with physiological saline followed by phosphate buffer ( pb , 0.1 m ; ph 7.4 ) containing 3% paraformaldehyde and 2% glutaraldehyde . brains were removed and post - fixed in the same fixative for 12 h , then hippocampi were excised and cut with a vibratome into 30-m thick , parasagittal sections . briefly , hippocampal sections were postfixed in 2.5% glutaraldehyde in pb , and then for 1 h in oso4 1% . after dehydration in graded series of ethanol , sections were cleared in propylene oxide , flat - embedded in epon - spurr between acetate sheets ( aclar ; ted pella , redding , ca , usa ) , and polymerized at 60c for 72 h. embedded sections were examined under a dissecting microscope , and hippocampal areas containing ca1 subfield were excised with a razor blade and mounted on cured resin pyramidal blocks utilizing a cyanoacrylic glue . from each sample , one semithin ( 1 m ) section was cut with a reichert ultramicrotome and mounted on glass slides for light microscopic inspection . ultrathin ( 70 - 100 nm - thick ) sections were cut either from the surface or from the edge ( i.e. , perpendicular to plane section ) , counterstained with uranyl acetate and lead citrate , and examined with a philips cm 10 electron microscope . identification of neuropilar profiles ( presynaptic axon terminals , dendritic spines , astrocytic processes ) was based on established morphological and morphometrical criteria . counts of synaptic neuronal profiles and astrocytic processes were performed in single plane ultrathin sections . in each rat from the two experimental groups ( n=6 for each group ) , 70 squared areas ( 100 m ) from different mesh nickel grids were scanned at the electron microscope , and all asymmetric , axo - spinous synaptic profiles were sampled for quantitative morphometric analyses . neuronal and astrocytic profiles were measured in a semiautomatic mode , using the imagej software for image analysis . all the counts and morphometric analyses have been performed by two distinct observers ( ss and ap ) . in each scanned area from ca1 stratum radiatum of both ctrl and treat rats , the density of axo - spinous synapses was calculated as number of synapses per m . in all synapses , we measured : i ) the perimeter ( in nm ) of presynaptic axon terminal and dendritic spine profiles ; ii ) the number and length ( in nm ) of postsynaptic specializations ; iii ) the extension ( in nm ) of bouton - spine contact . all glial profiles that were in direct apposition to pre- and/or postsynaptic profiles ( perisynaptic glial processes ) were also counted . perisynaptic glial processes were classified in two distinct sets : i ) processes that contacted axon terminals or dendritic spines , but were located at some distance from bouton - spine interface ( extrasynaptic glia ) , and ii ) processes contacting the axo - spinous synaptic junction ( intrasynaptic glia ) , thus directly facing the cleft , i.e. , the very site of neurotransmitter release . the length ( in nm ) of membrane apposition between glial processes and pre / postsynaptic profiles was measured , and the ratio between this glia - covered length and the total perimeter of axon terminal and spine contours ( after subtracting the length of boutonspine contact ) was calculated . finally , the percentage of synapses contacted by intrasynaptic glia over the total synapses was calculated . visual details on the methodological approach used for morphometric analyses are given in figure 1 . for all morphometric indices considered , average values were calculated in each single scanned area and filed for further statistical analyses . all data are presented as mean standard deviation of the mean ( sem ) . student s t - test was applied for statistical analyses to compare results obtained in ctrl vs treat rats ; p=0.05 was considered as significance threshold . sprague - dawley rats ( charles river , calco , lc , italy ) were used in this study . animal care and experimentations were performed in compliance with the italian laws on animal experimentation and with the guidelines of the local ethical committee of the university of urbino carlo bo . virgin female rats ( 200 - 250 g ) were housed individually with free access to food and water and maintained at a room temperature of 21c with 12-h light / dark cycle ( light on at 6 a.m. and off at 6 p.m. ) . dams were randomly divided into two groups : a control group ( ctrl ; n=4 ) , fed a commercial standard diet , and a supplemented group ( treat ; n=4 ) , fed a diet highly enriched in the natural rrr--tocopherol form ( 15 g / kg pellet ; laboratori dottori piccioni , gessate , mi , italy ) , as previously described . briefly , -t - enriched diet was administered constantly from 2 weeks before mating throughout pregnancy and lactation . after weaning , supplementation protocol did not affect course of pregnancy , newborn survival rate , weight of pups , and litter size , and no obvious teratogenic effects were observed in treat newborn . no significant intergroup difference in ponderal growth as well as in the timing of major maturational steps was found , neither obvious abnormalities were observed in older pups . for the present experimental purposes , ctrl and treat offspring ( 6 rats for each group , from 3 different litters ) were sacrified at adult age ( postnatal day 60 , p60 ) . rats were deeply anesthetized with sodium pentothal ( 45 mg / kg ) and perfused through the ascending aorta with physiological saline followed by phosphate buffer ( pb , 0.1 m ; ph 7.4 ) containing 3% paraformaldehyde and 2% glutaraldehyde . brains were removed and post - fixed in the same fixative for 12 h , then hippocampi were excised and cut with a vibratome into 30-m thick , parasagittal sections . briefly , hippocampal sections were postfixed in 2.5% glutaraldehyde in pb , and then for 1 h in oso4 1% . after dehydration in graded series of ethanol , sections were cleared in propylene oxide , flat - embedded in epon - spurr between acetate sheets ( aclar ; ted pella , redding , ca , usa ) , and polymerized at 60c for 72 h. embedded sections were examined under a dissecting microscope , and hippocampal areas containing ca1 subfield were excised with a razor blade and mounted on cured resin pyramidal blocks utilizing a cyanoacrylic glue . from each sample , one semithin ( 1 m ) section was cut with a reichert ultramicrotome and mounted on glass slides for light microscopic inspection . ultrathin ( 70 - 100 nm - thick ) sections were cut either from the surface or from the edge ( i.e. , perpendicular to plane section ) , counterstained with uranyl acetate and lead citrate , and examined with a philips cm 10 electron microscope . identification of neuropilar profiles ( presynaptic axon terminals , dendritic spines , astrocytic processes ) was based on established morphological and morphometrical criteria . counts of synaptic neuronal profiles and astrocytic processes were performed in single plane ultrathin sections . in each rat from the two experimental groups ( n=6 for each group ) , 70 squared areas ( 100 m ) from different mesh nickel grids were scanned at the electron microscope , and all asymmetric , axo - spinous synaptic profiles were sampled for quantitative morphometric analyses . neuronal and astrocytic profiles were measured in a semiautomatic mode , using the imagej software for image analysis . all the counts and morphometric analyses have been performed by two distinct observers ( ss and ap ) . in each scanned area from ca1 stratum radiatum of both ctrl and treat rats , the density of axo - spinous synapses was calculated as number of synapses per m . in all synapses , we measured : i ) the perimeter ( in nm ) of presynaptic axon terminal and dendritic spine profiles ; ii ) the number and length ( in nm ) of postsynaptic specializations ; iii ) the extension ( in nm ) of bouton - spine contact . all glial profiles that were in direct apposition to pre- and/or postsynaptic profiles ( perisynaptic glial processes ) were also counted . perisynaptic glial processes were classified in two distinct sets : i ) processes that contacted axon terminals or dendritic spines , but were located at some distance from bouton - spine interface ( extrasynaptic glia ) , and ii ) processes contacting the axo - spinous synaptic junction ( intrasynaptic glia ) , thus directly facing the cleft , i.e. , the very site of neurotransmitter release . the length ( in nm ) of membrane apposition between glial processes and pre / postsynaptic profiles was measured , and the ratio between this glia - covered length and the total perimeter of axon terminal and spine contours ( after subtracting the length of boutonspine contact ) was calculated . finally , the percentage of synapses contacted by intrasynaptic glia over the total synapses was calculated . visual details on the methodological approach used for morphometric analyses are given in figure 1 . for all morphometric indices considered , average values were calculated in each single scanned area and filed for further statistical analyses . all data are presented as mean standard deviation of the mean ( sem ) . student s t - test was applied for statistical analyses to compare results obtained in ctrl vs treat rats ; p=0.05 was considered as significance threshold . in adult rats born to dams supplemented with supranutritional doses of -t , the density of axo - spinous , asymmetric synapses in hippocampal ca1 stratum radiatum was significantly higher as compared to that found in age - matched , control animals ( 0.70 0.04/m and 0.49 0.03/m in treated and control rats , respectively ; p=0.02 ; figure 2 ) . instead , quantitative analyses of basic morphometric features of synapses failed to reveal major between - groups differences : as reported in table 1 , the perimeter of axon terminals , the length of postsynaptic specializations and their number per synapse , as well as the linear extension of bouton - spine interfaces were similar in the two groups ( table 1 ) . interesting changes were observed in the morphological organization of neuron - glia relationships at hippocampal synapses . as an index reflecting the extension of glial coverage of synapses , we calculated , both for axon terminals and dendritic spines , the ratio between the contour length that was in direct apposition with distal astrocytic processes and the total perimeter ( excluding the length of bouton - spine apposition ) . between - groups differences were found for axon terminals , with adult offspring from tocopherol - supplemented dams showing significantly higher ratio ( 0.250.008 vs 0.190.013 in treated and control rats , respectively ; p=0.02 ; figure 3a ) , thus indicating a more extended glial coverage of presynaptic boutons at axo - spinous ca1 synapses . in contrast , ratio between glia - surrounded and total perimeter length was unchanged when calculated for postsynaptic spine profiles ( 0.150.013 vs 0.150.011 in treated and control groups , respectively ; p=0.98 ; figure 3b ) . in addition , possible changes in the proportion of tripartite synapses in ca1 stratum radiatum were assessed by calculating the percentage of synapses contacted by astrocytic endfeet at their bouton - spine junction ( thus considering only intrasynaptic glia , directly facing the site of presynaptic release ) over the total synapses . this percentage was significantly higher in treated animals respect to controls ( 360.02 vs 250.02 ; p=0.03 ; figure 3c ) , thus pointing to a surplus of hippocampal tripartite synapses in adult offspring of -t - supplemented mothers . as a main outcome of present ultrastructural study , we show that the hippocampus of adult rats born to dams fed with supranutritional doses of -t , as compared to age - matched controls , displays : i ) higher density of asymmetric ( thus excitatory ) axo - spinous synapses ; ii ) larger astrocytic coverage of presynaptic terminals ; iii ) higher number of axo - spinous synapses contacted by astrocytic endfeet at bouton - spine interface , i.e. , tripartite synapses . these anatomical findings extend our previous data obtained in the very same experimental model , showing that maternal dietary loads of -tocopherol promote permanent deficits in hippocampal synaptic plasticity and spatial learning in adult offspring . in maternally - supplemented rats , modifications of redox state in embryonic and postnatal brain induced by -tocopherol acting as antioxidant might occur , thereby possibly playing a role in promoting the observed ultrastructural , long - lasting effects in adult hippocampus . endogenous reactive oxygen species ( ros ) have been reported to affect proliferation of neural stem / progenitor cells derived from hippocampus of embryonic mice , with antioxidant compounds attenuating the proliferative activity of cultured cells ; moreover , ros and antioxidant treatment have been shown to influence morphological and physiological differentiation of cortical cultured neurons from embryonic rats ; in addition , postnatal treatment ( from p2 to p25 ) with antioxidant compounds , such as nadph oxidase inhibitors , has been recently shown in rats to decrease the levels of endogenously produced ros at all developmental ages , in association to macroscopic changes in cerebellar anatomy ( i.e. , changes in foliation ) and alterations in motor behavior at juvenile ages . in our previous work , we failed to detect major effects of maternally - delivered tocopherol on morphological maturation in developing hippocampus . in fact , no gross anatomical alterations in hippocampal structure and layering were observed at any ages ; in addition , we found no changes in morphological differentiation of hippocampal pyramidal neurons , whose dendritic extension and complexity at p14 was similar to that measured in untreated , age - matched control rats . collectively , these findings make unlikely that early developmental changes in redox status could be seen as a major mediating factor in provoking the ultrastructural effects observed in the hippocampus of adult rats maternally - exposed to high doses of -tocopherol . during brain ontogenesis , overproduction of synapses and their subsequent elimination through activity - dependent processes are critical for correct refinement and normal functioning of neural circuits . moreover , dynamic processes of assembly and disassembly of synapses persist into adulthood ; in rodent hippocampus , including ca1 , synapse density has been shown to increase into adult age , nearly doubling between postnatal day 15 and 48 , thus pointing to a highly protracted period of synapse formation and refinement . the increase of axo - spinous synapses observed in ca1 stratum radiatum of adult rats maternally exposed to excess of -t may reflect an aberrant gauging of synapse production / elimination balance during hippocampal maturation . we have previously shown that maternal supranutritional intake of -t does not affect developmental expression of important pre- and postsynaptic markers : at p14 , in fact , the hippocampal expression level , topographical distribution and cellular localization of synatophysin and spinophilin were virtually normal . given that synaptogenesis largely outpaces synapse elimination at early postnatal ages , more plausibly , the increase of synapse density observed in adult offspring from -t - supplemented mothers could be due to reduced synaptic pruning . maternal loads of tocopherol deeply influence postnatal pattern of hippocampal pkc activity in offspring , depressing pkc phosphorylation and reducing the expression of pkc - dependent , developmental - related proteins . a crucial role for pkc activity in regulating synapse pruning was proposed in studies showing that adult pkc mutant mice display defective elimination of surplus climbing fibers onto cerebellar purkinje cells , accompanied by motor coordination impairment . down - regulation of hippocampal pkc phosphorylation levels in tocopherol - treated animals persists into late phases of postnatal maturation , at least up to weaning , thus possibly affecting synapse elimination processes occurring at juvenile age . alterations in the establishment , maintenance , or pruning of synapses have been postulated to occur in several neurodevelopmental disorders , including autism , epilepsy , learning disabilities , schizophrenia ; in this context , present findings supporting the view that manipulations of early nutritional environment as an excessive -t maternal intake and delivery to offspring - can lead to long - lasting changes in synapse density and neural circuits refinement , gain particular relevance . maternal dietary loads of -t differentially influence distinct kinds of hippocampusdependent learning in adult offspring , since spatial learning in morris water maze is impaired , whereas contextual fear conditioning is improved . these two learning types are supported by different extra - hippocampal regions and neural pathways : in particular , contextual fear conditioning requires the integrity of the amygdala , whose substantial projections to various levels of hippocampal formation , including ca1 , have been documented in rats . although the origin of surplus synapses in ca1 stratum radiatum was not determined , an outgrowth of fibers coming from amygdala could potentiate the amygdaloid input to the hippocampus , thus selectively favoring the improvement of fear conditioning behavior . such a selective circuit remodeling upon early life nutritional insult may be possibly framed into the general concept of the region - specific morphological and functional adaptative plasticity shown by amygdaloid neurons in response to a variety of environmental and behavioral manipulations . present results also show that basic morphometric features of axo - spinous synapse are not affected by maternal supplementation with supranutritional doses of -t . the perimeter of pre - synaptic axon terminals , the length of postsynaptic specializations and their number per synapse , as well as the extension of bouton - spine contacts were virtually unchanged respect to controls . these findings indicate that hippocampal synapses in adult offspring of tocopherol - supplemented dams , although increased in number , retain normal morphological and geometrical characteristics , thus suggesting that long - term effects on synapse formation / elimination balance are not associated with major alterations of synapse maturational processes . yet some caution is needed , since structural dimensions and morphometric characteristics of dendritic spines , due to their highly variable shapes , could not be systematically quantified by single ultrathin sections approach used in our study . interestingly , major changes were found in glia - synapse anatomical relationships . in adult rats maternally exposed to excessive -t intake , the linear portion of axon terminal profiles that was surrounded by astrocytic processes was more extended as compared to control animals , thus pointing to a heavier glial coverage of presynaptic boutons ; in addition , the percentage of synapses that were contacted by astrocytic endfeet at the bouton - spine junction , i.e. , the very site of presynaptic neurotransmitter release , was remarkably higher , thus suggesting a surplus of axo - spinous tripartite synapses . present anatomical observations indicate that early exposure to supranutritional tocopherol intake can result in permanent alterations of neuron - glia morphological organization at synapses . the concomitant occurrence of synapse outnumber and increased glial ensheathing of synapses is particularly relevant at the light of evidence showing that astrocytes can actively participate in the formation and maintenance of synapses , as well as in controlling synapse turnover required for neural development and plasticity . adding astroglia into neuronal cultures from rat hippocampus was reported to increase the density of synapses and promote gamma - aminobutyric - acid ( gaba ) and glutamate receptor clustering , thus facilitating spontaneous network activity ; moreover , studies in organotypic hippocampal cultures have shown that astrocytic contacts play an important role in regulating development of individual synapses , increasing the lifetime of dendritic protrusions and influencing the stability of synaptic connections . in fact , perisynaptic astrocytes respond to neurotransmitters released during neuronal activity and release neuroactive substances that can influence neuronal excitability and synaptic transmission ; moreover , glia - mediated glutamate uptake via glast / glt-1 transporters regulates glutamate clearance in the cleft and glutamate spillover . several mechanisms underlying astrocytic effects on synaptic plasticity have been described in diverse brain regions including hippocampus , having either facilitating or inhibiting , context - dependent roles : glia - derived transmitters , such as atp / adenosine or d - serine , can modulate glutamate receptor activity and trafficking , while glial transport influences temporal and spatial patterns of nmda receptor activation by controlling glutamate concentration at synapse . at the light of this evidence , alterations in perisynaptic glial coverage and tripartite synapse function are likely to influence synaptic plasticity and behavioral learning performance . it is thus tempting to speculate that the increased astrocytic coverage of ca1 stratum radiatum synapses could contribute to the impairment of ltp induction and alterations in hippocampus - dependent learning abilities previously reported in the same model of adult rats born to -t - supplemented mothers . recent findings seem in contrast with this view : in transgenic mice expressing an inositol - triphosphate absorbent that attenuates astrocytic calcium signaling , behavioral deficits in hippocampus - dependent spatial and contextual fear memory were associated with a reduction , instead of an increase , of glial coverage at ca1 asymmetric synapses . however , given the fundamental role played by intracellular calcium transients in glial physiology and excitability , blunting calcium signaling is supposed to affect most , if not all , aspects of activity - related astrocytic functions , thus deeply and globally disrupting neuron - glia communication processes . in our model , the increased glial ensheathing of synapses might potentially affect ltp induction mainly by enhancing glutamate uptake capacity . ceftriaxone - mediated enhancement of glt-1 gene transcription changes glt-1 expression and distribution , and impairs activity - dependent synaptic plasticity at mossy fibre - ca3 hippocampal synapses . moreover , knockout mice lacking ephrin - a3 , a molecule involved in neuron - glia communication processes regulating synapse morphology and function in the hippocampus , display impaired ltp induction and deficits in hippocampus - dependent tasks , mainly ascribed to post - translational up - regulation of glutamate transporters in astrocytes and more efficient glutamate removal from the cleft . in conclusion , adult offspring born to dams that were fed with supranutritional doses of -t over pregnancy and lactation display permanent hippocampal changes in axo - spinous synapse density and in neuron - glia morphological relationships at synapses , together with deficits in ltp induction and spatial memory . these findings confirm and extend previous evidence supporting the general concept that early life environmental stress , including those deriving from nutritional insults , can lead to long - lasting effects in adult brain . it is worth noting that the increase in vitamin e doses used for pregnant women in most supplementation trials , ranges 40 - 100 times the standard recommendation , which is comparable to the increase applied to the nutritional intervention of this study . therefore , present results emphasize the need to carefully evaluate the safety of developmental exposure to high doses of -t in animal species and particularly in humans . | an increased intake of the antioxidant -tocopherol ( vitamin e ) is recommended in complicated pregnancies , to prevent free radical damage to mother and fetus .
however , the anti - pkc and antimitotic activity of -tocopherol raises concerns about its potential effects on brain development .
recently , we found that maternal dietary loads of -tocopherol through pregnancy and lactation cause developmental deficit in hippocampal synaptic plasticity in rat offspring .
the defect persisted into adulthood , with behavioral alterations in hippocampus - dependent learning . here , using the same rat model of maternal supplementation , ultrastructural morphometric studies were carried out to provide mechanistic interpretation to such a functional impairment in adult offspring by the occurrence of long - term changes in density and morphological features of hippocampal synapses .
higher density of axo - spinous synapses was found in ca1 stratum radiatum of -tocopherol - exposed rats compared to controls , pointing to a reduced synapse pruning .
no morphometric changes were found in synaptic ultrastructural features , i.e. , perimeter of axon terminals , length of synaptic specializations , extension of bouton - spine contact .
gliasynapse anatomical relationship was also affected .
heavier astrocytic coverage of synapses was observed in tocopherol - treated offspring , notably surrounding axon terminals ; moreover , the percentage of synapses contacted by astrocytic endfeet at bouton - spine interface ( tripartite synapses ) was increased.these findings indicate that gestational and neonatal exposure to supranutritional tocopherol intake can result in anatomical changes of offspring hippocampus that last through adulthood .
these include a surplus of axo - spinous synapses and an aberrant gliasynapse relationship , which may represent the morphological signature of previously described alterations in synaptic plasticity and hippocampus - dependent learning . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the 5-ht1a receptor is one the most abundant subtypes
of the 5-ht receptor family ; it is highly enriched in the raphe nucleus ,
cerebral cortex , hippocampus , septum , and amygdale . because of its
presence in the brain regions whose functions are heavily involved
in mood and anxiety disorders , the 5-ht1a receptor has
been actively explored as a target for antipsychotic , anxiolytic and
antidepressant drug discovery . several 5-ht1a receptor
agonists such as buspirone and tandospirone are medications approved
to treat anxiety and depression . some of the atypical antipsychotic
drugs , such as aripiprazole , clozapine , and olanzapine , are also partial
5-ht1a receptor agonists and are sometimes used in low
doses in combination with standard antidepressants to achieve faster
symptom relief and greater overall efficacy . furthermore ,
5-ht1a receptors have been actively studied as potential
drug targets for treating the cognitive deficits in schizophrenia : the activation of the 5-ht1a receptor
has been linked to the increased dopamine release , which could improve
certain symptoms of schizophrenia . besides being
traditionally explored as targets for psychiatric disorders , 5-ht1a receptors have recently received considerable attention
as targets to develop treatments for neurodegenerative diseases . recent discoveries have shown that the modulation
of 5-ht1a receptor may present a novel mechanism for treating
the alzheimer s disease or help relieve the symptoms of parkinson s
disease . although several drugs
are available on market that acting via
a 5-ht1a receptor - mediated mechanism , only a few were originally
developed to selectively target 5-ht1a receptors . in addition ,
current 5-ht1a modulators exhibit various side effects
or even some severe side effects , preventing
their widespread clinical use . moreover , some patients
have been reported to be nonresponders or poor - responders to current
medications . virtual screening ( vs ) is a common and efficient approach for the
discovery of new lead compounds . structure - based
vs has been the most popular approach to identify putative receptor
actives in chemical libraries , but in
recent years , ligand - based cheminformatics approaches have been used
widely in vs applications as well . in
the case of 5-ht1a receptors , when the experimental structure
of the receptor is unknown , ligand - based approaches can be explored
for vs. our group has been investigating the use of quantitative structure
activity relationships ( qsar ) models as effective vs tools , and several
successful applications have been reported . the qsar approach
explores existing chemical databases with biologically activities
to establish statistically significant and externally predictive models
that allow one to predict biological activity of untested compounds
from their chemical structures . once a qsar model has been developed ,
it can be used to search libraries of chemical structures with the
aim of finding new , structurally different hit(s ) with the desired
biological activity . indeed , the size
and diversity of chemical libraries available for both virtual and
experimental screening have been growing rapidly in recent years ,
providing growing opportunities to use vs methods to identify novel
hits among available chemicals . in this paper , we report on
the development of rigorously validated
qsar models of the 5-ht1a receptor binders using previously
reported bioactivity data for the receptor ligands . we employ these
models for virtual screening to arrive at a small number of 15 prioritized
computational hits that have been subject to experimental validation . we show that 9 of these 15 hits show appreciable binding affinity
ranging between 10 m and 2.3 nm . this study confirms that qsar - based
virtual screening is an effective tool to discover novel bioactive
compounds that can be further pursued as novel antipsychotics . the data for 5-ht1a activity were retrieved
from the national institute of mental
health ( nimh ) psychoactive drug screening program ( pdsp ) ki database . in this study ,
we used 10 m as the cutoff value to define actives versus inactives ,
and we only retrieved the experimental radioligand binding data with
cloned human cell lines using the ligand [ h]-8-oh - dpat .
by submitting such queries , 180 unique compounds were designated as
5-ht1a actives with binding affinities ranging from 0.115
nm to 8.41 m . we also retrieved 78 inactives , which were shown
experimentally to have no binding affinity to the 5-ht1a receptor at 10 m concentration . an additional
66 putative 5-ht1a actives were extracted from the world
of molecular bioactivity ( wombat ) database . this commercial database is a collection of chemical annotations
published in top medicinal chemistry journals ; therefore , the binding
data therein are considered as highly reliable . compounds extracted
from wombat were regarded as 5-ht1a actives when they satisfied
all of the following criteria : ( 1 ) compounds were tested on cloned
human species cell lines ; ( 2 ) [ h]-8-oh - dpat was used as
hot ligand ; ( 3 ) their binding affinities were higher than 10 m . notably , all 66 compounds were different structurally from compounds
in the modeling set . prior to qsar
model development ,
chemical structures were curated following the guidelines we published
earlier . first , all molecules were cleaned
using the wash molecules module in molecular operating
environment ( moe , version 2009.10 ) . second ,
the routine standardizer was used for structure canonicalization and
transformation , jchem 5.2 , 2009 , chemaxon ( http://www.chemaxon.com ) . finally , duplicates were detected by the analysis of the normalized
molecular structures . for chemicals extracted from the pdsp ki database , 75 duplicate compounds for 5-ht1a actives and 17 for inactives , i.e. , different salts or isomeric
states , were detected . the functional data for duplicates were verified
to be identical , so in each case a single example was removed . the
curated subset of the 5-ht1a ligands from pdsp included
166 unique organic compounds ( 105 actives and 61 inactives ) . drug - like libraries are
collections of currently marketed drugs or drug candidates in the
approval process . for our study , we used the world drug index ( wdi )
database and the prestwick chemical library ( pcl ) ( http://www.prestwickchemical.fr/ ) . the wdi library is maintained by derwent publications and our
version contains 59 000 pharmacologically active compounds ,
including all marketed drugs and those in the development . the pcl
contains 1200 small molecules , all of which are marketed drugs . by
design , compounds in the pcl feature a high chemical and pharmacological
diversity as well as a high degree of bioavailability and safety in
human . the 5-ht1a receptor
belongs to the large family of gpcrs ; therefore , gpcr - targeted libraries
such as timtec ( http://www.timtec.net/ ) antitarg - g library
and asinex ( http://www.asinex.com/ ) synergy gpcrs cns library
were used for virtual screening to identify new putative 5-ht1a ligands . the timtec antitarg - g library is a plated screening
set of 2300 molecules that contain chemical scaffolds present in compounds
reported in the technical or patent literature to bind gpcrs . similarly ,
the asinex synergy gpcrs cns library is composed of 3233 compounds
rich in gpcrs drug - like pharmacophore fragments . the diversity libraries
were also from timtec and asinex , namely timtec diversity set 10k
and asinex diverse set - platinum 5k . the timtec diversity screening
set contains 10 000 samples selected from the company s
stock of over 180 000 compounds as the most structurally diverse
and competitively priced collection . the assorted set stands out as
having a diverse selection of singletons identified in the timtec
stock of readily available compounds . in addition , it is also a compound
collection that complies with lipinski s rule of five . asinex
diversity set - platinum 5k , which contains 5072 compounds , is an assortment
of all asinex libraries based on the compounds structural
diversity . this set of compounds is claimed by asinex to be a great
starting point that requires a diverse chemical collection . we
have followed the rigorous qsar modeling workflow for model building ,
validation , and virtual screening ( figure 1 ) developed in our laboratory . this
workflow requires that an external predictive power should be established
for every qsar model . thus , we have employed the external 5-fold cross - validation
protocol where the modeling set is randomly split into five subsets
of approximately equal size ( 20% of compounds ) . each time , one subset
is used as an external validation set , and the union of four other
subsets is used as the modeling set , i.e. , each modeling set contains
80% of compounds . modeling sets were further partitioned into multiple
pairs of representative training and test sets of different sizes
using the sphere exclusion algorithm developed in our laboratory , which ensures the closeness in chemical spaces within the paired
data sets . the workflow of qsar model building , validation and virtual screening
as applied to the 5-ht1a data set of 105 actives and 61
inactives from pdsp . the smiles strings of each compound in the 5-ht1a data set were converted to 2d chemical structures using the moe
software package . the dragon software
( version 5.5 ) was used to calculate a wide range of topological indices
of molecular structures . dragon descriptors with zero values or zero
variance were excluded , whereas the remaining descriptors were range - scaled
within the interval of 01 prior to distance calculations and
model building because the absolute scales for the variety of dragon
descriptors can differ by orders of magnitude . the knn classification
qsar method is based on the idea that the
class that a compound belongs to can be predicted by the class membership
of its nearest neighbors ( i.e. , most similar compounds ) , taking into
account weighted similarities between the compound and its nearest
neighbors . because our implementation of knn approach
includes variable selection , the similarity is evaluated using only
a subset of all descriptors , which is optimized by a simulated annealing
( sa ) approach to achieve the best correct classification rate ( ccr):1 where n1total and n2total are the number of actives
and inactives in the data set , and n1corr and n2corr are the number of known actives correctly predicted as
actives ( true positives ) and the number of inactives correctly predicted
as inactives ( true negatives ) , respectively . unlike total accuracy ,
ccr inherently took into account the imbalance in class membership
of objects in the data set , which was important because the 5ht1a data set was imbalanced containing 105 actives and 61 inactives . the accuracy of the models was characterized by the leave - one - out
cross - validation ( loo - cv ) ccrtrain for the training sets
and predictive ccrtest for the test sets . models with high ccrtrain and ccrtest were
used to predict compounds included neither in the training nor in
the test set as a matter of external validation . theoretically , any
compound represented by the corresponding chemical descriptors can
be assigned to a class ( predicted class ) using the classification knn approach . however , if the distances between the query
compound and all of its k nearest neighbors in the
training set are higher than some threshold , the query compound is
considered as highly dissimilar from all of the training set compounds ,
and the prediction of its activity using the knn
approach is considered unreasonable . therefore , a similarity threshold
( or model applicability domain , ad ) was introduced to avoid making
predictions for compounds that differ substantially from the training
set molecules . the distance threshold
is defined as follows:2here , y is the average
euclidean distance between each compound and its k nearest neighbors within the training set , is its standard
deviation of these distances , and n is an arbitrary
parameter called the n - cutoff to control the significance
level . typically , we set n to 0.5 , which places the
boundary for deciding whether a compound is within or outside of the
ad at one - half of the standard deviation from y. it is important to notice that increasing the value of n would increase the number of compounds in the external
set that are considered within the ad but could decrease the accuracy
of the prediction due to the inclusion of dissimilar nearest neighbors . random forest
is a machine learning technique that consists of many decision trees
and outputs the consensus prediction from the individual trees . in this study , the implementation of an rf algorithm available in the r project ( version 2.14.1 ) was used . in the rf modeling
procedure , n samples ( modeling set compounds ) are
randomly drawn with replacement from the original data set . these
samples were used to construct n training sets and
to build n trees . in these studies , predictions were made by averaging predicted activities
over all trees in the final forest . the original version of svm was
developed by v. vapnik and the description
of the svm algorithm can
be found in many publications . briefly , molecular
descriptors are first mapped onto a high dimensional feature space
using various kernel functions . then , svm finds a separating hyperplane
with the maximal margin in this high dimensional space to separate
compounds with different activities . models built with svm allow for
the prediction of the target property using a set of descriptors solely
calculated from the structure of a given compound . in this study ,
we used the winsvm program developed in our group ( freely available
for academic laboratories upon request ) implementing the open - source
libsvm package . the winsvm program provides
users with a convenient graphical interface to prepare input data ,
perform svm modeling , and select models for external evaluation . the
program also allows one to visualize molecular structures and produce
various plots , making the use of svm easier and more appropriate for
qsar modeling to obtain robust and predictive models and apply them
to virtual libraries . this
test includes rebuilding the training set models using randomized
activities ( y - vector ) of the training set and comparing the resulting
model statistics with that of the models built with original data . it is expected that models built with randomized activities should
have significantly lower ccr values for both the training and test
sets . the one - tail hypothesis testing was applied to confirm the robustness
of qsar models . in this approach , two alternative hypotheses are formulated :
( 1 ) for h0 , h = ; ( 2 ) for h1 , h > , where is the average
value of ccrtrain for y - randomization models and h is that for the actual models . the null hypothesis ( h0 ) states that the qsar models for the actual data set are
not significantly better than random models , whereas the h1 hypothesis assumes the opposite , suggesting that the actual models
are significantly better than the random models . hypothesis rejection
is based on a standard one - tail test , which involves the following
three steps : ( 1 ) determine the average value of ccrtrain ( ) and its standard deviation ( ) for random models ;
( 2 ) calculate the z score that corresponds to the average value of
ccrtrain ( h ) for the actual models using
the following equation:3(3 ) compare this z score
with the tabular critical values of zc at different levels of significance ( ) to determine
the level at which h0 should be rejected . if the z score is higher than tabular values of zc , one concludes that at the level of significance that
corresponds to that zc , h0 should
be rejected while h1 should be accepted . the y - randomization
test was applied to all data sets considered in this study , and the
test was repeated twice in each case . as illustrated
in the workflow of figure 1 , qsar models that
passed both internal and external validation were employed for virtual
screening . a global applicability domain ( calculated using all descriptors )
was applied first to filter out compounds that were structurally highly
different ( beyond the ad threshold calculated with n - cutoff = 0.5 , cf . all 105 known 5-ht1a actives extracted
from pdsp were used as probes in the chemical similarity calculations . then , all acceptable models obtained with various machine learning
techniques , knn , rf , and svm , were applied in consensus
to predict the class of compounds in the external library that were
found within the global applicability domain . furthermore , the results
were accepted only when the compound was found within the applicability
domains of more than 50% of all models used in consensus prediction
and the standard deviation of estimated means across all models was
small . during the consensus prediction of knn , we
restricted ourselves to the most conservative ad for each model using
the n - cutoff = 0.5 in eq 2 . all the modeling and virtual screening calculations were carried
out at a 352-processor beowulf linux cluster of the its research computing
division of the university of north carolina at chapel hill ( unc ch ) . the cpu nodes are intel xeon ibm bladecenter of dual intel xeon 2.8
ghz , with 2.5 gb ram on each node . the cluster runs the red hat enterprise
linux 4.0 ( 32-bit ) and the nodes communicate via a gigabit ethernet
network . in addition ,
the data processing speed was found to be able to scale linearly with
the size of the screening library . the chemical similarity
search was conducted with the moe 2006.08 package using the standard
protocol . the maccs structural keys were utilized with the tanimoto
coefficient ( tc ) as the similarity metric . in the case that the hits
from individual searches were in duplicate , a special scientific vector
language ( svl ) script was employed to remove them by considering both
chemical topology and chirality . the experimental tests were
performed by the national institute of mental health pdsp program
( http://pdsp.med.unc.edu/indexr.html ) . the five computational
hits from prestwick library were purchased from sigma - aldrich , and
the ten additional compounds were purchased from timtec llc ( cf . competition binding assays were performed using transfected or stably
expressing cell membrane preparations as previously described and all experimental details are available online ( http://pdsp.med.unc.edu/pdspw/binding.php ) . dragon software ( version 5.5 ) was used ; initially ,
880 chemically
relevant 0d-2d descriptors were calculated . a total of 672 descriptors
were eventually used for 5-ht1a data set after removing
descriptors with zero value or zero variance . furthermore , all descriptors
were range - scaled to fall between the values of zero and one . the knn qsar
method with variable selection afforded multiple models with
optimal accuracy characterized by ccr for both training and test sets . for the five internal modeling sets ( each one is comprised of approximately
80% of the entire 5-ht1a data set ) generated after applying
the external 5-fold cross - validation protocol ( cf . methods ) , there
were a total of 838 models with both ccrtrain and ccrtest equal to or higher than 0.80 . most models with ccrtest 0.80 also had corresponding ccrtrain 0.80 , but the opposite was not always true . the high accuracy
of the models implied that these models could correctly identify the
majority of actives and inactives in both the training and test sets . the rf qsar classification method
was applied for the same five modeling sets used for knn modeling . for each modeling set , the decision trees were tuned
and selected under the rf algorithm and only the final model ( a set
of classification trees ) was reported ( cf . the external 5-fold cross - validation procedure ( same as
when using knn ) was employed using the same division
of the data set into five folds ; the resulting external accuracy ranged
between 0.68 and 0.84 ( cf . n(1 ) = number of
actives , n(2 ) =
number of inactives , tp = true positive ( actives predicted as actives ) ,
fp = false positives ( inactives predicted as actives ) , fn = false
negatives ( actives predicted as inactives ) , tn = true negative ( inactives
predicted as inactives ) , se = sensitivity = tp / n(1 ) , sp = specificity
= tn / n(2 ) , en = the normalized enrichment , en(1 ) = ( 2tp n(2))/(tp
n(2 ) + fp n(1 ) ) , en(2 ) = ( 2tn n(1))/(tn
n(1 ) + fn n(2 ) ) , and ccr = correct classification rate . some n(1 ) actives of and n(2 ) inactives
were out of application domain of all consensus models , thus having
no prediction . the same five sets of modeling compounds
were also used to build svm qsar classification models . due to the
limited number of models selected by using 0.80 as the cutoff for
both ccrtrain and ccrtest , and the potential
unreliable predictions on external compounds by using only few models , the models with both ccrtrain and ccrtest
0.65 were considered acceptable and were selected for consensus prediction ;
a total of 207 of such models were retained . in addition to the internal
validation of knn , rf , and svm models using test
sets , y - randomization and external validation are the critical steps
of our qsar workflow ( figure 1 ) . only models
that have been validated by these two steps can be employed for external
prediction and virtual screening . furthermore ,
a data set of 66 5-ht1a actives from wombat was used as
the independent external validation set . the binary annotations of 5-ht1a as actives or inactives
in the training set were randomly
shuffled , and knn , rf , and svm classification models
were built with the same parameter settings as used in the original
data modeling . the y - randomization test was performed once for each
training / test set split and all its runs showed that almost all models
had both ccrtrain and ccrtest around 0.50 , which
is equivalent to random guess ( cf . figure 2 ) . because no classification rules or hyperplanes can be identified
by svm classification methods after the random shuffling of the original
5-ht1a annotations , no prediction could be further made
for the test set compounds , thus no statistics were reported in figure 2 . moreover , the one - tail hypothesis was applied ,
and a z score of 2.17 was calculated . after comparing this z score with the tabular critical values of zc at different levels of significance ( ) , we concluded that with 98.48% confidence the null hypothesis
h0 should be rejected , implying that the difference of
ccr for models built with the original data versus those built with
the data subjected to y - randomization was significant . box plots for the external
prediction accuracy ( ccrevs ) of different qsar classification
models for 5-ht1a actives . lower horizontal line of the box , 20th quantile ; middle line , median ;
upper line , 80th quantile . lower vertical line , range of data between
20th quantile and the minimum ; upper vertical line , range of data
between the 80th quantile and the maximum . the external 5-fold cross - validation
approach was employed for the external prediction , i.e. , for each
split , models were built using 80% of the 5-ht1a data set to predict the remaining randomly excluded 20%
of compounds . exactly
the same external sets were employed for validation of knn , rf , and svm classification models , and the results are compared
and summarized in both figure 2 and table 2 . it is noticed that only 19 out of 20 5-ht1a actives in the first external set were predicted by knn classification models ; the remaining 5-ht1a active compound could not be predicted by our consensus knn models because it was outside of the models
applicability domain . the consensus score , in terms of the average
class number in classification qsar , was calculated by the fraction
of models that predicted a compound as inactive divided by the total
number of models used for prediction plus one ; this formula is based
on the annotation of actives as having class label of 1
and inactives as those with the class label of 2 .
thus , if a set of classification qsar models is used to predict a
compound s activity , the mean predicted class could range between
1 ( when all models predict this compound as active ) and 2 ( when all
models predict this compound as inactive ) . obviously , when models
disagree , the mean class label may take any value between 1 and 2 .
under n - cutoff = 0.5 ( cf . eq 2 ) , most of the external validation set achieved a rather high prediction
accuracy . the highest accuracy for the knn classification
models across all external validation folds was observed for the fourth
external set split ( table 1 ) , with sensitivity
of 95% ( for actives ) and specificity of 77% ( for inactives ) , leading
to ccrevs = 0.86 . increasing n - cutoff
( eq 2 ) raised the model coverage for predicting
of both active and inactive compounds because of the extended applicability
domain for individual models . however , the prediction with extended
applicability domain for consensus models also comes with lower confidence
level ( data not shown ) . generally speaking , to have reliable predictions
but also broad model coverage , the full ic50 curve was
generated in further experiments and the ki value was determined . the consensus scores for each of the compounds in the external
sets predicted by all three ( knn , rf , and svm ) classification
models are shown in table 1 . the models with
qualifying ccrtrain and ccrtest values in excess
of 0.80 and the highest ccrevs resulting for a single split
of the data in the 5-fold validation protocol were used in consensus
for virtual screening . notably , the knn models chosen
for the prediction had relatively small ncutoff ( 0.5 ) and relatively broad coverage ( 50% ) for compounds
in external data sets . we used models built
for a pdsp data set of 166 5-ht1a active / inactive compounds
to predict the class label for 66 known 5-ht1a actives
from wombat . we should emphasize that these latter compounds had unique
structures that were different from the existing pdsp actives . among
the 66 actives ( all were within the applicability domain ) , 62 were
accurately annotated by knn consensus prediction
( se = 0.94 , table 1 ) . the only
four incorrectly predicted compounds had the consensus prediction
scores of 1.51 , 1.54 , 1.55 , and 1.67 , respectively . as illustrated
above , consensus prediction is based on the results obtained by all
validated predictive models . the closer the value is to 1.0 , the greater
the confidence in the prediction of a compound being 5-ht1a active , whereas the value closer to 2.0 implies greater confidence
in predicting a compound to be inactive . because the predicted class
labels for the four false negative 5-ht1a actives did not
exceed 1.67 , and compounds were within the applicability domain of
only 70 models ( i.e. , approximately 30% of all models ) , the knn prediction is considered as of low confidence . when
rf and svm were applied , the prediction accuracy for the additional
66 actives from wombat was also high , ranging from ccrevs = 0.94 to 0.96 ( table 1 ) . the success
of this independent external validation highlights the power of our
qsar models in predicting the possible 5-ht1a binding classifications ,
so that these models can be reliably applied for virtual screening
to identify novel 5-ht1a receptor actives . models with the highest
predicted ccrevs for each machine learning method , i.e. ,
217 knn models with both internal and external ccrtrain and ccrtest equal to or greater than 0.80
and ccrevs equal to 0.86 , one rf model with ccrevs equal to 0.84 , and 47 svm models with ccrevs equal to
0.95 , were used in consensus for virtual screening . initially , 55 , 384
compounds from the prestwick and wdi libraries were screened to identify
putative 5-ht1a actives ; the number of compounds falling
within the ad when the n - cutoff ( cf . eq 2 ) was varied are shown in figure 4 . it should be noted that there is a big overlap between compounds screened
by prestwick and wdi libraries , and hits from wdi also share the same
or highly similar structures with compounds in the modeling set . therefore ,
only the screening statistics from the prestwick library are shown
in figure 4 . the compounds within the ad defined
by n - cutoff = 0.5 were further predicted by knn consensus models . 234 compounds from the prestwick library
were predicted as actives by at least one of the knn consensus models . to narrow the hit list and obtain the higher
confidence level for each prediction , we took both the consensus score
( average class number ) and model coverage into consideration ; model
coverage was defined as a fraction of models for which a compound
was found to fall within the respective applicability domain . in particular ,
only hits with an average class number between 1.0 and 1.1 and the
model coverage over 50% were selected . in total , 125 compounds from
pcl and 181 from wdi were identified that satisfied both criteria . the majority of these virtual hits were found to be highly similar
to the compounds already known ( compounds in the qsar modeling set ) ,
so it would be less interesting to test these hits experimentally .
to verify the diversity of those virtual hits , each compound was represented by a fingerprint
of 166 substructure keys ( maccs structural keys ) , indicating the presence or absence of a particular chemical
substructure . the pairwise similarity was assessed by tanimoto coefficients ( tc ) between pcl hits , between pcl hits and
each hit s nearest neighbor from the actives in the modeling
set ( identified by the lowest euclidean distances calculated with
the dragon descriptors ) , and between pcl hits and all actives in the
modeling set . the majority of compound pairs formed by prestwick s
virtual hits with each hit s nearest neighbor within the modeling
set had tc over 0.90 , whereas other pairwise similarity scores show
a normal distribution , suggesting that the virtual hits are structurally
dissimilar from each other but highly similar with known 5-ht1a actives ( figure 5 ) . furthermore ,
the principal component analysis ( pca ) was also conducted and it could
be easily seen that there is a big overlap between the distribution
of chemical space occupied by the prestwick library and modeling set
compounds ( figure 3 ) . pca plot of three types
of virtual screening libraries along with
the modeling set ; the plots were generated from calculating the top
three principal components by using multiple chemical descriptors
( dragon 5.5 , 0d-2d descriptors ) of compound in respective libraries . compounds from the modeling set are colored in red ; compounds from
the prestwick library are colored in green ; compounds from the timtec
gpcr - targeted library are colored in blue and compounds from the timtec
diversity library are colored in orange . to explore more structurally diverse 5-ht1a compounds ,
we further screened gpcr - targeted libraries and diversity libraries
from the commercial chemical sources of timtec and asinex . thus , the
additional collection of 24 000 compounds was screened , which
included the timtec actitarg - g ( gpcr - targeted ) library of about 2300
compounds , the asinex synergy gpcrs cns ( gpcr - targeted ) library of
about 7000 compounds , the timtec diversity set 10k ( diversity library )
of 10 000 compounds and the asinex diversity set - platinum 5k
( diversity library ) of about 5100 compounds . the putative hit rates
for different screening libraries are shown in figure 4 with various n - cutoff values ( representing different applicability domain ) ,
and the exact numbers of compounds chosen from these libraries are
also available in the supporting information ( table s1 ) . it is obvious that many more chemicals were selected
from the gpcr - targeted library than from the diversity library by
applying the same n value , verifying that the diversity
library has much more structural - varied compounds than the gpcr - targeted
library . the hit rates of putative 5-ht1a actives identified
in five different types of screening libraries by the global similarity
search at three different values of the applicability domain n - cutoff ( 0.5 ; 0 ; + 0.5 ) . the binding potential for compounds within the ad of n = 0.5 was further predicted by knn consensus
models . 445 compounds from the timtec actitarg - g library , 487 from the timtec
diversity set 10k , 2177 from the asinex synergy gpcrs cns library
and 782 from the asinex diversity set - platinum 5k were predicted as
actives by at least one of the knn consensus models .
to narrow the hit list and obtain the higher confidence level for
each prediction , both the consensus score and model coverage were
taken into consideration . in particular , only the hits with average
class numbers between 1.0 and 1.1 and the model coverage over 50%
were selected . we found that there were 64 compounds from the timtec
antitarg - g library and 40 from the timtec diversity set 10k that satisfied
both criteria . as for asinex libraries , there were still hundreds
of compounds that met those strict criteria , but we decided not to
pursue these compounds at this time . several structural classes
were observed by screening different
libraries according to the tc values . notably , many of the 64 virtual
hits from the timtec antitarg - g library were found to be structurally
similar to actives used in model building , whereas the 40 virtual
hits from the timtec diversity set 10k had highly different structures .
the pairwise similarity measured by tc values was also compared between
virtual hits , hits versus their nearest neighbor within the modeling
set compounds , virtual hits versus modeling set compounds , and between
modeling set compounds ( figure 5 ) . it is clearly seen that the virtual hits from
the timtec antitarg - g library had chemical structures with a much
lower similarity ( figure 5ac ) to the known
5-ht1a actives than prestwick virtual hits ( figure 5bc ) . the average tc value between timtec anti - targ - g
library hits and their nearest neighbors in the modeling set was 0.60
compared to 0.90 for the hits screened from prestwick . it should be
noted that the cutoff value for tc to be defined as the hits by major
commercial packages for virtual screening is 0.80 , when combined with
the 166 maccs structural keys . for our virtual hits screened from
the timtec diversity set 10k , the tc value between hits and their
nearest neighbors in the modeling set was as low as around 0.45 , suggesting
that they are highly structurally different . although these hits are
also predicted to be 5-ht1a actives with a high confidence
by our consensus models as well as rf and svm , it would be interesting
and exciting to test them experimentally , in a hope of revealing 5-ht1a actives with a new scaffold . moreover , the visualization
of the pca plot also confirmed the broader distribution in chemical
space for compounds from the timtec diversity library compared with
timtec gpcr - targeted library ( cf . figure 3 ) . the distribution
of the pairwise structural similarity within the
sets of screening hits from three types of libraries in comparison
to modeling sets using the kernel density plot for tc distribution :
aa , between hits from the prestwick library ; ab , between the prestwick
hits and all modeling set compounds ; ac , between the prestwick hits
and their nearest neighbors in the modeling set . ba , between timtec
gpcr - targeted library hits ; bb , between the timtec gpcr - targeted library
hits and all modeling set compounds ; bc , between the gpcr - targeted
library hits and their nearest neighbors in the modeling set . ca ,
between timtec diversity library hits ; cb , between the timtec diversity
library hits and all modeling set compounds ; cc , between the timtec
diversity library hits and their nearest neighbors in the modeling
set . from all virtual screening hits
chosen by knn ,
15 chemicals were further selected for the experimental testing , including
five compounds from pcl , five from timtec antitarg - g library , and
five from timtec diversity set 10k . the following selection criteria
were used : ( 1 ) high confidence of consensus prediction by rf and svm ;
( 2 ) low structural similarity between hits and the 5-ht1a actives we already known ; ( 3 ) commercial availability . the validations of our in silico
hits by the nimh pdsp yielded many actives that were subsequently
confirmed to have appreciable 5-ht1a binding activity . we should stress that only binary qsar models were used for virtual
screening so no prediction of exact binding affinities ( ki values ) could be made . nine out of 15 in silico hits
were found to have the percent of inhibition equal to or higher than
50% ( i.e. , mesoridazine , clozapine , risperidone and fluphenazine from
pcl ; st030580 from gpcr targeted library ; st023860 , st074311 , st057540
and st066677 from the diversity library ) and five of them displayed
> 95% inhibition at 10 m . the five in silico hits from pcl showed the highest
success rate ( 80% ) , though most of them were similar to the modeling
set compounds ( tc ranged from 0.80 to 0.99 , with an average tc value
of 0.86 ) and no novel core scaffolds were found . mesoridazine
( ki = 33.1 nm ) and fluphenazine ( ki = 145.7 nm ) belong to the typical antipsychotic
class whereas clozapine ( ki = 104.8 nm )
and risperidone ( ki = 427.5 nm ) are atypical
antipsychotics ; all four drugs are commonly used in the treatment
of schizophrenia and bipolar disorder . the known mechanism of action
for mesoridazine as a phenothiazine antipsychotic is through its potent
binding with 5-ht2a and dopamine receptors , whereas the binding with human 5-ht1a receptors have not been reported before . the ki of fluphenazine , clozapine and risperidone for 5-ht1a have been reported elsewhere ; however , this
data was not included in pdsp and was unknown to us at the time of
our computational studies . thus , these observations may be considered
as ( re)discovery of the known mechanism of action for known antipsychotics . to our surprise , only one in silico hit from the gpcrs targeted
library was found to be active ( ki = 243.8
nm ) . this compound , st030580 , showed a quite different ring arrangement
from its nearest neighbor compound in the modeling set , while maintaining
the azaspiro - bicyclic structural element . among the three confirmed
hits from the timtec diversity set 10k , compound st057540 ( also known
as lysergol ( [ ( 8)-6-methyl-9,10-didehydroergolin-8-yl]methanol ) )
yielded 98.20% binding inhibition against 5-ht1a receptor
at 10 m and its ki value was found
to be 2.3 nm afterward ( figure 6 ) . furthermore ,
the tc between this compound and its nearest neighbor in the modeling
set ( i d : 27405 , with dibenzo[de , g]quinolone structure ) is only 0.69 ,
suggesting low structural similarity between this molecule and any
compound in the modeling set . lysergol is an alkaloid of the ergoline
family that occurs as a minor constituent in some species of fungi . lysergol is sometimes also utilized as an intermediate in the manufacturing
of some ergoloid medicines ( e.g. , nicergoline ) . lipinski s rule of five , with a logp value of 1.76 , which is considered
to be ideal for both oral absorption and cns penetration . it was also
predicted to have a very low probability of rapid biodegradation by
epi - suite . literature search indicates
that lysergol binds mainly to the gpcr targets and shows more selectivity
toward 5-ht1 versus 5-ht2 receptors . thus , our
discovery that lysergol is a potent low nanomolar 5ht1a binder confounded by its known high - affinity binding to 5-ht1b and 5-ht1d suggests
that lysergol may find application in treating schizophrenia as was
suggested independently ( e.g. , by groo and palosi ) . two other active hits , compounds st023860 and st074311 ,
also feature relatively different scaffolds in comparison to modeling
set compounds with tc of 0.75 and 0.69 , respectively . in addition ,
one confirmed hit from the diversity set , i.e. st066677 , showed the
tc as low as 0.53 to its most similar compound . these compounds represent
unique scaffolds opening opportunities for the further investigation
of them as well as their close chemical analogs as novel antipsychotic
agents . in summary , the above results once again prove the predictive
power of our binary knn , rf and svm classification
qsar models built from 5-ht1a actives / inactives . these
studies illustrate that qsar models generated by following the validated
qsar workflow , as employed in this paper , could be used as a general
tool for identifying promising hits by the means of virtual screening
of various types of chemical libraries . the full dose response
curve for hit compounds st057540 ( arrow - up
triangles , ki = 2.3 nm ) , st074311 ( arrow - down
triangles , ki = 8194 nm ) and the positive
control , methysergide ( solid squares , ki = 26 nm ) measured by human 5-ht1a receptor radioligand
binding assay . the radioligand is [ h]-8-oh - dpat at the
concentration of 0.5 nm with the standard binding buffer . we should emphasize that
rigorous model validation is an inherent
feature of our qsar modeling workflow . this issue of model validation
has been given a lot of attention by the qsar research community . in the past , most practitioners merely presumed
that internally cross - validated models built from available training
set data should be externally predictive . we and others have demonstrated
that internal validation techniques such as leave - one - out ( loo ) or
even leave - many - out ( lmo ) cross - validation applied to the training
set are insufficient to ensure the external predictive power of qsar
models . thus , we used external 5-fold cross - validation
approach as well as the y - randomization test in this study to ensure
the robustness and predictive power of our qsar models . needless to
say , the use of externally validated models and applicability domains
is especially critical when the models are employed in virtual screening . a set of unique 66 5-ht1a actives from different resources ,
i.e. , wombat library , were further validated by our consensus models . all three qsar methods ( knn , rf and svm ) could accurately
annotate the majority of compounds , with ccrevs ranged
from 0.94 to 0.96 . the success of this independent external validation
reassured us that our qsar models were indeed externally predictive ,
robust , and applicable to virtual screening . we have employed
our qsar models for virtual screening of several
chemical libraries , including two drug - like libraries , two gpcr - targeted
libraries and two diversity libraries . both the global similarity
search ( using ad ) and the subsequent qsar model predictions confirmed
our expectations that drug - like libraries and gpcr - targeted libraries
had a much higher computational hit rate than diversity libraries
when the same n - cutoff values were applied . the screening
hits from drug - like libraries had much higher structural similarity
to our modeling set compounds than hits from gpcr - targeted or diversity
libraries . as described in methods , 15 top hits ( five best from each
of the diversity library , gpcr - targeted library , and drug - like library )
were chosen for the experimental validation and nine out of these
15 compounds suggested by our qsar models were confirmed to be 5-ht1a actives . interestingly , overall the number of screening
hits was higher for the gpcr - targeted library than for the diversity
library ( cf . figure 4 ) as could be expected .
however , the number of experimentally confirmed hits was much higher
for the diversity library ( four out five screening hits were validated
experimentally ; cf . table 2 ) than that for
the gpcr - targeted library ( only one of five screening hits was confirmed ;
cf . interestingly , the most potent
nanomolar 5-ht1a active compound ( compound no . 14 in table 2 ; ki = 2.3 nm ) was identified
from the timtec diversity library , sharing very low structural similarity
( tc = 0.69 ) with its nearest neighbor compound in the modeling set . these findings verified that model - based virtual screening yielded
hits that would not be detected using simple similarity search because
of their structural novelty as compared to the training set compounds . our studies demonstrate that binary classification qsar models
built with dragon descriptors can accurately differentiate true 5-ht1a actives from inactives . a state - of - the - art qsar modeling
scheme was applied , and the models were rigorously validated using
both internal ( multiple training / test set divisions and y - randomization )
as well as external ( 5-fold cv sets ) validation approaches . we have
demonstrated that this strategy afforded multiple qsar models with
high internal as well as external predictive power . the predictors
were further validated on the wombat hits ( 66 literature extracted
compounds tested for 5-ht1a binding ) . we found that our
predictions agreed highly with the experimental annotation of 66 compounds
as 5-ht1a actives as reported in various publications . furthermore , our models used in the most conservative way , i.e. , in
consensus fashion and with the strictest applicability domain criteria ,
identified 43 putative 5-ht1a actives by mining three major
types of screening libraries including drug - like libraries ( wdi and
pcl ) , gpcr - targeted libraries , and diversity libraries . fifteen of
them were tested experimentally in the nimh pdsp at unc - chapel hill
and nine showed significant inhibition activities ( 50% inhibition )
in a single concentration . interestingly , the five virtual hits identified
from the timtec diversity library showed higher success ratio ( 60%
versus 20% ) than the other five from the timtec gpcr - targeted library ;
slightly better results were also reported for the pcl drug - like library
( 80% ) . one compound ( st057540 ) was found to have the highest ki of 2.3 nm among all hits , whereas the tc values
between this compound and its nearest neighbor in the modeling set
( i d : 27405 ) was only 0.69 . it was of great interest to find out that
this compound , lysergol , though used as an intermediate in manufacturing
of some ergoloid medicines , had unclear pharmacological indication ,
but many drug - like chemical properties suggesting that we may have
identified a yet unknown antischizophrenic drug candidate . in summary ,
we have demonstrated that qsar models can be successfully used for
finding promising and structurally diverse hits by the means of virtual
screening of chemical libraries . all qsar models developed and validated
in this study as virtual screening tools to identify 5ht1a ligands
are available from the chembench portal ( chembench.mml.unc.edu ) . | the
5-hydroxytryptamine 1a ( 5-ht1a ) serotonin receptor
has been an attractive target for treating mood and anxiety disorders
such as schizophrenia .
we have developed binary classification quantitative
structure activity relationship ( qsar ) models of 5-ht1a receptor binding activity using data retrieved from the pdsp ki database .
the prediction accuracy of these
models was estimated by external 5-fold cross - validation as well as
using an additional validation set comprising 66 structurally distinct
compounds from the world of molecular bioactivity database .
these
validated models were then used to mine three major types of chemical
screening libraries , i.e. , drug - like libraries , gpcr targeted libraries ,
and diversity libraries , to identify novel computational hits .
the
five best hits from each class of libraries were chosen for further
experimental testing in radioligand binding assays , and nine of the
15 hits were confirmed to be active experimentally with binding affinity
better than 10 m .
the most active compound , lysergol , from
the diversity library showed very high binding affinity ( ki ) of 2.3 nm against 5-ht1a receptor .
the novel
5-ht1a actives identified with the qsar - based virtual screening
approach could be potentially developed as novel anxiolytics or potential
antischizophrenic drugs . |
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use of fixed orthodontic appliances often results in increasing levels of periodontal pathogens in supragingival and subgingival bio - films , causing gingival inflammation during orthodontic treatment [ 1 , 2 ] . during fixed orthodontic treatment , plaque accumulates around the brackets and causes demineralization of the adjacent enamel , which is clinically important . a higher demand for orthodontic treatment has been observed in the past decades in both children and adults . orthodontic therapy is very popular in iran as well and an epidemiological study on iranian schoolchildren showed that about 36.1% of them needed orthodontic treatment . orthodontic procedures were meant to be noninvasive ; but orthodontic therapy using fixed and removable buccal appliances causes iatrogenic side effects like periodontal disease . human oral microflora is highly diverse and may be changed by several endogenous and exogenous factors . literature shows that presence of fixed or removable orthodontic appliances in the oral cavity provides retentive spaces for accumulation of microbial plaque and initiation of caries , which in turn compromise oral health and result in subsequent periodontal infection [ 711 ] . isolation of aerobic and anaerobic bacteria from blood samples of patients with orthodontic appliances highlighted the role of dental biofilm accumulated on these appliances in this respect . three methods are conventionally used for the attachment of wire to brackets : ligation with steel wires , elastomeric ligatures , and self - ligating brackets . all these methods have their own advantages and disadvantages ; but literature suggests that elastomeric ligatures enhance the retention of dental biofilm . candida albicans , known as the most prevalent fungus in oral normal flora , is frequently isolated from dental plaque and carious lesions of children with early childhood caries . streptococcus mutans also appears to be the most commonly involved acidogenic microorganism in development of caries . enterococcus faecalis is an enteric facultative gram - positive bacterium that grows independently in the root canal system causing endodontic infections . forsberg et al . showed that elastomeric rings resulted in accumulation of a greater number of microorganisms in dental plaque around brackets than steel ligatures . using elastomeric rings in orthodontic therapy results in significant accumulation of microorganisms such as s. mutans , c. albicans and a few others in dental plaque around brackets causing enamel demineralization . failure in maintaining proper oral hygiene by orthodontic patients may result in oral conditions like tooth decay and periodontal disease . therefore , cariogenic microorganisms in dental biofilm must be eliminated as much as possible during orthodontic treatment . normally , elastomeric rings are cut by scissors and placed in the mouth without prior disinfection or sterilization . this act has been questioned by the recently suggested biohazard measures , as they may become contaminated during processing and manipulation by dental assistants or orthodontists before final insertion . the rough surface and the absorption properties of elastomeric rings further contribute to the formation of bacterial plaque on their surfaces , resulting in accumulation of higher number of microorganisms on tooth surfaces [ 17 , 18 ] . in order to control dental plaque formation , several chemical mouthwashes are available in the market ; chx is known to be highly effective for prevention of dental plaque formation and elimination of pathogenic microorganisms like s. mutans and c. albicans . in most studies on mouthwashes , chx is used as the gold standard or positive control for evaluation of the efficacy of other products and is routinely prescribed for use by orthodontic patients . however , chx has several side effects such as undesirable tooth discoloration , unpleasant taste and causing dryness and burning sensation in the mouth , leading to patient dissatisfaction [ 22 , 23 ] . left : washing of contaminated ligatures ; right : s. mutans colonies isolated from ligatures recently , use of herbal mouthwashes such as persica and garlic extract has increased by orthodontic patients . persica was reported to be ineffective and garlic extract enhanced biofilm formation by s. mutans on orthodontic wires . streptococcus mutans , which is the major pathogen causing enamel demineralization around orthodontic brackets , has not been fully investigated in terms of its susceptibility to different mouthrinses . it grows in iran , pakistan and afghanistan and is known as avishan shirazi in iran . the main components of the essential oil of this plant include phenolic compounds such as carvacrol , thymol and eugenol . using herbal disinfectants is safer than chemical compounds such as chx . to the best of our knowledge , literature is scarce on the antimicrobial activity of zataria multiflora extract and its application for disinfection of elastomeric ligatures . however , some previous reports have demonstrated the antimicrobial efficacy of z. multiflora extract against yeasts and gram - positive and gram - negative clinical isolates of s. aureus , especially methicillin - resistant staphylococcus aureus ( mrsa ) [ 26 , 27 ] . therefore , this study aimed to evaluate and compare the antimicrobial activity of zataria multiflora extract and chx against microbial colonization on experimentally contaminated iranian and foreign - made elastomeric ligature modules . however several side effects such as undesirable tooth discoloration , unpleasant taste , dryness and burning sensation in the mouth , are reported which discouraged patients to use this mouthwash [ 22 , 23 ] . recently , the use of herbal mouthwashes such as persica and garlic extract by orthodontic patients is increasing . persica was reported non - effective and garlic extract showed to increase biofilm formation by s. mutans to orthodontic wire . streptococcus mutans , which is the major pathogen for demineralization on the enamel surrounding orthodontic brackets , has not been fully investigated . zataria multiflora is an herbal plant belonging to the laminaceae family that geographically grows in iran , pakistan and afghanistan known as avishan shirazi ( in iran ) . the main components of the essential oil of this plant are phenolic compounds such as carvacrol , thymol and eugenol . to our best knowledge , there are nt observed any comprehensive and comparative study on comparison of antimicrobial activity of zataria multifelora essence solution and chlorhexidine for disinfection of elastomeric ligatures in literature . however there are reports those z. multifelora essential oils has significant antimicrobial activity against yeast , gram positive , gram negative , clinical isolates of s. aureus , especially mrsa [ 26 , 27 ] . therefore , the main aim in this study was to evaluate and compare the antimicrobial property of zataria multifelora essence and chlorhexidine on reduction of microbial colonization on experimentally contaminated iranian and foreign elastomeric ligature module . streptococcus mutans ( strain ptcc
1683 ) , enterococcus faecalis ( ptcc 1394 ) , and candida albicans ( ptcc 5027 ) were obtained from iranian research organization for science and technology ( irost ) . in order to have fresh microbial cultures , s. mutans was seeded on mitis salivarius agar ( merck , germany ) containing 1% potassium tellurite , e. faecalis was cultured on blood agar ( merck , germany ) and c. albicans on sabouraud dextrose agar ( oxoid , uk ) plates . fresh colonies of bacterial species and candida were inoculated in brain heart infusion ( bhi ) broth and incubated for 46 hours , to the point when growth was considered to be in the logarithmic phase . the density of the bacterial suspension was adjusted with sterile pbs to that of 0.5 mcfarland standard concentration and used for the experiments . the 0.5mg / ml zataria multiflora extract solution ( barij essence pharmaceutical co , kashan , iran ) was used as mouthwash for evaluation of its antimicrobial effect on tested microorganisms in comparison with 0.02% chx ( shar daroo , iran ) . to evaluate the effect of zataria extract on microbial biofilm formation , we used sterile iranian conventional orthodontic elastomeric polyurethane ligatures ( m.i.b co , iran ) and foreign - made orthodontic elastomeric ligatures with the same shape and composition ( ortho technology , usa ) . several rapid and easy methods have been developed for detection of bacteria among those , viable microbial cell counting ( 28 ) was used in this study . for viable cell counting , 31 iranian and 31 ortho technology ligatures were immersed in s. mutans , e. faecalis and c. albicans suspensions on a reciprocal shaker ( 100rpm ) at 37c for 2 hours . ten rings of each group were randomly selected to determine the adhered viable microbial cells before using any mouthwash and each one was separately transferred to a sterile microtube containing 1 ml of sterile pbs and washed 3 times with pbs and sonicated at 45 kh ( elma , germany ) for 5 minutes . finally , 10 l of sonicated pbs was spread on mitis salivarius agar ( s. mutans ) , blood agar ( e. faecalis ) and sabouraud dextrose agar ( c. albicans ) plates . after incubation for 23 days , bacterial and fungal colonies were counted on each plate , and the number of colony - forming units ( cfus ) was calculated as a percentage of biofilm before performing the disinfection protocol on 21 remaining contaminated rings . the broth macro - dilution test evaluated by clsi ( clinical and laboratory standards institute ) was used to compare the antimicrobial activity of z. multiflora and 0.2 chx as follows : three remaining 21 contaminated iranian and ortho technology orthodontic ligatures were divided into 3 sterile falcon tubes ( n=7 ) containing 2ml of zataria multiflora extract ( as the test ) , 0.2% chx ( as the positive control ) and sterile pbs ( as the negative control ) and incubated on a shaker ( 100 rpm ) for 1 hour . each ligature was then separately transferred to a sterile microtube containing 1ml of sterile pbs , washed for 3 times and then sonicated as explained earlier . finally , 10 l of washed pbs was spread on specific agar plates as explained earlier . the relative number of cfus was calculated and the mean number of viable microbial cells remained on 7 rings decontaminated by each solution after performing the disinfection protocol was calculated and analyzed using wilcoxon , kruskal - wallis and the mann - whitney tests by spss version15 software . streptococcus mutans ( strain ptcc
1683 ) , enterococcus faecalis ( ptcc 1394 ) , and candida albicans ( ptcc 5027 ) were obtained from iranian research organization for science and technology ( irost ) . in order to have fresh microbial cultures , s. mutans was seeded on mitis salivarius agar ( merck , germany ) containing 1% potassium tellurite , e. faecalis was cultured on blood agar ( merck , germany ) and c. albicans on sabouraud dextrose agar ( oxoid , uk ) plates . fresh colonies of bacterial species and candida were inoculated in brain heart infusion ( bhi ) broth and incubated for 46 hours , to the point when growth was considered to be in the logarithmic phase . the density of the bacterial suspension was adjusted with sterile pbs to that of 0.5 mcfarland standard concentration and used for the experiments . the 0.5mg / ml zataria multiflora extract solution ( barij essence pharmaceutical co , kashan , iran ) was used as mouthwash for evaluation of its antimicrobial effect on tested microorganisms in comparison with 0.02% chx ( shar daroo , iran ) . to evaluate the effect of zataria extract on microbial biofilm formation , we used sterile iranian conventional orthodontic elastomeric polyurethane ligatures ( m.i.b co , iran ) and foreign - made orthodontic elastomeric ligatures with the same shape and composition ( ortho technology , usa ) . several rapid and easy methods have been developed for detection of bacteria among those , viable microbial cell counting ( 28 ) was used in this study . for viable cell counting , 31 iranian and 31 ortho technology ligatures were immersed in s. mutans , e. faecalis and c. albicans suspensions on a reciprocal shaker ( 100rpm ) at 37c for 2 hours . ten rings of each group were randomly selected to determine the adhered viable microbial cells before using any mouthwash and each one was separately transferred to a sterile microtube containing 1 ml of sterile pbs and washed 3 times with pbs and sonicated at 45 kh ( elma , germany ) for 5 minutes . finally , 10 l of sonicated pbs was spread on mitis salivarius agar ( s. mutans ) , blood agar ( e. faecalis ) and sabouraud dextrose agar ( c. albicans ) plates . after incubation for 23 days , bacterial and fungal colonies were counted on each plate , and the number of colony - forming units ( cfus ) was calculated as a percentage of biofilm before performing the disinfection protocol on 21 remaining contaminated rings . the broth macro - dilution test evaluated by clsi ( clinical and laboratory standards institute ) was used to compare the antimicrobial activity of z. multiflora and 0.2 chx as follows : three remaining 21 contaminated iranian and ortho technology orthodontic ligatures were divided into 3 sterile falcon tubes ( n=7 ) containing 2ml of zataria multiflora extract ( as the test ) , 0.2% chx ( as the positive control ) and sterile pbs ( as the negative control ) and incubated on a shaker ( 100 rpm ) for 1 hour . each ligature was then separately transferred to a sterile microtube containing 1ml of sterile pbs , washed for 3 times and then sonicated as explained earlier . finally , 10 l of washed pbs was spread on specific agar plates as explained earlier . the relative number of cfus was calculated and the mean number of viable microbial cells remained on 7 rings decontaminated by each solution after performing the disinfection protocol was calculated and analyzed using wilcoxon , kruskal - wallis and the mann - whitney tests by spss version15 software . mean and standard deviation ( sd ) of viable microorganisms attached to iranian and ortho - technology ligatures before and after disinfecting protocol are shown in tables 1 and 2 . as are shown in these tables , a higher density attached viable cells for both tested bacteria on iranian is present in comparison with ortho - technology ligatures before disinfection ( p=0.001 ) , but a statistically significant difference ( p=0.061 ) for attached viable c. albicans on both ligatures before entering to disinfection protocol . mean ( sd ) of attached microorganisms on iranian ligature rings before and after disinfection mean ( sd ) of attached microorganisms on ortho - technology ligature ring before and after disinfection chlorhexidine mouth wash , which used as gold standard in the present study , completely cleaned both ligatures . zataria essence solutions also completely cleaned c. albicans from both iranian and a ortho - technology ligature as there were nt any viable candida cells after decontamination with zataria . there were seen statistically significant differences between both bacterial viable cell counts on both rings before and after disinfection with zataria essence ( p=0.0001 ) . the mean and standard deviation ( sd ) of viable microorganisms attached to iranian and ortho - technology ligatures before and after applying the disinfection protocol are shown in tables 1 and 2 . as shown , viable bacterial cells attached to iranian ligatures had a higher density than those attached to ortho technology ligatures before disinfection ( p=0.001 ) , but attachment of viable c. albicans colony count was not significantly different ( p=0.061 ) before the disinfection protocol . chlorhexidine mouthwash , which was used as the gold standard in the current study , completely disinfected both ligatures . zataria extract solution completely eliminated the c. albicans colonies from both iranian and ortho technology ligatures as there was no viable c. albicans colony after disinfection with zataria extract . statistically significant differences were seen in the viable bacterial cell counts on both rings before and after disinfection with zataria extract ( p=0.0001 ) . however pbs , used as the negative control in the current study , decreased the mean viable microbial count but not significantly ( table 3 ) . p value for comparison of mean viable microbial cells isolated from iranian and ortho - technology ligature rings before and after disinfection greater emphasis has been placed on oral hygiene measures during orthodontic treatment in the recent years . immunocompromised patients and those with systemic conditions receiving orthodontic treatment require additional care because of the risk of bacteremia and infection . results of the current study showed that iranian elastomeric ligatures are more susceptible for attachments of all tested microorganisms in comparison with ortho technology ligatures . the retention of microbial plaque to three different elastic ligatures was determined by condo et al . in an in vivo study . they reported no statistically significant difference among the elastic ligatures ; while a higher microbial colonization was seen on iranian polyurethane ligatures than on polyurethane foreign - made ligatures in the current study . arun et al . compared the s. mutans and lactobacilli counts on stainless steel and elastomeric ligature rings using the same method applied in the current study . they reported significantly higher colonization of both bacteria on elastomeric rings than on stainless steel rings . also , they reported higher colonization of s. mutans than lactobacilli on elastomeric ligatures . in the current study , a higher colonization of s. mutans was seen in comparison with e. faecalis and c. albicans , similar to arun s study . reported a significantly greater number of both aerobic and anaerobic microorganisms on teeth ligated with elastomeric rings than with steel ligatures . forseberg et al . also reported higher colonization of s. mutans on elastomeric rings than on stainless steel ligatures . they also reported a significant increase in the salivary level of s. mutans and lactobacilli after the insertion of fixed orthodontic appliances with elastomeric ligatures . however , two types of elastomeric ligatures were compared in the current study ; our findings showed a higher colonization of s. mutans , which is in agreement with the earlier findings by forseberg et al . turkkahraman et al . also reported higher colonization of microorganisms on teeth ligated with elastomeric rings than steel ligatures ; however , this difference was not statistically significant . one limitation of the current study may be the underestimation of bacterial colonization due to the use of non - elastic rings in the current in vitro study . there are several studies evaluating the effect of different mouthwashes and dentifrices containing various antimicrobial agents on salivary s. mutans and dental plaque of patients with fixed orthodontic appliances with controversial results [ 3 , 24 , 25 , 32 ] . magnusson et al . showed that dentifrices with antimicrobial agents only resulted in small or no change in s. mutans count in the saliva and dental plaque of orthodontic patients . zataria multiflora extract has carvacrol , thymol and linalool in its composition , which have antimicrobial activity . this is an herbal , affordable extract and can be easily accepted by orthodontic patients for disinfection purposes . several reports are available about the antimicrobial activity of zataria multiflora extract against clinical isolates of s. aureus , especially mrsa , e. coli , and yeasts [ 26 , 27 , 35 ] . this study also showed significant antimicrobial activity of zataria extract on c. albicans , s. mutans and e. faecalis . antibacterial activity of zataria solution on s. mutans in comparison with chx in the current study was also confirmed by yaghotti et al . they compared the efficacy of a thymol - based mouthwash ( with constituents similar to those of zataria ) with that of chx against s. mutans and s. sanguinis . gel for treatment of denture stomatitis and showed a significantly greater reduction in surface erythema of the palate than miconazole gel . disinfection of elastomeric ligatures in 5% glutaraldehyde solution is recommended , but literature shows that repeated disinfection of elastomeric ligatures can damage these ligatures [ 37 , 38 ] . evangelista et al . also reported a decrease in tensile strength of elastomeric ligatures , disinfected with phenol and glutaraldehyde . comparison of these results to those of other studies was impossible because there are no published studies assessing bacterial contamination of conventional elastomeric rings and efficacy of herbal disinfection . our findings suggest that further investigations are required to provide detailed information about the commercially available conventional and modified elastomeric rings and the efficacy of herbal mouthwashes available in the market . colonization of s. mutans and e. faecalis on iranian and ortho technology ligatures was significantly different . | objectives : long - term use of orthodontic appliances and fixation ligatures creates a favorable environment for the accumulation of oral normal microflora and increases the risk of enamel demineralization and periodontal disease.the aim of this study was to compare the antimicrobial effects of zataria multiflora extract and 0.2% chlorhexidine ( chx ) mouthwash on experimentally contaminated orthodontic elastomeric ligatures.materials and methods : in this lab trial study , iranian and foreign - made elastomeric ligatures were experimentally contaminated in streptococcus mutans , enterococcus faecalis and candida albicans suspensions .
ligatures were then decontaminated using 0.2% chx as the control , 0.5 mg / ml zataria multiflora extract mouthwashes as the test and phosphate buffered saline ( pbs ) as the negative control for one hour .
antimicrobial properties of both solutions were evaluated by comparing the mean viable bacterial cell count on both rings after decontamination , using spss version 15 software.results:the mean viable bacterial cell count on iranian ligatures was greater than that on foreign - made ligatures before disinfection ( p=0.001 ) , however this difference for c. albicans was not statistically significant ( p=0.061 ) .
chlorhexidine mouthwash completely eliminated all tested microorganisms attached to both elastomeric rings , but zataria extract was only capable of completely eliminating c. albicans from both ligatures .
statistically significant differences were found in viable bacterial counts on both ligatures before and after disinfection with zataria extract ( p=0.0001).conclusion : zataria multiflora extract has antimicrobial properties and can be used for disinfection of elastomeric ligatures . in vivo studies are required to evaluate the efficacy of the incorporation of this herbal extract in mouthwashes for orthodontic patients . |
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thymomas are the most common primary neoplasm of the anterior mediastinum but account for less than 1% of all adult malignancies . thymomas are usually slow - growing tumors manifesting with local extension , which tend to spread along the serosal surfaces , i.e. , along the pleura and pericardium , whereas extrathoracic metastases are uncommon . however , some proposals of revision of the staging system have been offered to identify significant differences in survival between each stage . a large - scale study , which collected thymic epithelial tumors from multiple institutions in japan , demonstrated that the yamakawa and masaoka tnm system classification ( 1991 ) is an excellent predictor of the prognosis of thymic epithelial tumors , including thymic carcinoma , in both overall and tumor - specific survival and that the n and/or m factors influence the prognosis more than the t factor . for the subclassification of the n and/or m factors , large - scale studies , including resectable and unresectable tumors , are necessary . over the past 12 years , the lung disease site group ( ldsg ) has produced 25 evidence - based guidelines on various aspects of lung cancer . despite the advances in the treatment of thymoma that have occurred in the last two decades , there is little definite evidence to form guidelines of best clinical practice . given the rare nature of these tumors and the multiple acceptable approaches to treatment , it was difficult for the ldsg to make definite recommendations . the current data do not adequately address certain controversial topics , which include : ( 1 ) the role of adjuvant treatment in stage ii disease , ( 2 ) the choice , timing , and sequencing of combined modality therapy ( i.e. , surgery , radiotherapy , and chemotherapy ) in stage iii disease , ( 3 ) identifying for whom surgery is appropriate , and whether debulking surgery is appropriate in unresectable or disseminated disease as there is limited evidence to refute or support it , and ( 4 ) determining the best treatment approach to recurrent disease . at present , there is individual and institutional variation in the timing and sequencing of treatment and what extent of disease is regarded as being resectable . a 73-year - old caucasian male , heavy smoker ( 120 pack - years ) , was admitted to the intensive care unit ( icu ) with acute respiratory distress , sofa score evaluation 14 , and curb-65 score 4 . the patient had a history of stage iii copd ( fev1 , 35%pred ) ( gold ers / ats ) and an undiagnosed mediastinal mass of 5 cm in diameter , which had been first diagnosed by ct scan 3 years before . at that time , the patient had refused further investigation of the newly discovered undiagnosed mass . upon admission , his temperature was 39c , blood pressure : 90/70 mm hg , heart rate : 60 beats / min , respiratory rate : 32 breaths / min and blood arterial gas was po2 : 45 mm hg , pco2 : 80 mm hg , ph : 6.50 , and hco3 : 45 mmol / l with fio2 of 21% . the laboratory findings were as follows : white blood count : 20,000/l , c - reactive protein : 22 mg / dl , erythrocyte sedimentation rate : 25 mm , procalcitonin : 2 ng / ml , urea : 59 mg / dl , creatinine : 0.8 mg / dl , k : 4.1 mmol / l , na : 138 mmol / l , sgot : 23 u / l , sgpt : 39 u / l , ldh : 204 u / l , cpk : 15 u / l , alp : 196 u / l , g - gt : 103 u / l . his x - ray presented a small pleural effusion on the left hemithorax , but the significant finding was the dilated upper mediastinum . the undiagnosed mass had an increased size , measuring 13 cm in diameter ( fig . initially , he was treated with a double antibiotic regimen consisting of ceftriaxone and ciprofloxacin for 7 days . after the 7-day regimen , he became afebrile , and we decided to perform a fine needle aspiration ( fna ) biopsy ( fig . 2 ) , instead of choosing the usual surgical method [ 3 , 6 , 7 ] . the biopsy result was positive for egfr expression by immunohistochemistry , and was consistent with the diagnosis of thymic carcinoma . according to the masaoka staging system , the anesthesiologists and thoracic surgeons of our hospital decided that the patient was not a candidate for surgery : based on the stage iii copd with fev1 ( 35%pred ) and an elevated post - intubational pco2 of 60 mm hg during daytime , he would not be able to wean off the mechanical ventilation after surgery . although , multiple chemotherapy regimens with or without radiotherapy have been well established or administered experimentally , we decided to treat the patient with the tk inhibitor gefinitib [ 5 , 8 , 9 ] . the patient had a successful weaning from the mechanical ventilation , but he remained on noninvasive positive airway pressure ventilation , with a full face mask with continuous positive airway pressure ( cpap ) ( 17 cm h2o pressure/24 h daily ) . , he received gefinitib ( 250 mg / daily ) and his disease remained stable as judged by ct scan evaluation ( fig . 3 ) . we can not be certain whether the tumor did not increase in size due to tk inhibitor administration or due to its slow growth , we can only speculate that the tk inhibitor administration had a positive effect because of the positive expression of the egfr . we decided that the patient should receive anticoagulation therapy with low - molecular weight heparin systematically in a daily prophylactic dose , since he would remain bedridden for a prolonged time . unfortunately , the patient died at home on the fifth month after his hospital discharge , and the autopsy revealed massive pulmonary embolism . thymomas usually present in the fourth and fifth decades of life [ 10 , 11 ] . one third to one half of patients present with an asymptomatic anterior mediastinal mass on chest radiograph , one third present with local symptoms ( e.g. , cough , chest pain , superior vena cava syndrome , and/or dysphagia ) , and one third of cases are detected during the evaluation of myasthenia gravis . distant metastases are distinctly uncommon at initial presentation of this tumor . however , when present , the most common metastatic site is the pleura , with involvement of the kidney , bone , and liver ; brain metastases are infrequently seen . the diagnostic work - up after ct scan usually includes surgical excision , which provides the precise histological and staging information necessary for the next therapeutic steps . the most common tumors that the clinician must include in the differential diagnosis of an anterior mediastinal tumor are lymphomas and germ cell tumors [ 11 , 12 ] . it is less often used to make the primary diagnosis of tumors , particularly thymic neoplasms . this is due to problems of sampling error , rarity of thymic tumors , multiplicity of lesions in the mediastinum , and inexperience on the part of the cytopathologist . a common problem that clinical physicians and pathologists have to face when using fna for biopsy is the small amount of material obtained . small sample problem will be encountered more and more nowadays , since less invasive procedures like fna are more frequently preferred and , like in our case , they are successful in providing information regarding egfr - expression status . the same problem is also encountered for lung cancer biopsies , where the obtained biopsy specimens are very small , and in most of the cases , the egfr - positive mutation depends entirely on the right area of the tumor where the biopsy is performed . nevertheless , egfr mutations are rare and a second biopsy should not be repeated unless the first is not positive for malignancy . targeted therapy with tk inhibitors ( erlotinib - tarceva and gefinitib - iressa ) is a well - established therapy for egfr - positive mutation tumors . however , a question remains to be answered : which of the two inhibitors is more efficient ? furthermore , patients who present intense rush due to the tk inhibitor therapy seem to respond positively to this kind of treatment in comparison to patients who do not present intense rush . these patients show better results with smaller doses of tk inhibitors [ 13 , 14 ] . our patient did not present rushes , and unfortunately , although he was under low - molecular weight heparin , a massive pulmonary embolism occurred . this kind of event is expected to occur in patients with an extensive tumor burden . further studies with larger patient series evaluating whether this form of treatment is applicable to this type of cancer are needed . small case studies or published case reports present conflicting data : some demonstrate stable disease or response to tk inhibitors , while others report disease progression [ 14 , 15 ] . in addition to egfr , other parameters could be evaluated in histopathologic specimens such as raf , vegfr , pdgfr , c - kit , flt-3 , hdacs and p38 , and as a result , more treatment modalities could be explored and established as therapy . in conclusion , we are convinced that this form of treatment could be applied in thymic tumors , as shown in published data . however , studies with larger patient series are yet to be completed . in addition , more parameters from systematic analyses of the genetic aberration profiles of these tumors remain to be explored to evaluate the efficacy of other treatment modalities . written informed consent was obtained from the patient upon discharge for publication of this case report and all accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal . written informed consent was obtained from the patient upon discharge for publication of this case report and all accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal . | thymic malignancies are rare intrathoracic tumors that may be aggressive and difficult to treat in advanced stage .
surgery is the cornerstone of the management of thymomas : it is significant for the definite histopathological diagnosis and staging , and in most cases , it constitutes the first step of the treatment strategy . for patients with primary unresectable thymomas , the multimodal treatment schedule nowadays includes neoadjuvant chemotherapy , extensive surgery , adjuvant radiotherapy , and in some cases , adjuvant chemotherapy .
a patient with a history of stage iii copd and an undiagnosed thoracic mass was admitted to the intensive care unit with acute respiratory distress .
a radiologic evaluation by ct scan revealed a mass of 13 cm in diameter at the mediastinum .
fine needle aspiration was performed and revealed a thymoma . due to poor performance status ,
the patient was not able to undergo surgery .
he refused to be treated with neither chemotherapy nor radiotherapy , but due to egfr overexpression , treatment with tk inhibitor was suggested .
fine needle aspiration biopsy is commonly used to identify metastasis to the mediastinum . however , it is less often employed as a primary diagnostic tool for tumors , particularly thymic neoplasms .
the use of targeted therapies for the treatment of thymic malignancies has been described in the literature . over the past years
, significant efforts have been made to dissect the molecular pathways involved in the carcinogenesis of these tumors .
insights have been obtained following anecdotal clinical responses to targeted therapies , and large - scale genomic analyses have been conducted . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
intravitreal injections of dexamethasone ( dex ) and triamcinolone ( taa ) are a mainstay in a clinical retinal practice to treat a wide range of pathologies , such as cystoid macular edema secondary to retinal vein occlusion , diabetic macular edema , choroidal neovascularization in macular degeneration , and noninfectious uveitis [ 16 ] . glucocorticoids have powerful anti - inflammatory action , but the exact underlying mechanism of this effect remains poorly defined . additionally , intravitreal administration of glucocorticoids may lead to side effects such as cataract progression , development of steroid - induced glaucoma , and central serous chorioretinopathy [ 7 , 8 ] . clinical decision making may be greatly enhanced with a better appreciation of the differential gene expression induced by intravitreal steroids . in a previous study using a mouse model , we demonstrated that intravitreal steroids affect the expression of several genetic pathways and alter the balance between neuroprotective and neurodegenerative processes in retina . in this sequel study we used the same model to examine the differential gene expression effects of intravitreal injections of dex and taa in rpe / choroid tissue in vivo , since they too are affected by both disease pathogenesis and intravitreal steroidal treatment at clinically relevant time points of 1 week and 1 month postinjection . the purpose of this study was to evaluate the gene expression effects that intravitreal dex and taa have on the rpe / choroid and the differences between them at clinically relevant time points . this study protocol was approved by the institutional animal care and use committee at the university of california davis before initiation . the study was conducted according to the approved protocol and in accordance with the arvo statement for the use of animals in ophthalmic and vision research . six- to eight - week - old male c57bl/6j mice ( jax - west , sacramento , ca ) were used for all experiments . mice were housed at 21c , under a 12-hour light/12-hour dark cycle , with food and water supplied ad libitum . one ml of ketamine ( 100 mg / ml ) , 0.1 ml of xylazine ( 100 mg / ml ) , and 8.9 ml of sterile water were mixed to prepare a final solution for injection . animals were anesthetized by injecting a 0.1 ml/10 g of body weight intraperitoneally . intravitreal injections were performed transconjunctivally in anesthetized c57bl/6j mice with hamilton 33 g needles delivering 1 l of solution . in group 1 ( n = 4 ) animals received balanced salt solution ( bss ) , group 2 ( n = 4 ) dexamethasone ( 10 g ) , and group 3 ( n = 4 ) taa ( 25 g ) . the solvent for dex and all mice included in this study were injected on the same day at the same time . at weeks 1 and 4 after the injections , the eyes were harvested for ocular dissection and total rna isolation . at days 7 and 30 postinjection mice from each group were sacrificed using a co2 chamber and the rpe / choroid tissue was harvested using published procedures . sample collections were done in the morning , in the period of 1 h. briefly , mouse eyes were removed with curved forceps , the eye glued to the petri dish with superglue , and a circumferential cut was made dorsal to limbus , removing the front of the eye . retinal tissue was removed after severing the optic nerve and the outside of the back of the eye was cleaned of muscles and connective tissue . dissected rpe / choroid was collected within one minute after the sacrifice of the animal , immediately placed in 300 l of rnalater , and stored at 20c . qiagen 's rneasy isolation kit was used to isolate total rna . as a first step tissue was placed in qiazol and pushed through syringe with 17 g needle several times so the pigmented cells of rpe would be released , while sclera stayed intact as a clear sheet and was removed from the tube with needle . since the choroid tends to be firmly attached to the inner sclera , it is likely that most of it stayed attached . rna samples were run on an agilent bioanalyzer microfluidics chip rna nano 6000 to assess quality and quantity . out of four samples , three samples having the highest quality ( rna integrity number ( rin ) 7 value ) were labeled as probes for the affymetrix genechip microarrays ( affymetrix , santa clara , ca ) . rna samples were labeled using 0.100 ug of total rna , following the manufacturer 's protocol for genechip target labeling 3 ivt express kit ( affymetrix , santa clara , ca ) . after labeling , this array contains 45,101 probe sets , analyzing the expression of 39,000 transcripts and variants from over 34,000 well - characterized mouse genes . hybridization was performed in the uc davis genome center microarray core facility using a standard procedure ( affymetrix , santa clara , ca ) . microarray data were analyzed using genespring gx 12.5 microarray analysis software and ingenuity pathway analysis ( ipa ) software for biologically relevant changes of expression in genes and related pathways of the rpe / choroid . one - way anova was used in genespring to identify statistically significant genes at a significance level of p 0.01 . the benjamini - hochberg post hoc correction method was too conservative for microarray results with limited number of biological replicas such as this . no probe sets met this significance level of testing . therefore , a different set of stringency requirements was applied to reduce the risk of false positives . to identify biologically relevant gene expression changes for each of the time point / treatment conditions , volcano plot analysis was performed , using a combination of unpaired t - test and asymptotic p value computation . the volcano plot is a scatter plot of the fold change versus the p value ( in log10p scale ) . it is commonly used to simultaneously depict the p value and the fold change for gene selection cutoff schemes in microarray data . it may be viewed as a summary of statistical significance and biological significance over a large number of genes . we employed the standard approach of using a p value ( p 0.05 ) as the primary criterion followed by fold change ( 1.5 fc 1.5 ) as the secondary criterion to select differentially expressed genes . this approach ensures control of false - positive error and preserves the desired biological significance . the data discussed in this publication have been deposited in ncbi 's gene expression omnibus [ 13 , 14 ] and are accessible through geo series accession number gse49872 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=%20gse49872 ) . supplementary material ( available online at http://dx.doi.org/10.1155/2016/9856736 ) attached to this paper contains the complete list of the differentially expressed genes identified by anova at p 0.01 and volcano plot at ( 1.5 fc 1.5 ) p 0.05 . pathway analysis was performed using ingenuity pathway analysis ( ipa ) software ( qiagen , valencia , ca ) . the lists of candidate genes identified by genespring analysis were uploaded to the ipa site and ipa core analysis was applied to give us the list of activated pathways and gene networks with the highest degree of significance . quantitative pcr ( qpcr ) was used as a second method for validation of the results of microarray data analysis for several candidate genes ( ahrr , arntl , grik1 , lcn2 , per1 , and snap25 ) . all gene expression assays ( taqman , applied biosystems , foster city , ca ) were conducted in the real - time pcr research and diagnostic core facility at university of california davis . control genes ( b2 m , gapdh , and hprt1 ) were also included in order to validate the results of the qpcr . for the genes used for qpcr validation of the microarray data , threshold cycle ( ct ) values were used to determine the quantity of gene copies . for each gene , the fold change for each gene was determined compared to the mean of all 3 control genes used . raw data was imported as .cel files and underwent rma normalization with using baseline values of median of all samples . next step was filtering by expression on the raw data for the signals between 20% and 100% , to eliminate too weak signals which were on the borderline of background noise . the signals for the probes were retained only if they were present in at least 1 out of 6 conditions within the range . the number of 36,136 probe sets passed out of total of 45,101 probe sets on the genechip . statistical analysis by one - way anova identified 1,868 out of 36,136 probe sets differentially expressed at a p value of p 0.05 and 430/36,136 at p 0.01 when the entire 18-chip sample set was considered . comparison of treatment versus control for each time point using an unpaired t - test identified a set of common genes similarly regulated by both steroids , as well as , a set of unique genes differentially regulated by each steroid . figures 1(a ) and 1(b ) represent venn diagrams detailing the numbers of genes in each category per each time point for fold change ( fc ) 1.5 and p 0.05 . a list of common genes and unique genes for each steroid / time point / regulation group is presented in supplementary tables s1s6 . the full lists of differentially expressed genes by anova at p 0.01 and p 0.05 , as well as t - tests 1.5 fc 1.5 , p 0.05 , can be found in the supplementary tables s7 and s8 . biological networks and pathway analysis was performed utilizing ingenuity pathway analysis ( ipa ) software to identify integral biological pathways that are commonly or uniquely affected by the steroids . statistical analysis ( anova ) of the entire 18-chip sample set identified 430 probe sets differentially expressed at p 0.01 . from this data set the top candidate pathway affected was circadian rhythm signaling figure 3 represents the canonical ipa pathway circadian rhythm signaling . in figure 4 the circadian rhythm signaling pathway is overlaid with expression data from each of the time point / steroid combinations . table 1 lists the genes that were identified by ipa analysis as key members of the pathway and their p values . mammalian circadian clock generates molecular circadian rhythm through coupled transcription / translation feedback loops which involves core clock genes : period ( per ) 1 and per 2 , cryptochrome ( cry ) 1 and cry 2 , clock , and aryl hydrocarbon receptor nuclear translocator - like ( arntl or bmal1 ) genes . recently , it has been shown that the transcription factor npas2 ( mop4 ) has been able to functionally substitute for clock in the mouse forebrain , thus adding the total number to 7 genes . the circadian rhythm generation is tissue specific , and changes of the rate of transcription of mrnas or the stability of per and cry proteins affect clock speed . prokineticin receptor 1 ( prokr1 ) , with an alternative name eg - vegfr1 , is a 7-transmembrane receptor protein from rhodopsin family , an antiapoptotic gene , involved in the signaling pathway in neovascularization . the glutamate receptors ( i.e. , grik1 , grm7 , and grin ) are the predominant excitatory neurotransmitter receptors in the mammalian brain . light sets off pacemakers mediated by glutamate ( glu ) , leading to glutamate receptor activation . of note is that most of the clock proteins have been reported to be localized in both the nucleus and cytoplasm [ 17 , 18 ] . at dex 1 week postinjection per1 , per3 , arntl , clock , cry , and grm7 were upregulated , while per2 , grik1 , grin , and prokr were downregulated ( figure 4(a ) ) . treatment with taa 1 week postinjection resulted in per1 , arntl , and clock upregulated , while grik1 , grm7 , prokr , and partially grin were downregulated ( figure 4(b ) ) . at dex 1 month postinjection most of these genes were downregulated , except arntl , which was still upregulated ( figure 4(c ) ) , while with taa 1 month postinjection most of the genes were downregulated except per2 , which was still upregulated ( figure 4(d ) ) . molecular timekeeping in cells ingenuity pathway analysis is a hypothesis generating tool that helps interpret the data in the context of biological processes , pathways , and networks . while each gene can be interpreted as a one - dimensional representation of data , we can think of a pathway ( linked list of interconnected genes ) as a two - dimensional representation of data , while gene networks represent a multidimensional representation of the data . figure 5 is the multidimensional representation of connections between circadian rhythm pathway with glucocorticoid , dopamine , calcium , gaba , glutamate , and vegf signaling pathways via common genes or molecules . because of that interdependence , regulatory changes in one pathway can reflect in many others , in this case , neurotransmitter signaling pathways , causing widespread consequences , some of which are discussed in section 4 . an alternative analysis by volcano plot in genespring ( 1.5 fc 1.5 , p 0.05 ) taking into account only genes that have the highest fold change was done for each steroid / time point to identify additional signaling pathways unique for each steroid / time point . in this section are highlighted changes in gene expression of genes of interest for ophthalmic diseases , neurodegeneration and neuroprotection , and related signal transduction pathways . the top 6 relevant canonical pathways for each time point with the significance of representation identified by ipa are presented in figures 6(a)6(d ) . the top 16 genes with altered expression in this group are all involved in visual perception and phototransduction . most of these genes were upregulated compared to controls ( snap25 , sag , rho , cnga1 , gnat1 , rcvrn , neurod1 , pde6 g , guca1a , cacna2d4 , pde6b , tulp1 , fscn2 , rdh12 , and olfm3 ) , and one ( grik1 ) was downregulated ( table s1 ) . the most upregulated gene in dex 1 week postinjection was snap25 with a fold change of 12.73 . this protein is regulated by optineurin , a pathogenic gene associated with primary open angle glaucoma ( poag ) . the most affected pathway for taa in week ca signaling has a role in neurotransmitter signaling , molecular transport , and vitamin and mineral metabolism . modulation of migration and ca signaling in retinal pigment epithelium cells has been observed as an initial step of proliferative vitreoretinopathy and in angiotensin mediated ca signaling during pathologic neovascularization and inflammation . the genes involved in ca signaling that were affected were nachr , nicotinic acetylcholine receptor , which was downregulated , ccl28 , chemokine ( fc = 1.56 ) , trdn , triadin ( fc = 2.09 ) , asph , aspartate - beta - hydroxylase ( fc = 1.87 ) , ptger3 , prostaglandin e receptor 3 ( fc = 2.25 ) , sln , and sarcolipin ( fc = 2.56 ) . interestingly , the most upregulated gene was lcn2 , lipocalin 2 , an innate immunity protein , with a role in cellular response to oxidative stress , drug insult , il-1 response , and tnf - alpha response ( fc = 19.3 ) . the most downregulated ones were crystallins ( cryba1 , crygs and crybb2 , cryaa , cryba4 , crygb , and crygc ) , genes whose protein products play a role in development of the eye , in visual perception , and have a role as heat - shock proteins ( table s2 ) . the genes arntl , per1 , gnb3 , and arrdc2 were upregulated ( fc = 1.552.33 ) , while abra , ptger3 , dpp4 , chrna1 , ifi204 , murc , itsn1 , prokr1 , and ddit4l were downregulated ( fc = 2.42 to 1.56 ) . only three genes were found in common for both dex and taa : myh8 , tpm3 , identified as self - antigen in patients with behcet 's disease with posterior uveitis , and elov1 , with a role in fatty acid biosynthesis . ten genes had significantly altered expression following dex at 1 month postinjection ( table s4 ) . one of the upregulated genes was slc18a2 , a solute carrier with a role in neurotransmitter transport , as well as serotonin and dopamine receptor signaling [ 23 , 24 ] . the second gene was foxs1 , a member of the notch signaling pathway , which has a role in vasculogenesis , as well as insulin receptor and vegf receptor signaling . downregulated was nsg2 , neuron specific gene , part of the dopamine receptor signaling pathway . there were eight affected genes in this group ( table s5 ) , the most interesting being the downregulation of glutamate receptor grm7 . this receptor has a role in neuroprotection , synaptic transmission , and regulation of ca ion transport via voltage gated ca channel activity . as a general observation and contrary to the data of steroid effects in the retina , there were far fewer genes with an altered expression at 1 month than at 1 week postinjection . only one gene was common for both steroids after a t - test with fc = 1.5 , p < 0.05 ( table s6 ) . that gene was fibrillin2 ( fbn2 ) , an extracellular matrix constituent , with ca ion binding capabilities , mildly upregulated for both steroids ( fc = 1.51.6 ) . independent qpcr validation of representative differentially expressed genes was performed using commercial gene expression assays ( taqman , applied biosystems ) . the fold changes and their direction were confirmed for the chosen genes , thus corroborating the microarray data analysis results . rpe dysfunction plays a role in many ocular disorders including age - related macular degeneration , central serous chorioretinopathy , white - dot syndromes , and retinitis pigmentosa , which can lead to permanent visual distortion and blindness . one of the main functions of the rpe is regulation of disc shedding by photoreceptors , which is a rhythmic process essential for photoreceptor health and proper function . the mammalian retina has its own circadian clock mechanism , originally thought to be located in the photoreceptors [ 2931 ] . more recent studies have reported the presence of a circadian clock in rpe cells [ 3234 ] . it has been shown that signaling is mediated by the dopamine d1 receptor and gaba [ 35 , 36 ] . the mammalian retinal circadian clock mechanism is autonomous from the central master circadian clock , which in mammals resides in the hypothalamus . the cyclic pattern of expression of circadian proteins is dependent on a number of external cues or the primary and the strongest zeitgeber is light , which starts the process , and then there are many secondary ones , conveying the information to peripheral tissue . secondary ones start with serotonin and melatonin and then there are more downstream ones , such as insulin and glucocorticoids [ 38 , 39 ] . glucocorticoids have been demonstrated to be implicated in the entrainment of circadian rhythms [ 4042 ] . they were shown to be capable of overcoming other entrainment factors regulating the peripheral oscillators in the kidney and lung and also act as strong entraining signals for peripheral circadian oscillators and even feedback on central oscillators . in our study this finding suggests that intravitreal injection of steroids has a significant effect on the rpe / choroid , causing dysregulation of the retinal circadian clock , which is observable at 1 week postinjection and continues through 1 month postinjection . disruption of circadian rhythm has been demonstrated to promote inflammation [ 4547 ] and has been implicated as a contributing factor to disease pathogenesis [ 48 , 49 ] . have shown in a diabetic mouse model that dysregulation of circadian rhythmicity resulted in increased retinal inflammation . since both inflammation and the physiological glucocorticoid metabolism are involved with circadian rhythm , it is not surprising that this is a major pathway influenced by steroid administration . although the clinical importance of this finding is not clear , we suggest that the effect of steroids on the expression of circadian rhythm genes may be part of their therapeutic anti - inflammatory action and is not necessarily an adverse effect . on the other hand , it has been reported that the release of glucocorticoid hormones happens in an hourly or ultradian rhythm in a pulsating manner and that levels of free glucocorticoid hormones are highly synchronized between blood , subcutaneous tissue , and the brain . by administering a bolus of steroids , this fine regulation is disrupted , and the loss of the ultradian rhythm may subsequently result in gene expression changes in downstream target tissues . our results demonstrated that the effect of both steroids on circadian rhythm gene expression was much more marked after 1 week than after 4 weeks . this may be reflecting the initial effect following the administration of a single intravitreal dose , which is subsequently waning . accordingly , it is possible that the use of a sustained - release implant , such as ozurdex or iluvien , may achieve a more stable concentration and lead to prolonged effects on rpe / choroid gene expression . these implants have already proven their longer - lasting therapeutic effect in a variety of diseases [ 2 , 4 , 5255 ] . the most affected pathway for dex week 1 postinjection is phototransduction pathway ( figure 6(a ) ) . why genes that belong to the phototransduction pathway change in the rpe / choroid was puzzling . we do not believe that retinal tissue contamination was the cause of this , since mouse retinal tissue is very loosely attached to rpe and upon dissection separates very easily and cleanly , so there is very little , if any , chance of contamination of rpe with retinal cells . to verify whether some of the rod and cone genes are actually expressed in rpe , we checked for the presence of several phototransduction genes listed in the tables from newman et al . ( table s7 ) and found several of them differentially expressed in rpe of amd versus normal ( gngt1 , guca , pde6 , both cone and rod specific , and rgs9 ) . our data suggests that their altered expression levels are a result of the dex treatment of the eye . we hypothesize that apparent upregulation of expression of these genes may have possibly resulted from accumulation of these mrnas due to slowed down phagocytic and autophagic processes that are dysregulated by steroid therapy . another one of the top five activated pathways in dex 1 week postinjection is gaba receptor signaling ( figure 6(a ) ) . the gaba transporter gat , located in the plasma membrane of nerve terminals and glial cells , plays an important role in the termination of synaptic transmission . this neurotransmitter has also been implicated in effecting the circadian rhythm [ 35 , 36 ] , and therefore its activation may reflect the significant alterations demonstrated in the gene expression of this pathway . since both dex and taa were shown to alter the gene expression of the circadian rhythm pathway , it is not surprising that they both altered the gaba receptor signaling pathway as well . the main signal transduction pathways that appear to be activated by steroids in the rpe are glutamate and ca signaling . light sets off pacemakers mediated by glutamate ( glu ) , leading to glutamate receptor activation [ 57 , 58 ] . a recent paper by suwanjang et al . shows how those two pathways are interconnected with glucocorticoid signaling : glucocorticoids reduce the intracellular ca concentration in neurons and astrocytes and protect neurons against glutamate toxicity . l - glutamate is the major excitatory neurotransmitter in the central nervous system that activates both ionotropic ( nmdar ) and metabotropic ( grm ) glutamate receptors . glutamanergic neurotransmission is involved in most aspects of normal neural function , such as intracellular signal transduction , axonal targeting , and synaptic clustering . our results show upregulation of grm7 with dex and downregulation with taa at 1 week . at 1 month grm7 , glutamate receptor 7 , with a role in neuroprotection , synaptic transmission , and regulation of ca ion transport via voltage gated ca channel activity , has been shown to confer susceptibility to many neuropathologic conditions such as age - related hearing impairment [ 57 , 60 , 61 ] , attention - deficit / hyperactivity disorder [ 58 , 62 ] , and schizophrenia [ 6365 ] . two genes that were consistently downregulated at both time points for taa were rbfox1 and neuronal mrna splicing factor fox-1 , which splices mrnas encoding proteins important in synaptic transmission and membrane excitation . a similar protein in the mouse may be involved in regulating the effects of dopamine in the basal ganglia . at dex 1-month postinjection time point , two members of dopaminergic neurotransmission were dysregulated , slc18a2 and nsg2 . our results from differential gene expression of over 34,000 well - characterized mouse genes of the rpe / choroid of c57bl/6j mice after intravitreal steroid injections at 1 week and 1 month postinjection suggest that dysregulation of glutamatergic and dopaminergic neurotransmission signaling may represent a novel target for therapeutic approaches in the treatment of steroid - induced glaucoma . in conclusion , in our previous study , we have reported that intravitreal dex and taa resulted in gene expression changes which potentially altered the balance between neuroprotective and neurodegenerative processes in the retina . in this study , using the same mouse model , we investigated the effects of these steroids on gene expression in the rpe / choroid and found that the major influenced pathways are circadian rhythm and several neurotransmission pathways . comparative analysis of differential gene expression that takes place in different tissues following the administration of dex and taa suggests tissue - specific changes . the activation of signal transduction pathways in the rpe / choroid may mirror or complement the changes in neuroprotection and neurodegeneration processes that occur in the retina following intravitreal steroid administration . the effect on circadian rhythm pathway the effect of both dex and taa was more significant at 1 week than at 4 weeks , which implies that a more controlled slow - release form of administration may achieve longer - lasting cellular and genetic effects . in conclusion , our findings have the potential to contribute to a more complete understanding of the mechanisms by which the steroids dex and taa regulate gene expression in the eye and to the development of improved therapeutics . | purpose . to identify retinal pigment epithelium ( rpe)/choroid genes and their relevant expression pathways affected by intravitreal injections of dexamethasone and triamcinolone acetonide in mice at clinically relevant time points for patient care
. methods .
differential gene expression of over 34,000 well - characterized mouse genes in the rpe / choroid of 6-week - old c57bl/6j mice was analyzed after intravitreal steroid injections at 1 week and 1 month postinjection , using affymetrix mouse genome 430 2.0 microarrays .
the data were analyzed using genespring gx 12.5 and ingenuity pathway analysis ( ipa ) microarray analysis software for biologically relevant changes .
results . both triamcinolone and dexamethasone caused differential activation of genes involved in
circadian rhythm signaling pathway at both time points tested .
triamcinolone ( taa ) uniquely induced significant changes in gene expression in
calcium signaling ( 1 week ) and glutamate receptor signaling pathways ( 1 month ) .
in contrast , dexamethasone ( dex ) affected the
gaba receptor signaling ( 1 week ) and serotonin receptor signaling ( 1 month ) pathways .
understanding how intraocular steroids affect the gene expression of rpe / choroid is clinically relevant . conclusions .
this in vivo study has elucidated several genes and pathways that are potentially altering the circadian rhythms and several other neurotransmitter pathways in rpe / choroid during intravitreal steroid injections , which likely has consequences in the dysregulation of rpe function and neurodegeneration of the retina . |
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pneumonia is defined as being nosocomial or hospital - acquired ( hap ) if it becomes apparent > 48 h after admission to the hospital . pneumonia in ventilated patients is defined as ventilator- associated pneumonia ( vap ) and considered as a subset of hap . the risk of developing pneumonia is particularly high in patients requiring mechanical ventilation . in intensive care patients , pneumonia is the most common hospital - acquired infection . according to data of the european point - prevalence study epic , nosocomial pneumonia accounts for 47% of all icu - acquired infections . during the past two decades , gram - positive bacteria ( mostly staphylococci and enterococci ) have been becoming increasingly prevalent in the spectrum of nosocomial pathogens besides gram - negative bacteria . among gram - positives , this increase in gram - positive bacteria is related to demographic changes as well as to an increase in the number of high - risk patients and intensive care measures . extensive use of broad - spectrum antibiotics with predominantly gram - negative coverage and the emergence of multi - drug resistant staphylococci and enterococci in the community setting further contribute to this trend . according to the data of the kiss study performed in germany , gram - positive pathogens cause a major fraction of nosocomial pneumonias , staphylococcus aureus being the most important pathogen in ventilator - associated pneumonia , followed by pseudomonas aeruginosa , klebsiella pneumoniae and escherichia coli . of note , in that large study ( n = 16,351 ) there was no difference in the rank of pathogens between early onset hap ( up to day 4 after hospitalization ) and late onset vap . predictabilities of the occurrence of pathogens were similar and independent on pre - defined time frames for " early " and " late " onset . primarily due to issues in definition , the exact attributive mortality of hap remains a matter of debate . however , it has been confirmed by large observational studies that pneumonia is the major cause of sepsis . sepsis can progress to severe sepsis and septic shock , which is associated in a drastic rise in mortality . thus , early and appropriate treatment of infection should interrupt this progression and improve patient outcome . indeed , clinical studies have univocally shown for both , sepsis and hap , that i ) inadequate ( i.e. insufficient level of agent at the site of infection ) , ii ) inappropriate ( i.e. not covering the right pathogen or pathogen resistant to agent ) or iii ) delayed antimicrobial therapy is associated with increased pneumonia mortality as well as increased length of hospital stay and costs . a recent study by kumar et al . investigating 5,715 patients with septic shock ( leading focus pneumonia ) in three countries found that staphylococcus aureus was the leading individual bacterial pathogen accounting for 20% of all cases of inappropriate antimicrobial treatment , which was associated with dramatic decrease in survival . staphylococcus aureus was the only pathogen that correlated with mortality in a multiple logistic regression analysis carried out in a large retrospective cohort study of inpatients with culture - positive pneumonia in the usa . mrsa is growing in prevalence and is now endemic in many healthcare facilities and communities . in 2003 , > 60% of staphylococcus aureus isolates from us - icus were methicillin - resistant . in europe , there is a north to south trend in the proportion of s. aureus that is methicillin- resistant , ranging from 0% in northern european countries to > 50% in more southern countries ( european antimicrobial resistance surveillance system . some strains of mrsa , particularly those of community origin [ community - acquired mrsa ( ca - mrsa ) ] , produce the panton - valentine leukocidin ( pvl ) toxin , which is associated with necrotizing infections , often in previously healthy individuals . pvl - producing strains may become of increasing importance if camrsa strains continue to invade the hospital setting but they will not be discussed further in this article in order to maintain a focus on nosocomial pneumonia in which pvl - producing strains are much less prevalent . background : vancomycin is a glycopeptide antibiotic that disrupts cell wall synthesis in gram - positive bacteria by inhibiting peptidoglycan biosynthesis . an auc / mic ratio of 400 has been advocated as a target to achieve clinical effectiveness with vancomycin . animal studies and limited human data appear to demonstrate that vancomycin is not concentration dependent and that the auc / mic is a predictive pharmacokinetic parameter for vancomycin for review see . pros : vancomycin is currently ( still ) the treatment of choice in mrsa pneumonia . complete resistance to vancomycin is conferred by the vana determinant and has yet been detected only in individual patients . cons : however , there are some clinically important disadvantages regarding the use of vancomycin for the treatment of pneumonia : i ) mics have been observed to be increasing over time ( mic creep ) . such isolates are still considered to be susceptible per definition since the mic is below defined breakpoints . nonetheless , relationship between increased mic and treatment success or failure in serious staphylococcus aureus infections has recently been established . failure rates exceeding 60% for staphylococcus aureus displaying a vancomycin mic value of 4 mg / l prompted recommendations in 2006 from the clinical and laboratory standards institute to lower the breakpoint for susceptibility from 4 to 2 meanwhile , a number of studies have established a relationship between vancomycin treatment failures and infections in patients with methicillin - resistant staphylococcus aureus displaying an mic of 2 mg / l ( for review see . this trend has urged the idsa to issue " vancomycin therapeutic guidelines " that recommend to aim for trough serum vancomycin concentrations of 15 - 20 mg / and to maintain trough serum vancomycin concentrations always > 10 mg / l to avoid the development of resistance . further , alternative treatment is recommended for isolates with mic > 2 mg / l , because a targeted auc / mic of 400 is not achievable with conventional dosing . ii ) vancomycin penetrates poorly into the lung , only < 12% of plasma levels are detected in pulmonary epithelial lining fluid at therapeutic doses which is associated with pneumonia treatment failure despite the in vitro susceptibility of the bacterial isolates [ 25 - 27 ] . iv ) vancomycin is associated with nephrotoxicity and ototoxicity , although the frequency of these adverse reactions was higher in early reports and is attributed to the low purity of early formulations . nephrotoxicity due to vancomycin is of great concern due to the contribution of acute kidney injury to poor clinical outcome in critically ill patients in the icu , a population particularly vulnerable to mrsa infection [ 30 - 33 ] . the disadvantages of vancomycin can in part be compensated by adding a second antibiotic with mrsa activity and good tissue penetration , e.g. rifampin . in vitro studies combination of vancomycin with rifampin has been used since the 1970s and has been advocated by some opinion leaders despite the lack of data from randomized controlled trials ( rct ) . recently , the first rct from korea with 83 enrolled cases demonstrated superiority of vancomycin / rifampin versus vancomycin alone in patients with mrsa pneumonia : the clinical cure rate in the modified intention - to - treat population was 53.7% ( 22 of 41 ) in the vancomycin plus rifampin group , and 31.0% ( 13 of 42 ) in the vancomycin - only group ( p = .047 ) . however , it has to be considered that resistance to rifampin evolves easily and broad use of this combination may result in a fast increase of resistance to rifampin . another drawback of rifampin is drug interactions that limit its use in critically ill patients and patients under immunosuppressive therapy . while macrolides , tetracyclines , and aminoglycosides inhibit the elongation of already initiated peptide chains , linezolid prevents the initiation of protein synthesis by blocking the assembly of a functional initiation complex . this mechanism of action precludes cross - resistance with other protein synthesis inhibitors ( for review see . in vitro studies have shown that the antibacterial activity of linezolid is concentration - dependent rather than time - dependent with auc / mic as the best parameter predicting the in - vivo efficacy . linezolid has activity against gram - positive pathogens , including bacteriostatic in vitro activity for staphylococci , but has limited activity against gram - negative bacteria . pros : linezolid is besides vancomycin currently the only of the novel drugs with mrsa - activity that is approved for the treatment of nosocomial pneumonia in the usa and europe , including cases caused by mrsa . in the usa it represents the only alternative to vancomycin for this indication . pulmonary penetration of linezolid is good : 415% of plasma levels were recovered in epithelial lining fluid . in contrast to other antibacterial drugs used in the treatment of severe gram - positive infections ( glycopeptides , lipoglycopeptides , quinupristin / dalfopristin , daptomycin , tigecycline , cephalosporins with mrsa - activity ) , linezolid is available for both intravenous and oral administration . the bioavailability of oral formulations approaches 100% , while food has no relevant effect on drug exposition . the elimination half - life is in the 4 to 5 h range requiring twice daily administration . resistance was first observed in a clinical staphylococcus aureus isolate in 2001 , although the leader surveillance program has shown that 99.55% of isolates remained susceptible to linezolid in the usa in 2006 . cons : thrombocytopenia is a commonly observed adverse reaction to linezolid therapy , with occurrence rates of around 30% [ 49 - 51 ] , a rate much higher than that reported in phase 3 trials . thrombocytopenia is more common following prolonged treatment ( > 14 days ) and in patients with renal insufficiency . the inhibition of mitochondrial protein synthesis by linezolid - studied in one case by de vriese - can result in potentially severe clinical effects including optic / peripheral neuropathy and lactic acidosis . these events are not frequently observed and are mostly reversible following termination of linezolid treatment , but there are reports of severe irreversible effects , such as permanent blindness , in patients treated only for a short time . as a reversible , non - selective monoamine oxidase inhibitor , linezolid in combination with serotonergic agents has been associated with serotonin syndrome . the linezolid licence recommends that treatment should be restricted to a maximum of 28 days . two retrospective subgroup analyses of ventilated and non - ventilated patients with mrsa from nosocomial pneumonia clinical trials showed that linezolid - treated patients had higher survival and clinical cure rates than vancomycin - treated patients . it has been suggested that this may be due to the favourable intrapulmonary distribution of linezolid . however , the viability and validity of these subset analyses has been questioned , such that further trials are required before linezolid can be recommended to be used preferentially over vancomycin for the treatment of mrsa pneumonia . a recent trial of patients with mrsa - vap , which was designed and powered to demonstrate " non - inferiority " of linezolid to vancomycin demonstrated numerically better values for linezolid - treated patients compared with van - comycin - treated patients with respect to microbiological eradication ( 56.5% and 47.4% , respectively ) , clinical cure ( 66.7% and 52.9% , respectively ) , survival rate ( 86.7% and 70.0% , respectively ) and length of hospitalization ( 18.8 and 20.1 days , respectively ) , ventilation ( 10.4 and 14.3 days , respectively ) and icu stay ( 12.2 and 16.2 days , respectively ) . for the small number of subjects in this study these differences were not statistically significant . a recent meta - analysis including 9 trials comparing glycopeptides ( 7 vancomycin and 2 teicoplanin ) with linezolid found that the linezolid versus glycopeptide analysis showed a clinical cure relative risk of 1.01 ( 95% confidence interval , 0.93 - 1.10 ; p = .83 ; i(2 ) = 0% ) and a microbiological eradication relative risk of 1.10 ( 95% confidence interval , 0.98 - 1.22 ; p = 0.10 ) . in contrast , they found that the risks of thrombocytopenia ( relative risk , 1.93 ; 95% confidence interval , 1.30 - 2.87 ; p = 0.001 ) and gastrointestinal events ( relative risk , 2.02 ; 95% confidence interval , 1.10 - 3.70 ; p = 0.02 ) were higher with linezolid , but they did see no differences for renal dysfunction or all - cause mortality ( relative risk , 0.95 ; 95% confidence interval , 0.76 - 1.18 ; p = 0.63 ) . however , only 3 of these studies enrolled exclusively patients with pneumonia . despite using the same trials for calculation , a second meta - analysis found also no difference in treatment success for patients pneumonia ( or = 1.16 , 95% ci 0.85 - 1.57 ) but a trend for better eradication rates in all microbiologically assessed patients for linezolid ( or = 1.33 , 95% ci 1.03 - 1.71 ) . they found no difference in total adverse effects ( or = 1.14 , 95% ci 0.82 - 1.59 ) but more nephrotoxicity in patients receiving vancomycin ( or = 0.31 , 95% ci 0.13 - 0.74 ) . as clinical data on linezolid in hap / vap are inconclusive so are the recommendations of current guidelines . the ats / idsa guideline on hap / vap does not advocate to preferred use of linezolid in patients with hap / vap . in contrast , the german sepsis guideline recommends linezolid in proven mrsa pneumonia and expressively warns to rely on vancomycin mono - therapy in such cases . in order to resolve these questions pfizer initiated a - probably final - international phase 4 study to compare linezolid to vancomycin for the treatment of nosocomial pneumonia proven due to mrsa . the complete results of that study have not been published yet , but preliminary results were presented at the 48annual meeting of the infectious diseases society of america ( idsa ) in boston on october 23 , 2010 by jean chastre ( abstract # 5047 ) . the zephyr ( linezolid in the treatment of subjects with nosocomial pneumonia proven to be due to methicillin - resistant staphylococcus aureus ) study was the largest ever conducted in this population . investigators from 156 centers worldwide randomized 1,225 patients , of whom 448 patients had proven mrsa nosocomial pneumonia ( modified intent - totreat group ) ; 339 patients also met key protocol criteria at the end of study ( per - protocol group ) and were included in the primary analysis . in the per protocol group , 125 patients treated with linezolid ( 68.3% ) and 140 ( 74.5% ) treated with vancomycin were ventilated at baseline . mean vancomycin through level on day 3 was 14.1 mg / l ( 95% ci 2.8 - 50.8% ; n = 140 ) . interestingly , clinical response rates in the mitt were not influenced by vancomycin through levels . clinical success rates at the end of study were 57.6 percent ( 95/165 ) for patients treated with linezolid compared with 46.6 percent ( 81/174 ) for patients treated with vancomycin in the per - protocol group , the primary endpoint . these results demonstrated that linezolid achieved a statistically significantly higher clinical success rate compared to vancomycin ( 95% ci for the difference in response rates : 0.5% , 21.6% ; p = 0.042 ) . results were consistent for the per - protocol group at end of treatment and for all mrsa pneumonia subjects ( modified intent - to - treat ) at end of treatment and end of study . microbiologic success was also consistent in both the per - protocol and the modified intent - to - treat groups at both end of treatment and end of study . thrombocytopenia ( linezolid 1.3% , vancomycin 2.2% ) and renal failure ( linezolid 3.8% ; vancomycin 7.2% ) occurred at relatively low rates . there is growing evidence that vancomycin , despite low resistance rates is a suboptimal therapeutic option in critically ill patients , particularly in patients with pneumonia . disadvantages of vancomycin are i ) slow bactericide action , ii ) poor penetration into pulmonary tissue , iii ) the globally slowly increasing vancomycin mics ( " creep " ) that result in increased clinical failure despite being susceptible according to defined break points and iv ) nephrotoxicity . vancomycin has been shown to be less effective than -lactams for the treatment of mssa infections [ 26,67 - 69 ] and beta - lactams should be used when possible . in mrsa infections at least poor tissue penetration and bactericidal effect can be improved by combination with rifampin according to in vitro and animal studies and one prospective randomized controlled trial . however , this approach has serious limitations such as the risk of emerging of rifampin resistance and drug interactions . for mrsa pneumonia daptomycin is inactivated by surfactant and will not be investigated for pneumonia , tigecyclin ( ceftazidim ) was significantly inferior to the comparator imipenem ( vancomycin ) in a prospective study in patients with nosocomial pneumonia . quinupristin - dalfopristin is approved for the treatment of mrsa pneumonia in some european countries , but not in the usa . the ats / idsa nosocomial pneumonia guidelines do not recommend quinupristin - dalfopristin for the treatment of mrsa pneumonia due to clinical cure rates being lower than vancomycin in clinical trials . in fact , linezolid is currently the only licensed and well studied alternative to vancomycin in mrsa pneumonia . conflicting results of smaller prospective studies , meta - analysis of these studies and retrospective observations can now be replaced by the new findings of the zephyr study , advocating the superiority of linezolid over vancomycin in proven mrsa pneumonia . | the incidence of nosocomial pneumonia involving methicillin - resistant staphylococcus aureus strains ( mrsa ) is on the rise worldwide . for years
, vancomycin has been used as the drug of choice in the treatment of mrsa infections and was recommended as such by clinical guidelines .
there is growing evidence that vancomycin , despite low resistance rates is a suboptimal therapeutic option in critically ill patients , particularly in patients with pneumonia .
disadvantages of vancomycin are i ) slow bactericide action , ii ) poor penetration into pulmonary tissue , iii ) the globally slowly increasing vancomycin mics ( " creep " ) that result in increased clinical failure despite being susceptible according to defined break points and iv ) nephrotoxicity .
in contrast to other novel antibiotics with mrsa activity , linezolid is currently approved for the treatment of nosocomial pneumonia in the usa and europe .
several studies have compared vancomycin with linezolid for nosocomial pneumonia with conflicting results .
this review compares both substances regarding pharmacodynamics , resistance , safety and clinical efficacy and discusses preliminary data of the zephyr study .
this study compared linezolid versus vancomycin in patients with proven mrsa pneumonia and was the largest trial ever conducted in this population . |
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this genus comprises of gram - negative , strictly - aerobic , non - fermenting , non - fastidious , non - motile , catalase - positive , oxidase - negative bacteria with a dna g + c content of 39% to 47% . according to euzeby 's list of prokaryotic names with standing in nomenclature ( http://www.bacterio.cict.fr/a/acinetobacter.html ) acinetobacter junii was proposed by bouvet and grimont in 1986 ; it was isolated from human clinical specimens , with characteristics corresponding to those of the genus acinetobacter . the organism in this study is a. junii strain mtcc 11364 equivalent to dsm 14968 (= cip 107470 ) isolated from wastewater treatment plant . this organism was previously known as acinetobacter grimontii
, and was further re - classified as a later synonym of a. junii by vaneechoutte et al . in 2008 . a. junii strain mtcc 11364 was obtained from mtcc and grown on tryptic soya agar medium ( tsa ; himedia ) at 30 c . genomic dna was extracted from 36 hour old culture using zr fungal / bacterial dna miniprep as per manufacturer 's instructions . amplification and sequencing of 16s rrna were performed as described by mayilraj et al . in 2006 . to determine the phylogenetic relationship of strain mtcc 11364 , the 16s rrna sequence consisting of 1502 bp was compared with those of type strains of species of related genera and identification of phylogenetic neighbors and the calculation of pairwise 16s rrna gene sequence similarities were achieved using the eztaxon server and aligned using mega version 5.0 . bootstrap analysis was performed to assess the confidence limits of the branching ( fig . 1 ) . the genome of a. junii mtcc 11364 was sequenced using the illumina - hiseq 1000 technology . sequencing resulted in 29,216,732 paired - end reads ( insert size of 350 bp ) of length 101 bp . a total of 28,584,052 high - quality reads with approximately 810 coverage were assembled with clcbio wb6 ( word size 40 and bubble size 60 ) to obtain 248 contigs ( n50 , 24,956 bp ) with 3,549,566 bp and an average g + c content of 38.0% . the functional annotation was carried out by rast ( rapid annotation using subsystem technology ) , fig . 2 shows the subsystem distribution of strain a. junii strain mtcc 11364 , trna was predicted by trnascan - se 1.23 and rrna genes by rnammer 1.2 . the genome includes 3 rrna genes ( 5s , 23s , 16s ) and 64 aminoacyl - trna synthetase genes . a total of 3294 coding regions ( 1605 genes transcribed from the positive strand and 1689 from the negative strand ) were found in the genome , of which 2332 ( 71% ) could be functionally annotated . the genome coding density is 86% with an average gene length of 907 bp . the annotated genome has 77 genes responsible for resistance to antibiotic and toxic compounds including 10 genes for mdr efflux pumps . fifty one genes code for proteins that are involved in oxidative stress response , seven genes for osmotic stress response , fifteen genes for heat shock and several more genes for various other stress responses , to make a total of 96 genes involved in stress response . the functional comparison of genome sequences available on the rast server revealed the closest neighbors of a. junii mtcc 11364 as a. junii sh205 ( score 512 ) followed by acinetobacter baumanii acicu ( score 485 ) , acinetobacter haemolyticus atcc 19194 ( score 471 ) and a. baumanii ab0057 ( score 465 ) . the a. junii strain mtcc 11364 whole genome shot gun ( wgs ) project has been deposited at ddbj / embl / genbank under the project accession asyz00000000 of the project ( 01 ) that has the accession number asyz01000000 and consists of sequences asyz01000001-asyz01000248 . the authors declare that there is no conflict of interest on any work published in this paper . | the genus acinetobacter consists of 31 validly published species ubiquitously distributed in nature and primarily associated with nosocomial infection .
we report the 3.5 mb draft genome of the acinetobacter junii strain mtcc 11364 .
the genome has a g + c content of 38.0% and includes 3 rrna genes ( 5s , 23s , 16s ) and 64 aminoacyl - trna synthetase genes . |
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this is an acute trust providing a wide range of hospital services such as a and e , surgical and medical specialties , therapy services , maternity and pediatric care . patients aged between 18 and 60 , who have been diagnosed hypertension and are on anti - hypertensive ( at least one ) for last 6 months are included in this study . patients diagnosed with hypertension but of less than 6 months duration were also excluded . patients taking other drugs along with anti - hypertensive respondents were selected daily by convenience sampling from the hypertensive patients who attended outpatient clinic at nhs hospital . the data collection tool used was a questionnaire adapted from morisky self - reported medication adherence questionnaire relating to medication use and major reasons for non - adherence . it is 4-item questionnaire with a high reliability and validity , which has been particularly useful in chronic conditions like hypertension . it measures both intentional and unintentional adherence based on forgetfulness , carelessness , stopping medication when feeling better and stopping medication when feeling worse . the scale is scored 1 point for each no and 0 points for each yes . the total score ranges from 0 ( non - adherent ) to 4 ( adherent ) . the study was conducted for 4 months on participants who consented to participate in the study . it is important to consider the rights of respondents in every research ; therefore the rights of respondents during the interviews were well - respected . in morisky 's medication adherence scale questionnaire , a no answer was allocated a score of 1 while a yes answer was given a score of 0 . hence a score of 4 would designate patient as fully adherence while a score of 0 would tag him as fully non - adherent . similarly , a score of 1 , 2 and 3 would specify patient as 25% , 50% and 75% adherent respectively . patients who score 75% or more were considered as adherent while those who score less than 75% were termed as non - adherent . furthermore , written consent was obtained from the respondents prior to participation in the study . this is an acute trust providing a wide range of hospital services such as a and e , surgical and medical specialties , therapy services , maternity and pediatric care . patients aged between 18 and 60 , who have been diagnosed hypertension and are on anti - hypertensive ( at least one ) for last 6 months are included in this study . patients diagnosed with hypertension but of less than 6 months duration were also excluded . patients taking other drugs along with anti - hypertensive respondents were selected daily by convenience sampling from the hypertensive patients who attended outpatient clinic at nhs hospital . the data collection tool used was a questionnaire adapted from morisky self - reported medication adherence questionnaire relating to medication use and major reasons for non - adherence . it is 4-item questionnaire with a high reliability and validity , which has been particularly useful in chronic conditions like hypertension . it measures both intentional and unintentional adherence based on forgetfulness , carelessness , stopping medication when feeling better and stopping medication when feeling worse . the scale is scored 1 point for each no and 0 points for each yes . the total score ranges from 0 ( non - adherent ) to 4 ( adherent ) . the study was conducted for 4 months on participants who consented to participate in the study . it is important to consider the rights of respondents in every research ; therefore the rights of respondents during the interviews were well - respected . in morisky 's medication adherence scale questionnaire , a no answer was allocated a score of 1 while a yes answer was given a score of 0 . hence a score of 4 would designate patient as fully adherence while a score of 0 would tag him as fully non - adherent . similarly , a score of 1 , 2 and 3 would specify patient as 25% , 50% and 75% adherent respectively . patients who score 75% or more were considered as adherent while those who score less than 75% were termed as non - adherent . furthermore , written consent was obtained from the respondents prior to participation in the study . the age of majority of the participants were between 30 and 40 ( n = 78 , 43.3% ) while only 36 participants ( 16% ) belonged to the age group of 50 - 60 [ table 1 ] . the result also showed that majority of the participant were female ( n = 123 , 61.5 ) . the overall adherence rate was found to be 79% ( n = 158 ) , however only 36% ( n = 72 ) of participants were fully adhering to the prescribed medications . the results showed that women were higher in numbers but their adherence rate was low was compared to their male counterparts as only 74.7% ( n = 92 ) females were complying with the physicians order , of which those of fully adhere to the medicines were only 34.1% ( n = 42 ) . conversely , the rate of adherence among males were high ( 85.7% , n = 66 ) although those of completely adhere to antihypertensive medicines were moderate in numbers ( 39% , n = 30 ) as shown in table 2 . age distribution of study participants estimation of morisky 's medication adherence scale by participants gender moreover , when medication adherence was linked with age groups of participants it was revealed that majority of participants aged between 30 and 40 and this class of age also possesses the higher rate of adherence ( 82% , n = 64 ) . interestingly , it was noted that participant who showed 100% adherence were mainly the oldest ones ( i.e. 50 - 60 years ) where 52.7% of participants of that group were absolutely adherent . the age group where absolute adherence was minimum was 40 - 50 years ( 19% , n = 7 ) as mentioned in table 3 . estimation of morisky 's medication adherence scale by participants age the summary of the reasons of non - adherence is shown in table 4 . 69% ( n = 461 ) reasons mentioned by the participants who did not adhere to anti - hypertensive medicines were from intentional non - adherence and the major reason was the fear of side - effects ( 25.4% , n = 170 ) followed by inconvenience of taking medicines outside home ( 17% , n = 114 ) . the least affecting intentional reason was fear of taking too many drugs at the same time ( 3.2% , n = 21 ) . furthermore , unintentional reasons account for 31% ( n = 207 ) of non - adherence among hypertensive patients . the non - intentional reason which is most frequently quoted by the patients was forgetfulness ( 22.4% , n = 149 ) while the reason which is least mentioned by the participants was cost of medicines ( 2% , n = 13 ) . medication adherence is an important tool that can increase treatment effectiveness , however literature has shown that the rate of adherence in chronic disease like hypertension is very low and thus it is an important problem in the management of diseases which require long - term treatments . it is higher than the medication adherence to antihypertensive medications reported in egypt ( 74.1% ) , malaysia ( 44.2% ) , gambia ( 27% ) , pakistan ( 57% ) and korea ( 61.1% ) respectively . the difference is adherence rate could be due to cost of medical care and drugs , better care services and patient awareness about medication adherence . majority of the subjects participated in this study was between 30 and 40 years of age and the same age group shows maximum adherence ( n = 64 , 82% ) to antihypertensive medications . however study conducted in north america showed that participants aged 30 - 40 years have low adherence when compared to elder ones . this discrepancy in result could be due to the reason that people of old generation have strong false belief such as fear of taking medication , side - effects of medication and so they are more often surrounded by the myths about their disease and medicines which could enhance rate of non - compliance . another possible reason for this difference in results could be due to the poor understanding of hypertension by this age group and the reluctance of accepting hypertension as a major threatening disease . furthermore , in developing countries like pakistan it was reported that older patient showed better compliance and it was mainly due to better social support structure supported by extended family system which have resulted in improved medication adherence . another study reported that inadequate health literacy , impaired cognition and decline in functions make elderly patients more prone to non - adherence . this study also focused on associating medication adherence with gender and it was found that in male patients adherence to anti - hypertensive medications was on higher side as compared to their female counterparts ( 87.7% vs. 74.7% ) . this finding is in line with a study conducted in us , where sex was a significant predictor of adherence and men were more likely to be adherent then women . in contrast , another study reported that the adherence rate between men and women were almost equal ( 61.4% vs. 60.9 ) . similarly , few researches have shown that women are more likely to adhere to anti - hypertensive medications as most men are unaware to their hypertension and those whose who are aware are less likely to be taking their medication in a way as prescribed to them . furthermore , another study conducted on chinese hypertensive participants showed that female patients were positively associated with anti - hypertensive mediations . the disparity of results among studies highlights the issue of sex differences in barriers to anti - hypertensive medication adherence . poor sexual functioning and body mass index of 25 kg / m or more are the factors associated with low medication adherence to anti - hypertensive medications in males while dissatisfaction with communication with health care provider and depressive symptoms were associated with low adherence in females . the study revealed that side - effect of medications , a form of intentional non - adherence , was the most common factor reported by participants . many studies have supported this result by reporting that side - effect is one of the most important determinants of adherence in hypertensive patient . it is therefore needed that health care provider must increase patients knowledge and understanding about the disease and the pharmacotherapy to increase the likelihood of medication adherence . the other approach could be to design a regime which caters an individual need of a patient in terms of affectivity and tolerability . this could sometimes prove to be very hazardous for patients as they often rely on self - thought approaches and try to double the dose to compensate for the missed dose . the possibilities could be a regular follow - up with effective counseling and by using real time medication monitoring system . the urbanization effect has also influenced medication adherence as busy work schedules often force patient to omit doses while at work due to busy schedules and hence inconvenience is what they face while they are outside home . this study supports this statement as inconvenience of taking medicines outside home was the second most reported reason of intentional non - adherence . this result is also uniform with another past research which has shown that busy life - style is an important barrier to medication adherence in hypertensive population . this study has some limitations such as ; self - reporting was the only method employed in this study which is subjective in nature and may have under estimated the status of non - adherence when compared to objective measures of non - adherence such as pill count and prescription refills . however , morisky et al . have reported that the self - report approach of measuring adherence is simple , inexpensive and useful way of identifying non - adherence in clinical setting . this study did not include hypertensive patient from in - patient setting and also excluded those who are taking medication for other co - morbidities . hence the extent of generalizability is limited to similar patients ; nonetheless the study provides some useful results which would be helpful in improving the rate of medication adherence in hypertensive population . overall , more than three - fourth of the hypertensive participants were found to be adherent to their treatment . the study also revealed major factors such as side - effects , forgetfulness , busy schedule , inconvenience of taking medicine outside home are major barriers to anti - hypertensive medications . there is a need to tailor the therapy according to individual need of the patients to maximize the adherence and accomplish the eventual goal of controlling blood pressure . | background : hypertension is a silent killer , a time bomb in both the developed and developing nations of the world .
it is one of the most significant risk factors for cardiovascular morbidity and mortality resulting from target - organ damage to blood vessels in the heart , brain , kidney and eyes .
adherence to long - term therapy for chronic illnesses like hypertension is an important tool to enhance the effectiveness of pharmacotherapy.objective:the two objectives of this study were to evaluate the extent and reasons of non - adherence in patients attended national health service ( nhs ) hospital , sunderland.materials and methods : the study was conducted for 4 months in the out - patient department of nhs hospital .
a total of 200 patients were selected randomly for this study .
morisky 's medication adherence scale was used to assess the adherence rate and the reason of non - adherence .
data were entered and analyzed using microsoft excel 2010.results:the overall adherence rate was found to be 79% ( n = 158 ) .
adherence rate in females were low was compared with their male counterparts ( 74.7% vs. 85.7% ) .
the higher rate of adherence was found in age group of 30 - 40 years ( 82% , n = 64 ) .
the major intentional and non - intentional reason of non - adherence was side - effects and forgetfulness respectively.conclusion:overall , more than three - fourth of the hypertensive participants were found to be adherent to their treatment . on the basis of factors associated with non - adherence , it is analyzed that suitable therapy must be designed for patients individually to increase medication adherence and its effectiveness . |
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muscle contraction is due to cyclic interactions between
myosin ii
motor proteins and actin filaments powered by the cellular fuel adenosine-5-triphosphate
( atp ) . in in vitro assays one uses surface adsorbed
myosin motors , or preferentially myosin motor fragments such as heavy
meromyosin ( hmm ) , to propel fluorescent actin filaments . whereas such
assays have inspired a range of nanotechnological applications , actin and myosin have not previously been interfaced with vertical
nanowire arrays . we chose gap as our material system because
of the established biocompatibility of gap nanowires and the generally advantageous optical properties of the
iii v semiconductor materials . nanowires were produced by gold - particle - seeded metal
organic vapor phase epitaxy ( movpe ) ( figures 1a and s1 ) . because nanowire
growth is initialized by gap assembly at the interface of the substrate
to the gold particle , the wire size and array geometry ( figures 1a and 2a ) can be defined
by electron beam lithography ( ebl)-defined gold patterning . the use
of regular nanowire spacing enables the growth of arrays with uniform
wire length . after testing several surface derivatization procedures
( see supporting information , results section )
with respect to actomyosin motility quality ( speed and fraction of
mobile actin filaments ) we chose atomic layer deposition of al2o3 on nanowires ( figure 1a ) and surrounding areas , creating suitable surface contact angles
for motility without the need for prior
silanization . together with the gap nanowire core diameter ( 3050
nm in different arrays ) , the 60 nm of al2o3 coating
resulted in a total nanowire diameter of 150171 nm ( 160
nm unless otherwise stated ) . the wire length was either 0.96
0.03 or 5 0.2 m ( mean standard deviation ) in
different arrays . in vitro motility studies were performed by infusing hmm ( 120 g / ml ) into flow
cells formed by a chip with nanowire arrays ( figure 1 ) as the ceiling and a coverslip as the floor . irrespective
of the actual geometry , top and bottom
in the text below are defined as indicated in figure 1b . the hmm - induced sliding of alexa fluor 488 phalloidin labeled
actin filaments was observed after infusion of assay solution ( 1 mm
mgatp ; 2225 c ) . we used a nikon eclipse te300 inverted microscope with
hg - lamp illumination , nikon 100 1.4 na oil immersion objective ,
fitc filter set [ excitation , 465495 nm ; dichroic mirror cutoff ,
505 nm ; emission barrier filter , 515555 nm ] and a hammamatsu
emccd camera . further details of the experimental methods are given
in the supporting information , methods
section , e.g. , quantitative analysis of the motility quality of filaments
moving up and down wires ( supporting figures s2s3 ) . principle of experiments . a , nanowires are grown from gold seed
particles ( i ) by metal organic vapor phase epitaxy ( ii ) and coated
with al2o3 by atomic layer deposition ( iii ) . b , schematic illustration of fluorescently labeled actin filaments
( red ) that are propelled by hmm motors adsorbed to the al2o3 surface of nanowires . note , illustration not to scale . nanowire arrays . a , scanning electron microscopy
( sem ) image of
nanowires in 50 50 m array with 1 m
interwire distance and 5 m nanowire length . b , fluorescence micrograph from in vitro motility assay experiment
with the same type of array geometry as in panel a before the addition
of atp . actin filaments
( bright fluorescent lines ) are aligned with nanowire arrays being
attached to hmm on the tip of the nanowires . strongly fluorescent
spots on nanowires attributed to light - guiding from fluorescent actin
filaments interacting with the wires along their length . occasional
fluorescent spots outside the array in panel b ( arrows ) are attributed
to actin filaments attached to nanowires spuriously formed by parasitic
growth ( examples of such wires also indicated in panel a ) and are
therefore in focus . we found that actin filaments
can be bound to and propelled by
hmm adsorbed to al2o3-coated gap nanowires . first , we infused fluorescent actin filaments in the absence of atp
where hmm is expected to bind the filaments but not propel them . under
these conditions , filaments attached themselves horizontally across
the top ( figure 1b ) of a nanowire array , apparently
binding to multiple nanowire tips ( figure 2 ) . in addition , several strongly fluorescent spots were associated
with individual wires in sparse ( interwire distance 1 m ; figure 2 ) arrays and along the edges in denser arrays ( interwire
distance < 300 nm ; not shown ) . we attribute these spots to filaments
that are attached vertically along individual wires ( see below ) . upon
addition of atp , we observed actin motility on areas surrounding the
nanowire arrays ( figure 3a ) as well as on top
of arrays having interwire distance of 1 m ( figure 3b ; supporting information , movie 1 ) and < 300 nm ( figure 3c ; supporting information , movie 2 ) . the filaments
on the top moved from tip to tip , and , ultimately , some of them were
propelled vertically down the wires ( figure 3d ) . occasionally , filaments were also observed climbing up wires
from the surrounding surface ( figure 3e ) . however ,
observation of the latter phenomenon with 5 m long nanowires
required refocusing of the microscope because the underlying substrate
and the top of the nanowire array were located in different focal
planes ( figure 3f ) . this fact was important
as it also enabled us to unambiguously identify the plane in which
filaments were moving ( see also supporting information , figure s4 and movie 3 ) . a , motility on al2o3 surface outside
autofluorescent array illustrated by maximum pixel intensities over
50 consecutive images at 2.5 s frame rate . b , the maximum
pixel intensity in sequence of 220 images at 2.5 s frame rate for array with 1 m interwire distance and 5 m
nanowire length . arrows show starting points for some trajectories
of actin filaments sliding on top of the array . c , image sequence
of array with circular and square nanowire patterns connected by zigzag
lines with nanowires . scanning electron
micrograph in inset showing area on another array similar to that
in circle in main figure ) . d , e , time sequence of filaments climbing
down and up a nanowire , respectively . for dashed rectangles in panel
d , see figure 4c . the crosses in panel e indicate
tracking of the trailing filament end to estimate the length of the
filament segment that is vertically attached along the nanowire ( fluorescent
spot of increasing intensity ) at each given point in time . f , fluorescence
images where focus changed from the bottom surface ( left ) to the top
of the wires ( right ) . see also supporting information movie 3 and figure s4 for similar effects of change in focus on
another area . a climbing event , whether
up or down a wire , was associated with
an initial rapid increase in intensity of a fluorescent spot centered
on the wire ( figure 3 ; supporting information , movie 1 ) , followed by rapid decline
in fluorescence of this spot ( see further below ) . such intensity variations
( blinking ) were not observed in the absence of atp ,
and they were seen in all cases where the filament transition from
horizontal to vertical movement could be directly observed . by observing individual
actin filaments first approach and then climb up or down individual
nanowires , we could identify and quantify light guiding of fluorescent
emission from actin filaments specifically bound to nanowires . on
a sparse array ( nanowire length 5 m , interwire distance 1 m ) ,
filaments that climbed up or down wires were visible from the top
as strongly fluorescent spots ( figure 3b ; also
figure 2b , figure s2 and supporting information , movie 1 ) with a time - dependent intensity
proportional to the fraction of the filament length attached vertically
along the wire via hmm . importantly the number of fluorophores along
the wires , at each given point in time , is directly proportional to
the filament length along the wire in a well characterized way . this
follows because there is precisely one binding site for the aph fluorophore
per actin monomer . further , the distance between
the monomers is well - known giving 362
aph binding sites per m ( see further supporting
information , methods ) . thus , taking advantage of this homogeneous
fluorescence labeling , we were able to estimate , with good accuracy ,
the number of fluorophores on the filament segments along the wire
by tracking the trailing end of individual filaments whose leading
end progressed up or down the wire ( figure 3e ) . results of such tracking for three filaments climbing 5 m
long nanowires are illustrated in figure 4a , together with intensity
measurements that show progressive linear increase in intensity ( itip ) emitted from the nanowire tip as the filament
length along the wire increases . further , this intensity saturated
at low values ( figure 4b ) for nanowires with
length of less than 1 m ( supporting information , figure s5 ) , whereas the maximum intensity itip for filaments moving along 5 m long nanowires increased
in proportion to the maximum intensity ( ih ; proportional to length ) measured when the filaments moved horizontally .
the results for the two wire lengths show that only the part of the
filament held close to the wire surface contributes significantly
to the fluorescence signal . more importantly , however , the results
in figure 4a , b for 5 m long wires can not
be explained by , for example , the projection of fluorescence of vertically
aligned filaments because the depth of focus of our objective ( < 0.6
m ; figure 3f ; supporting
information , figure s4 and movie 3 ) , was much too limited to
collect light from all along the nanowire . instead , light - collection
and light - guiding by the nanowire is necessary to integrate the filament
fluorescence intensity into a nearly diffraction limited spot , in
the focal plane of the wire tip . this view is further supported by
the effects of changes in focus on the intensity ( supporting information , figure s4 ) measured within a 0.5
0.5 m roi centered on a nanowire . a , average background - subtracted pixel fluorescence intensity from
the tip of nanowires ( 0.5 0.5 m region of
interest ( roi ) ; 16 bit images ; intensity units , iu16 ) plotted
against the distance that three different filaments ( different symbols )
have moved down a wire of 5 m length . the nonzero intercept
on the vertical axis is due to the tip of horizontally moving filaments
entering the roi without movement of the trailing filament end that
is tracked . this may occur for filaments moving on top of a sparse
nanowire array as the number of strongly attached actin propelling
heads on the wire tips vary with time . thus , the leading filament
end would temporarily move faster than the tracked trailing end due
to buckling caused by previous faster sliding of the trailing end . b , total background - subtracted fluorescence
intensity ( itip ; average intensity
number of pixels ) observed from nanowire tip due to nanowire - climbing
filaments plotted against the total intensity ( ih ) of the same filaments when sliding on top of the array ( for
5 m high nanowires ; filled circles ) or on the surface between
nanowires ( 0.96 m wires ; open squares ) . measurements using
matlab after conversion to 8 bit images corresponding to units , iu8 . c , time course of the fluorescence signal at three different
locations as indicated in figure 3d , illustrating
the potential to use the light guiding effect for fluorescence detection
with increased signal / noise ratio . measurement using imagej and 16
bit images showing average intensity per pixel . for details of imagej
and matlab - based analyses , interestingly , the behavior
was different in denser nanowire arrays
( < 300 nm spacing ) where similar apparent light - guide effects were
mainly observed at edges ( indicated by strong fluorescent spots in
figure 3c ; see also supporting
information , movie 2 ) . quantitatively , we estimate that
50% of the light emitted
from fluorescent actin filaments attached vertically along wires in
sparse ( 1 m interwire distance ) arrays is emitted from their
tip . we draw this conclusion , first , from the measured slope near
unity ( 1.04 ; 95% ci [ 0.91 , 1.17 ] ) of the observed linear relationship
( r 0.91 ) between itip and ih for nanowires of 5 m
length ( figure 4b ) . second , we reasonably assume
that less than 50% of the light from a filament ( moving in an optically
homogeneous environment across the nanowire tips ) is available for
collection by the objective . we further note that our experimental
setup does not fulfill any geometrical conditions for intensity enhancement
( neither of itip nor ih ) by fluorescence interference contrast effects . our results are thus consistent with the notion
that the entire emitted fluorescence intensity from a fluorescence
labeled actin filament , held by hmm along a nanowire in a sparse array ,
couples into the wire and that 50% of the light intensity is emitted
at the wire tip , whereas 50% enters the surface substrate . in this
context it is important to note that zincblende gap has a direct band
gap of 2.78 ev , corresponding to 450 nm light wavelength . this should
be compared to excitation wavelengths in the range 465495
nm and emission in the range 500600 nm ( observed in range
515555 nm with the current microscope filter set ) . therefore ,
the gap nanowires and the underlying gap substrate should absorb and
reflect light only to a minor degree . our observations of efficient
coupling of light into the gap nanowires
and light guiding along these wires are consistent with previous findings
using other semiconductor materials . in those prior studies , photonic
confinement was shown for a range of nanowires ( e.g. , zno , cds , their high refractive index and the surrounding dielectric material
allow for wave - guiding with amplified output . furthermore , enhanced
light trapping and emission has been observed using light coupled
into tips of iii v semiconductor nanowires that are grown in
ordered arrays and coated with a dielectric material with a smaller
refractive index than the core ( e.g. , al2o3 ) . the degree of enhancement appeared to depend
on fine - tuning of the dimensions of the iii this is consistent
with our observations of different degrees of light guiding for different
interwire distances and suggests that there is appreciable room for
optimization of light collection and emission in future experiments . our demonstration of
light - guiding of gap nanowires in a biological buffer has a range
of interesting applications in biosensing ( see also ref ( 5 ) ) . thus , the nanowires would
perform well as optical sensors that capture analyte molecules via
recognition elements ( e.g. , antibodies or oligonucleotides ) immobilized
on their large surface areas . if fluorescent secondary recognition
molecules are sandwiched via the analytes to the nanowires , light
intensity proportional to the number of such recognition molecules
would be emitted from the wire tip . in the following , we illustrate
and quantify the advantages of this approach using a specific example
where the velocity and fraction of motile actin filaments are evaluated . high - throughput assays with this capability are of considerable interest
per se , e.g. , to evaluate effects of drugs on actomyosin motility . the efficient detection of actin filaments is also important in motor - driven diagnostics and biocomputation devices . the
achievable s / n ratio for the detection of actin filament speed using
nanowire arrays is much higher than that achievable on flat substrates
and automated read - out is greatly simplified ( see below ) . we quantify
the fraction of motile filaments by the ratio of the numbers of time - varying
and time - invariant fluorescent spots ( figure s2 , supporting information ) and determine the filament velocity
from the rate of change of the fluorescence intensity ( figure 4a ; see also figure s3 , supporting
information ) . the fraction of motile filaments and velocity
for nanowire - climbing actin filaments determined in this way was 81
3.8% ( mean standard error of the mean ; se ; n = 136 filaments ) and 2.18 0.38 m / s ( n = 10 filaments ) , respectively , compared to 84 3.9% ( n = 83 ) and 5.16 0.15 m / s ( n = 22 ) , respectively , for a standard trimethylchlorosilane ( tmcs)-derivatized flat surface . crucially , however , the time - varying
fluorescence intensity for a filament moving down a nanowire was detected
with considerably higher s / n ratio than for a horizontally moving
filament ( figure 4c ) . to exemplify , for two
different filaments the s / n ratio was 13 and 17 , respectively , when
moving down a wire ( using a region of interest [ roi ] of 0.5
0.5 m ; figure 4c ) compared
to less than 4 when the filaments moved on top of the nanowire array
( whether using small , 0.5 0.5 m or large
rectangular rois ; figure 3d ) . qualitatively
similar data were observed for several more filaments and also for
filaments that first moved on the flat underlying substrate and then
climbed up wires . under the present , far from optimized conditions
( see below ) , the
limit of detection ( lod ) was 50100 fluorophores on individual
wires as suggested by observation of the photobleaching of stationary
actin filaments attached via hmm to the wires , assuming 362 fluorophores
per m of an actin filament ( see above and supporting information , methods ) . here , the lod was defined
as the number of fluorophores that could be detected with s / n ratio
of 3 or greater . the number of 50100 fluorophores per wire
corresponds to the amount of protein accumulating on the surface of
a nanowire with 100 nm diameter in less than 1 h at 1 pm solution
concentration . however , importantly the
lod is probably somewhat lower than 50100 fluorophores as
there is most likely less than 362 monomers per m of the filament ,
e.g. , due to prior photobleaching events . averaging the signal
over a large number of wires ( nwire ) would
increase the s / n ratio in proportion to ( nwire ) . for instance , a 100-fold
increase in s / n ratio would be achieved by averaging over 10 000
nanowires , which could be readily fitted into < 100 100 m area . this would allow detection of analyte at 1 pm concentration
in minutes or 10 fm concentration in an hour . unlike in the present study where actin filaments were bound to
only a small fraction of the nanowires , recognition elements and analytes
in biosensor applications the averaging should therefore
be readily automated , e.g. , by appropriately adjusting array interwire
distance , objective magnification , and ccd pixel size to each other . several additional improvements would markedly increase the s / n
ratio and further lower the lod . this includes oblique illumination
of the observation area together with reduced flow - cell height for
reduced out - of - focus background and use of laser illumination instead
of an hg lamp to increase excitation intensity at 480 nm wavelength . furthermore , a major source of noise , absent in a general biosensor
application , is attributed to fluorescent actin filaments floating
in solution . these unbound model analytes were not eliminated by rinsing
as would be implemented for any other analyte , following specific
binding to recognition molecules on the wires . finally , it is likely
that a theoretical analysis of the underlying optical mechanisms would
allow optimization of the in- and out - coupling of light , further enhancing
s / n ratio . thus , the fluorescence wavelength , the nanowire material ,
length , diameter , spacing , and the thickness of the oxide shell might
affect coupling of light into the wire . this coupling may also depend
on the distance of the fluorophore to the nanowire surface ( 40 nm
for hmm - actin ) , the position of the emitting
fluorophores along the wire , and the polarization
of fluorescence . a second
key result of our study is the unique method to unambiguously demonstrate
specific analyte detector interaction using molecular motor
propelled cytoskeletal filaments . this was enabled by ( i ) measurement
of the length of overlap between a fluorescence labeled filament and
the long axis of the nanowire , ( ii ) a well - defined number of fluorophores
per m of the filament , and ( iii ) molecular - motor driven variation
of the filament nanowire overlap . here we used heavy meromyosin
propelled actin filaments labeled with a maximum of one fluorophore
per monomer ( 362 monomers/m ) via the strong phalloidin actin interaction . however , other
motor systems , such as the kinesin - microtubule system , may also be
used , enabling wide applicability due to different dependence of motor
function on surface chemistry ( cf . ref ( 36 ) ) for the two systems . to the best of our
knowledge , the present results also constitute the first demonstration
of actomyosin motility on al2o3 substrate and
on vertical nanowire arrays , opening for a range of opportunities . first , the observed climbing of filaments along nanowires enables
three - dimensional motor driven lab - on - a - chip devices . second , the
observation of actin filament propulsion across the tips of 160 nm
wide nanowires at 1 m interwire spacing opens for unique studies
of actomyosin function . for instance , it will allow testing of hypotheses
about the motility of actin filaments over a sparse layer of motors with minimal interference from surface they
are also of interest for studying contractile events at low myosin
densities on actin filaments as occurs physiologically at low activation
levels in muscle cells when only a fraction of the regulatory units
along the filament are turned on to allow myosin binding . for developments
of functional assays it may be beneficial to make the vertical parts
of the nanowires resistant to hmm binding in analogy to a previous
study where kinesin propelled microtubules were observed moving between
tips of micrometer wide poly(dimethylsiloxane ) ( pdms ) pillars . we have demonstrated
light guiding with
high efficiency along al2o3 coated gap nanowires
of appreciable relevance for biosensing applications . with further
improved s / n ratio as discussed , sensor elements based on individual
vertical nanowires could achieve fm pm sensitivity , and arrays
of such nanowires could perform appreciably better . the use of molecular
motor driven propulsion of fluorescent filaments along the nanowires
to detect specific molecular interactions may have broad applicability
in the evaluation of biosensor performance . in addition , our findings
lay the foundations for novel types of fundamental studies and nanotechnological
applications of molecular motors . | semiconductor nanowire arrays offer
significant potential for biosensing
applications with optical read - out due to their high surface area
and due to the unique optical properties of one - dimensional materials .
a challenge for optical read - out of analyte - binding to the nanowires
is the need to efficiently collect and detect light from a three - dimensional
volume .
here we show that light from fluorophores attached along several
m long vertical al2o3 coated gallium
phosphide nanowires couples into the wires , is guided along them and
emitted at the tip .
this enables effective collection of light emitted
by fluorescent analytes located at different focal planes along the
nanowire .
we unequivocally demonstrate the light - guiding effect using
a novel method whereby the changes in emitted fluorescence intensity
are observed when fluorescent cytoskeletal filaments are propelled
by molecular motors along the wires .
the findings are discussed in
relation to nanobiosensor developments , other nanotechnological applications ,
and fundamental studies of motor function . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
changes in cortical mapping begin seconds after the loss of the limb and can continue to change years after the injury ( kaas et al . , 1983 ; merzenich et al . , 1983 ; merzenich and jenkins , 1993 ) previous studies have found that following upper extremity amputation , cortical sensory representations for the adjacent intact areas extend to cortical regions corresponding to the absent hand ( elbert et al . , 1997 ; grusser et al . , 2001 ) . other studies have found that in human arm amputees , the cortical reorganization contralateral to the amputation spreads bilaterally to both hemispheres ( bjorkman et al . , 2007a , b ) . after immediate hand replantation and long - term hand transplants , although results related to motor cortical reorganization following repair are more readily available , few studies have reported sensory cortical reorganization on human subjects after nerve repair ( bjorkman et al . , 2007a , b ; blume et al . , 2014 ) . in previous studies from bjorkman et al ( 2007a , b ) , sensory cortical remapping on one patient who underwent an immediate surgical hand replantation was reported . twelve months after the surgery , the primary somatosensory cortex was found to reorganize with the activation pattern to be more bilateral compared to an able - bodied hand ( bjorkman et al . two years after the surgery , predominantly contralateral somatosensory cortex activation was reported ( bjorkman et al . , 2007b ) . similarly , animal studies reported that median nerve regeneration in adult owl monkeys showed the reestablishment of topographic representations for localized skin areas , although in a limited extent ( wall et al . , 1986 ) . these results provided evidence for reorganization of cortical sensory representation to a pattern closer to the a relatively new surgical approach following amputation , targeted reinnervation ( tr ) provides a novel and fundamentally different way for nerve repair in that it denervates specific muscles and skin regions ( kuiken , 2003 ; kuiken et al . , 2004 ; hijjawi et al . , 2006 ) , and then reinnervates them with the residual nerves of the amputated limb . after tr , the reinnervated muscles can act as a natural biological amplifier to enhance the efferent motor command signal , allowing for the control of a multi - degree of freedom myoelectric prosthesis . as compared to transplantation , tr is less costly , requires less time in hospital and for recovery , and can be applied to a wider amputee population ( see a review for the comparison between transplantation and tr ( agnew et al . , 2012 ) ) . it has been demonstrated that several months after tr , new functional connections between the nerves and muscles can be created , which restores motor function ( agnew et al . , 2012 ) . correspondingly , a close to normal motor cortical map for the missed limb related to motor task performance can be re - established ( chen et al . , 2013 ) . besides recovery of motor function , tr also causes return of sensations of touch , pressure , vibration and temperature for the missing limb of the skin overlying reinnervated muscles ( kuiken et al . if indeed cortical restoration of original spatial maps is an important metric for the effectiveness of a restoration method , it will be of great importance to test whether tr successfully restores the sensory cortical representation as well . however , both short - term and long - term cortical changes in spatial mapping associated with tr - induced sensory recovery are still unknown . to answer this question , we used high - density electroencephalography ( eeg ) to identify and quantify cortical activity in response to stimulating the middle finger of the intact limb and the residual median nerve of the amputated limb in one session before and three sessions after targeted reinnervation surgery in an individual who underwent tr . a 24-year - old , right hand dominant male , who sustained a traumatic injury and a left trans - humeral amputation in july 2007 , was recruited for this study . he wears it for several hours a day and uses it to help him hold objects , fold clothes , drive and open letters . initially post - operation , he reported that he had some phantom limb sensation . however , when we recruited him in 2008 , he said that the phantom limb pain was gone , and that he only occasionally experienced phantom limb sensation and that this was not bothersome to him . the first set of experimental data were collected in august 2008 , 1 week before his targeted reinnervation ( tr ) surgery . as shown in fig . 1 , during the tr surgery , the median nerve was transferred to the medial head of the biceps ; the distal radial nerve was transferred to the lateral head of the triceps ; and the ulnar nerve was transferred to the motor reinnervation point of the brachialis . subcutaneous fat was removed over the biceps and triceps to increase the emg signal magnitude for subsequent myoelectric control . the fat removal denervated some of overlying skin enabling hand afferents to reinnervate the participant s skin after several months . when this section of skin was touched after reinnervation occurred , he felt his missing hand . post - tr , three sets of data were collected in january and july of 2009 and june 2010 . to verify the reliability of our experimental protocol and data processing , we also recruited one age - matched able - bodied right hand dominant control participant ( age : 24 years old ) . two sets of data were collected in the able - bodied control participant on march 23rd , 2010 and april 13th , 2010 . all studies were approved by the institutional review board of northwestern university and in compliance with the principles of the declaration of helsinki with written consent provided by the participants prior to participation . in each of the participants , we first searched for the stimulation site and intensity using constant current square - wave pulses that were delivered by a compex ii stimulator ( compex medical sa , ecutens , switzerland ) with pulse width at 0.3 ms long , and interstimulus interval at 200 ms . when determining the stimulation site , we searched for the location where the subject felt the most focused sensation of one of the four fingers . then , the intensity was set to the highest amplitude that the participant could tolerate without losing focus or causing discomfort , pain , or detectable muscle contractions . the amputee subject reported the strongest and most focused sensation of the tip of his missing middle finger when stimulating the reinnervated median nerve site , although also with faint sensation of his thumb and elbow . as reference , we also recorded somatosensory - evoked potentials ( seps ) while stimulating the tip of his middle finger from the intact side . for each site , 20003000 trials of seps were recorded in 2 or 3 blocks of 1000 , with at least 5 min of resting time between each block . during all of the experiments , the amputee participant remained relaxed and the middle finger from the intact hand was stimulated via cutaneous stimulation , and the residual median nerve at the amputated side was stimulated using direct nerve stimulation via a needle electrode during all but the last experiment ( see table 1 for the summary of the experiments on different dates ) . because the participant was scheduled for surgery to remove a neuroma 2 days after the last experiment , we used cutaneous stimulation of his reinnervated median nerve close to the elbow to reduce the risk of infection . the exact stimulation sites via needle and cutaneous electrodes for the median nerve are shown in fig . , we also recorded seps associated with finger stimulation in one age - matched able - bodied control participant on two different days . this able - bodied participant reported most focused sensation when we stimulated the ulnar nerve in both arms via needle electrodes applied at the elbow as well as via cutaneous electrodes applied at the little finger ( see fig . 3 for the stimulation sites ) . therefore , we recorded seps while stimulating the two - side little fingers via cutaneous stimulation , and two - side ulnar nerves via need electrodes . high - density ( 128 channels ) scalp eeg data were collected at 4 khz . eeg electrode positions and the anatomical landmarks ( nasion and two preauricular points ) were recorded using a 3d magnetic digitizer ( polhemus , colchester , vt ) . the digitized electrode locations were used to co - register the eeg data with the participant 's anatomical mri . t1-weighted mr images were taken with a 3 t siemens magnetom trio scanner ( siemens ag , erlangen , germany ) at northwestern memorial hospital . approximately 176192 contiguous images in the sagittal plane were taken , with voxel dimensions of 1.0 1.0 1.0 mm and voxel matrix of 256 256 . eeg signals were screened for the presence of eye and muscle movement artifacts in any of the channels , which eliminated that signal in an individual trial from further analysis . the remaining trials were aligned by the down - phase of the stimulation artifact , segmented ( 2050 ms , with 0 respect to the down - phase of the stimulation artifact ) , baseline ( 20 to 5 ms ) corrected , and averaged for each channel . the averaged eeg signals were down - sampled to 256 hz and imported into the curry software environment ( version 5.0 , compumedics neuroscan , charlotte , nc ) for reconstruction of the cortical activity . in curry software , a subject - specific boundary element method ( bem ) model the bem model was composed of three compartments for the skin , skull , and brain with 10.0 , 9.0 , and 7.0 mm resolution , respectively . coefficients of conductivity used for each compartment were 0.25 s / m for skin , 0.017 s / m for skull , and 1.79 s / m for brain ( yao and dewald , 2005 ) . the input eeg data were baseline ( 20 to 5 ms ) corrected , and then co - registered to the reconstructed skin by superimposing the locations of anatomical landmarks ( nasion and two preauricular points ) . the low resolution electromagnetic tomography ( loreta ) method with the parameter lp = 1 was chosen as the inverse method to localize cortical generators from the scalp eeg potentials ( pascual - marqui et al . all the sources were constrained on the segmented cortex with the spatial resolution as 3 mm , and perpendicular to the surface of the cortex . this method has been shown to provide better source localization ability than moving dipoles and minimum norm methods ( yao and dewald , 2005 ; grova et al . current density reconstructions exported from curry were loaded into matlab ( the mathworks , natick , ma ) for further processing and analysis . in matlab , the region of interest ( roi ) , consisting of bilateral primary somatosensory cortices ( s1 ) for sensory representations , was manually chosen by a well - trained neuroscientist based on the participant 's anatomical mri data . the matlab routine then automatically extracted all sources from the current density reconstructions that resided in this region . bilateral s1 areas were further divided into segments from medial to lateral , each being 10 mm long . this processing allows us to represent the bilateral s1 using location indices , where negative and positive 110 indicate segments on ipsilateral and contralateral side s1 , respectively , all from medial to lateral ( see the top of fig . cortical activity on each of these segments was voxel - averaged and normalized to the maximum during the 1040 ms window . using the above method , a time - location distribution ( with time resolution of 3.9 ms and spatial resolution of 1 cm ) of cortical activity on bilateral s1 over 1040 ms time window was created . additionally , we quantified the peak location of cortical activity and whether it has a diffused or focused pattern . in an effort to do this , we first summed the distribution along the time axis , and thus obtaining a location curve along the bilateral s1 location indices . this location - curve showed the accumulated activity at each location during the whole 1040 ms time window . the peak location was defined as the location with the strongest accumulated strength over bilateral s1 cortices . the diffusion / focus of the activity was quantified on each side of s1 by the value of quality ( i.e. , the q - value ) . the q - value is commonly used in describing the quality of a band - pass filter , which is calculated as the peak value of a response divided by the bandwidth . a big q - value means a very focused response , while a low q - value indicates a diffused response . when applying the q - value for quantification of the diffusion / focus of cortical activity , we first normalized the location - curve ; then on each hemisphere 's s1 , we identified the peak and the bandwidth to calculate the q - value over one side of s1 . as shown in fig . 4 and the green open markers in fig . 6 , able - bodied participant 's results are consistent between the two different sessions and between different stimulation methods . 6 , the peak locations in the able - bodied participant ( green ellipses ) are consistently located in the range between 4 and 7 , which is in agreement with finger and hand areas on the contralateral s1 . the maximum error in the peak location , between different sessions but with the same method or between different methods but with the same day , is 1 location index ( 10 mm ) in the able - bodied participant . 6 also shows that cortical activity resulting from finger stimulation in the able - bodied participate has a more focused pattern in the contralateral s1 as compared to the ipsilateral s1 , as suggested by the averaged ellipse , in the most right part of fig . 6 having a larger size in the vertical direction than that in the horizontal direction . this averaged ellipse is located at the averaged peak location and with an averaged size from all different stimulations over two sessions in this able - bodied participant . this averaged result , marked by a green open ellipse , was used as the reference to compare results between the amputee and the able - bodied participant . results from the intact side in the amputee participant are shown in the left plots in fig . 6 . as shown in fig . 6 , the peak locations of cortical responses to stimuli from the intact middle finger in the amputee participant on different sessions were not as consistent as compared to the results from able - bodied control participant . at 6 months after tr ( session 2 ) , we observed medial - shifted activity for the intact middle finger from the contralateral s1 with a relatively lower contralateral q - value ( i.e. , the vertical size of the blue dotted ellipse in fig . 6 is smaller as compared to the vertical size of the averaged green ellipse ) . this q - value stayed low at 12 months after tr , but with the peak back to the two years after the tr , the peak stayed inside the hand area but now with a comparably normal q - value , again as compared to the average green ellipse obtained from the control subject . at 2 years after tr , minimal activity was observed over the ipsilateral s1 when stimulating the intact finger , as suggested by a low q - value from the ipsilateral s1 . results from the amputated side in the amputee participant are shown in the right plots in fig . d and the red solid ellipse in fig . 6 , which varied a lot on different sessions . results from stimulating the residual median nerve before tr showed a diffuse bilateral pattern without clear focus in either time or spatial domains , as shown in the right plot in fig . 5a , and also indicated by the low q - values from both ipsilateral and contralateral s1 . six months and 1 year after tr , the responses of stimulating the residual median nerve showed bilateral spatial patterns on s1 , with increased foci in both spatial and time domains as compared to before tr results . in the spatial domain , both distributions ( 6 and 12 months after tr ) showed two foci , and with the contralateral one on the the activity at the 6 months post surgical mark has a second peak in ipsilateral center at finger / hand position ( index 5 ) ; while at 1 year after tr , the second activity center shifted to the contralateral shoulder position ( index 1 ) . furthermore , activities from both sessions 2 and 3 still have a relatively larger q - value in ipsilateral s1 as compared to that from contralateral s1 ( i.e. , the ellipses have a longer horizontal size on sessions 1 , 2 and 3 ) , suggesting that the activity from contralateral s1 is more diffused than that from ipsilateral s1 . two years after tr , we observed even more focused cortical activity in both time and spatial domains ( fig . , we see strong activity during 2640 ms on bilateral s1 finger position ( index 5 and 5 ) with stronger activity found in the contralateral s1 . both the location and time delay of this strong contralateral activity are consistent with that obtained by stimulating the intact middle finger . however , the stimulation of the intact index finger shows activity only over the contralateral s1 , while results obtained by stimulating the corresponding location from the amputated side still showed moderate activity from the ipsilateral s1 . finally , 2 years after tr , for the first time a more focused activity pattern was obtained over the contralateral s1 , with the vertical size of the ellipse comparable to that of the averaged green ellipse and to the blue dotted ellipse resulting from stimulating the intact middle finger at that time . in short , 2 years after tr , both the peak location and the q - values fell inside the normal range . our results from one individual with a trans - humeral amputation provide a single case study for long - term plasticity of sensory cortical representations following peripheral injury and targeted reinnervation . about 1 year after amputation and before tr , we found that the cortical representation of the residual median nerve showed a diffuse pattern without clear focus in either spatial or time domains . all results are consistent with previous findings from animals and human studies ( wall et al . , 1986 ; bjorkman et al . , 2007a , b ) . focused cortical activity in the time domain was first observed 6 months after tr , and then consistently appeared in the results obtained 1 and 2 years post - tr . in the spatial domain , we observed a bilateral somatosensory representation evoked by stimulating the residual median nerve from all of the 3 post - tr experiments , with more diffused pattern during the first 2 post - tr experiments . the diffused pattern may explain why the participant , in addition to strongly feeling his missing middle finger , also faintly felt his elbow and thumb . two years post - tr , we observed bilateral cortical sensory representations on s1 , with strong and focused activity on the contralateral side and moderate and less focused activity on the ipsilateral side , all with centers over the finger / hand area ( indices : 5 and 5 ) , suggesting that sensory representation of the reinnervated median nerve returned to a close - to - normal pattern . besides the changes in the cortical representation for the amputated finger , we also found that cortical representation for the intact finger was altered during the first 2 years following tr . normal pattern , i.e. , the peak location getting closer to the normal position and with higher focus in the contralateral s1 . our results showed similar changes of the spatial pattern as that reported previously following hand replantation . in these previous reports , about 1 month after immediate surgical replantation that repaired the injured nerve , an ipsilateral cortical representation of the missing hand was observed . the sensory mapping of the replanted hand was then shifted to a bilateral pattern about 4 months after the replantation , and back to a predominantly contralateral representation about 8 months after the replantation ( bjorkman et al . , 2007a ) . this suggests that tr has a similar effect on the cortical reorganization as immediate replantation . however , the return of a predominantly contralateral representation took about 2 years after tr , which is slower than the progress after hand replantation . the longer recovery phase of sensory representation after tr , as compared to after replantation , may be due to the fact that hand replantation was performed immediately after injury , while tr was performed 1 year after amputation in this participant . in the former case , there may not have been enough time between the injury and replantation to generate significant cortical sensory remapping . however , in our case , we showed that the cortical representation of the missing finger was already difficult to identify in either the time or spatial domains . lastly , our results demonstrated that even over a year after the amputation , the sensory cortex still preserved its plasticity this means that cortical sensory mapping following amputation can potentially be reversed following interventional procedures such as tr . in short , these results demonstrate that tr - induced cortical sensory remapping starts to have clear foci on hand / finger area in s1 no longer than 6 months after tr . about 2 years after tr , the new sensory maps for the missing part ( e.g. the middle finger ) return closer to the original locations . this cortical reorganization is associated with the previous reported return of the cutaneous sensation following tr ( kuiken et al . the return to a normal pattern for the intact finger stimulation occurred about at 1 year after tr , which was earlier than that for the amputated side . the lack of a clear pattern of activation in both time and spatial domains after amputation and before tr may be related to the withdrawal of axons from thalamus and cortex in response to peripheral somatosensory denervation ( graziano and jones , 2009 ) . since the majority of somatosensory pathways terminate at contralateral s1 , previous work constrained their analysis in the contralateral hemisphere ( blume et al . , 2014 ) . although stronger and more focused activity in the contralateral s1 was obtained in the able - bodied subject , this was not the case in the amputee participant . bilateral cortical activity was also reported in another study of an amputee participant ( bjorkman et al . , none of the previous reports explain the neuroscientific basis for this bilateral activity over the sensory cortices . we believe that this bilateral activity may be related to activation of sensory pathways via the reticular formation , which results in bilateral projections to the sensory cortices . although we used stimulation with an intensity lower than the motor threshold , it may still have resulted in activation of some crude touch sensation via a - delta and c fibers when we stimulated the skin or the nerve ( del gratta et al . , 2002 ) . these a - delta and c fibers project via the reticular formation and the thalamus to bilateral somatosensory cortices , as supported by the existence of both uncrossed and modest crossed thalamocortical projections , as seen in primates ( preuss and goldman - rakic , 1987 ) . an additional explanation is that projections to the contralateral somatosensory cortex result in bilateral representations of hands / arms and shoulder via interhemispheric interactions ( iwamura et al . , 1994 , 2002 ) . concurrently , we also see medial activation in both able - bodied and amputee participants . medial activation may be caused by the overlap in representation of different parts of the limb on s1 ( woolsey et al . , 1979 ; nii et al . , 1996 ; maldjian et al . , 1999 ) due to this overlap , when examining the representation of a single part of the limb , it is possible that cortical responses in multiple parts of s1 can be seen . it is worth noting that results in the able - bodied subject from every single experiment showed a peak location at hand / finger area on contralateral s1 and with a more focused pattern at the contralateral s1 as compared to that over the ipsilateral s1 . such consistency is not seen in the amputee participant . shortly after amputation and tr surgery , the activity in the ipsilateral s1 via spinoreticular pathways may be enhanced , due to the reduced sensory stimulation via the dorsal columns and the spinothalamic sensory pathways . therefore , the enhanced ipsilateral activity may suggest an initial adaptive strategy following amputation and tr . less focused activity can also be caused by low signal to noise ratio ( snr ) . we compared snr of able - bodied participant from different methods and on different days ( ranging from 7.8 to 15 ) to that of the amputee participant ( ranging from 7.9 to 15 ) . therefore , the snr can not explain the consistency in the primary activity in the contralateral s1 in the able - bodied participant versus the highly varied locations and q - values in the amputee participant . however , the subject reported almost no phantom pain , thus this possibility is not a likely contributing factor either . furthermore , since we did not include a control participant who had a similar amputation but underwent no tr surgery , we therefore can not totally exclude the possibility that the return to normal representation is at least partially related to . however , most previous studies support a cortical remapping immediately following amputation , and not enough evidence suggests that a return of the normal cortical activity would occur naturally. it is also worth noting that our results are limited to only one single amputee participant and with the last experiment using a cutaneous stimulation versus the previous 3 experiments using direct nerve stimulation . in order to validate our methods , experiments using both stimulation methods were conducted in an age - matched able - bodied control participant on different experimental sessions , to evaluate errors caused by different methods and different sessions . results on this able - bodied control participant are consistent , with the resulted peak locations always within the finger / hand areas on the contralateral s1 ( indices from 4 to 7 ) . these results gave us confidence in the ability to evaluate changes of cortical sensory maps in the participant who underwent a trans - humeral amputation and subsequent targeted reinnervation . finally , it is interesting to note that the return to the normal hand / finger area over the contralateral s1 cortex mirror results obtained in the reconstitution of normal motor maps following tr as reported in a previous study ( chen et al . , | this case study demonstrates the change of sensory cortical representations of the residual parts of the arm in an individual who underwent a trans - humeral amputation and subsequent targeted reinnervation ( tr ) . as a relatively new surgical technique
, tr restores a direct neural connection from amputated sensorimotor nerves to specific target muscles .
this method has been successfully applied to upper - limb and lower - limb amputees , and has shown effectiveness in regaining control signals via the newly re - innervated muscles .
correspondingly , recent study results have shown that motor representations for the missing limb move closer to their original locations following tr . besides regaining motor control signals
, tr also restores the sensation in the re - innervated skin areas .
we therefore hypothesize that tr causes analogous cortical sensory remapping that may return closer to their original locations . in order to test this hypothesis , cortical activity in response to sensory - level electrical stimulation in different parts of the arm
was studied longitudinally in one amputated individual before and up to 2 years after tr .
our results showed that 1 ) before tr , the cortical response to sensory electrical stimulation in the residual limb showed a diffuse bilateral pattern without a clear focus in either the time or spatial domain ; and 2 ) 2 years after tr , the sensory map of the reinnervated median nerve reorganized , showing predominant activity over the contralateral s1 hand area as well as moderate activity over the ipsilateral s1 .
therefore , this work provides new evidence for long - term sensory cortical plasticity in the human brain after tr . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
coccidioidomycosis was initially identified and
classified incorrectly as a protozoan species in 1892 by
alejandro posadas,(2 ) it was reclassified as a fungus in
1900(3 ) but retained the name due to its close
resemblance to the protozoan coccidian . the two
species which cause human disease , c. immitis and c.
posadasii , are named for the seriousness of the disease
( immitis , latin : not mild ) and in homage to alejandro
posadas . alejandro posadas initial case , a 36 year old
argentinean soldier , eventually died from the disease in
1898 . lesions , at autopsy , were found in his lungs ,
adrenals , lymph nodes , liver , peritoneum , prostate ,
spleen , and testes.(4 ) disseminated coccidioidomycosis is a regional
disease which has national importance.(5 ) coccidioides
is a dimorphic fungus endemic to the southwestern
united states as well as central and south america.(6 )
outbreaks have also been described well outside the
generally accepted borders . some are due to travel to
endemic areas(7 - 15 ) , while others represent expansion
of the known borders of coccidioidomycosis ( 16 )
coccidioidomycosis causes a range of clinical presentations from asymptomatic disease , a mild selflimited
respiratory disease ( 17 ) to dissemination to
multiple systems and organs . dissemination , especially
fungemia , ( 18 - 20 ) can lead quickly to sepsis and death . once
inhaled the spores take 1 - 3 weeks to germinate and
cause disease . it is not spread via human to human
contact and outbreaks are highly dependent on
environmental conditions . dry , hot summers after a wet
winter and spring , increase the number of reported
cases.(21 ) it is common in the southern california
region and , in some endemic san joaquin valley areas ,
75% of the population has immunity against the fungus
as measured by skin testing ( 22 ) . disseminated disease is likely to be seen in certain
populations ; the infection and dissemination rate is
much higher among blacks and filipinos ( 23 ) . males
are infected more often than females ( 24 ) , which may
be exposure based , but may also be related to hormonal
or genetic factors . hiv patients ( especially with cd4
counts < 100)(25 - 28 ) as well as patients on chronic
immunosuppression for other medical conditions
( rheumatological and/or transplant related diseases ) and
those treated with immunosuppressive chemotherapy
are at higher risk of either reactivation of a latent
infection or disseminated disease ( 29 - 35 ) . diabetes ,
which does appear to increase the risk for symptomatic
infection and may increase the risk for dissemination
( 36 ) . the disseminated form of coccidioidomycosis may
present with evidence of involvement of the skin ( 19 ,
37 ) , prostate gland ( 38 - 43 ) , eye ( 44 , 45 ) , bone ( 46 ) ,
meninges ( 47 - 49 ) and liver ( 50 - 56 ) . splenic and hepatic
involvement , as diagnosed on autopsy , has been
described previously ( 4 ) but clinically evident
hepatosplenomegaly in a living adult patient has not
been widely reported . we present a case of a healthy
adult hiv seronegative patient with massive
hepatosplenomegaly from disseminated cocci . the patient was an incarcerated 24 year old african
american male who presented to the emergency
department ( ed ) from a correctional facility where he
had served four months of his sentence . he had been
participating in a work - release program during which
he was responsible for cleaning up camp sites , national
forest sites , and roadways , where it is suspected that he
came into contact with soil containing spores of
coccidioidomycosis . his chief complaint was anorexia and rare vomiting
for approximately two months . he had begun
experiencing worsening general malaise , a ten pound
weight loss and the patient stated that he used alcohol occasionally
and had an approximately three - pack - year history of
smoking , which he stopped a few years ago . the
patient also admitted to drug use ( marijuana and
methamphetamine ) before his incarceration . he had no
prior hospital admissions and had been in excellent
health until two months before admission . prior to his incarceration he
stated that he was in a stable relationship with his
girlfriend . his maternal family had sickle cell trait , but he and his
sister were negative for sickle cell trait or disease . on initial evaluation his blood pressure was 110/60 ,
respiratory rate 41 , temperature 38.3 , heart rate 134 ,
saturation 98% on 15 liters o2 on a non - rebreather
mask with bilateral crackles . his initial physical exam
was otherwise notable for an enlarged liver and spleen
with a spleen tip palpable at 8 centimeters below the
lateral rib margin . he had two cafe - au - lait spots on his
anterior abdomen but his exam was otherwise negative . his initial labs were as follows : sodium 126 ,
potassium 5.1 , chloride 98 , bicarb 18 , bun 25 ,
creatinine 1.4 , glucose 89 , calcium 7.7 , phosphorus 4.3 . hemoglobin 4.9 , hematocrit 16.1 , white blood cell
count 17.9 , platelet count 98,000 , mcv 72 . arterial blood gas : ph 7.49 , pco2 24 , po2 111 , bicarb 18 , co2
19 . bilirubin was 2.4 , direct bilirubin 1.2 , indirect bilirubin
1.2 , alkaline phosphatase 192 , alt 76 , ast 85 . further workup during his admission included a
negative elisa hiv test and his cd4 count was 384
( during his illness ) . a
hemoglobin electrophoresis was performed which
revealed no hgb s , hgb a1 90.5% , a2 3.7% , f 5.4% . sputum
acid fast bacilli staining was negative and the samples
were culture negative . a computerized tomography ( ct ) scan showed
hepatosplenomegaly with a miliary infiltrate in both
organs . he initially refused intubation but as he became more
fatigued from his tachypnea he eventually consented .
his tachycardia and tachypnea were initially thought to
be due to his severe anemia and burden of his
underlying disease . he was transferred to the intensive
care unit ( icu ) where he progressed to acute
respiratory distress syndrome ( ards ) . he was initially
given azithromycin 500 mg a day , piperacillin/
tazobactam 3.375 grams iv every 8 hours , and
vancomycin 1250 mg iv every 12 hours . his initial
hemoglobin and low platelets prompted a bone marrow
biopsy on day 3 which showed coccidioidomycosis
spherules . he also had a positive endotracheal aspirate
for methicillin sensitive staph aureus . when the
spherules were described on the bone marrow aspirate ,
and confirmed with serological testing , he was started
on fluconazole 600 mg every day and given nafcillin 2
grams iv every 4 hours . when he did not improve
after 4 days he was switched to amphotericin for one
dose then to liposomal amphotericin which caused
acute renal failure . his course was
also complicated by a ventilator associated
pseudomonas pneumonia which was treated with
piperacillin / tazobactam 2.25 grams every 6 hours and
levofloxacin 500 mg iv every 6 hours . his anemia was
recalcitrant to multiple transfusions and he had minor
bleeding from multiple sites . the patient developed a board - like abdomen and a
perforated viscus was suspected . no
perforation was identified but the spleen and liver
appeared irregular and were infiltrated with large fungal colonies on biopsy . given the massive splenic
involvement which was felt to be compressing his
diaphragm and affecting his pulmonary function , a
splenectomy was performed . it measured 25 cm by 18 cm by
14.5 cm , weighed almost 9 pounds and had visible
coccidioidomycosis colonies on macroscopic exam . the
pathology slides also confirmed colonies of coccidioides
( figures 4 - 5 ) . despite antibiotic and antifungal
treatment , after a total of 18 days he developed a cardiac
arrhythmia and died on ventilatory support . splenomegaly is an uncommon finding in
disseminated cocci , especially in an immunocompetent
person . our review of the literature was unable to find
any previously reported cases of clinically evident
splenomegally in an immunompetent host . we hope that
other similar cases will continue to be reported so that
further insight into this disease process may be gleaned . this case highlights many of the problems commonly
encountered in patients with coccidioidomycosis
infections : delay to diagnosis , low suspicion with
diagnosis suggested from peripheral tests , and
seriousness in african american males . the
uncommon features are also notable ; his rapid
progression to ards , sepsis and death , his negative
hiv elisa and his relatively appropriate cd4 count
along with no significant past medical history are not commonly encountered in a case this severe . this case
highlights the importance of considering
coccidioidomycosis and other mycotic diseases when
presented with a patient in respiratory failure , even
when they are not immunosuppresed . | coccidioidomycosis is a dimorphic fungus endemic to the southwestern united states ,
central and south america .
we report a case of a previously healthy person who presented with
respiratory failure and disseminated coccidioidomycosis who eventually had a fatal outcome .
coccidioidomycosis , or valley fever has been called the great imitator ( 1 ) as it can have a wide
variety of clinical presentations .
this case is unique as it represents the first described case of an
immunocompetent host with rapidly progressing , disseminated coccidioidomycosis with clinically
apparent splenomegaly and hepatomegaly . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the coexistence of cardiovascular diseases ( cvds ) and metabolic disorders is frequent in patients with chronic obstructive pulmonary disease ( copd).1 the prevalence of both these conditions is highest in patients with moderate - to - severe copd in particular and drops in patients with very severe copd.2 accordingly , cvd is a leading cause of death in the earlier stage of the disease.3 thrombosis , in turn , is the most frequent underlying mechanism of the three major cvds : ischemic heart disease , stroke , and venous thromboembolism,4 which raises the question whether blood markers involved in the pathogenesis of thrombosis are linked to copd . plasminogen activator inhibitor-1 ( pai-1 ) is a member of the superfamily of serine protease inhibitors and the principal inhibitor of fibrinolysis within the plasminogen activator system.5,6 high concentration of pai-1 plays a pivotal role in the pathogenesis of arterial and venous thrombosis and , therefore , precedes the occurrence of thrombotic events.7,8 in addition , higher level of pai-1 is associated with obesity , insulin resistance , diabetes , hyperlipidemia , and premature aging.5,9,10 all these conditions are prevalent in copd,11,12 and a potential influence of pai-1 on an enhanced thrombogenesis in copd has been hypothesized.13 indeed , a recent case control study demonstrated that pai-1 polymorphisms related to a higher pai-1 expression are associated with copd.14 furthermore , a study conducted on patients with predominantly mild - to - moderate copd found that higher serum pai-1 levels are related to airflow limitation.15 whether the observed elevation in pai-1 levels in copd is independent of metabolic and cardiovascular functions is unknown . in order to elucidate the clinical value of pai-1 as a potential biomarker in copd , the serum levels of pai-1 and a broad spectrum of potential confounding factors for pai-1 were analyzed in patients with mild - to - very severe copd and in control subjects . this cross - sectional study is nested within a prospective copd cohort study conducted at the pulmonary research institute , the lungenclinic grosshansdorf ( grosshansdorf , germany ) . details regarding the copd population and the methodology of the study have been published elsewhere.1618 in the present study , 74 stable outpatients with mild - to - very severe copd ( ie , n=16 , 20 , 18 , and 20 with global initiative for chronic obstructive lung disease [ gold ] stages i , ii , iii , and iv , respectively ) and 18 nonsmoking controls without any lung disease from the 3-year follow - up visit were included . the study was approved by the ethics committee of the medical association of schleswig - holstein , bad segeberg , germany ( iii / ek 116/05[i ] ; 185/08[i ] ) , and all the participants provided written informed consent . in this study , the serum levels of pai-1 were analyzed by using enzyme - linked immunosorbent assay ( elisa ; human serpin e1/pai-1 duoset elisa ; r&d systems , wiesbaden - nordenstadt , germany ; detection range = 0.31220 ng / ml ) ; metabolic and cardiovascular functions as possible confounders of pai-1 , including triglyceride levels , high - density lipoprotein ( hdl ) cholesterol , fasting plasma glucose , waist circumference , blood pressure , the presence of the metabolic syndrome ( based on the latter five variables according to the criteria of the international diabetes federation19 ) , smoking status , and history of coronary artery disease and diabetes , were determined ; high - sensitivity c - reactive protein ( hs - crp ) was measured as an established marker of systemic inflammation and adiponectin , which is involved in anti - inflammatory , anti - diabetic , and anti - atherogenic processes;20 in addition , global cardiac function by serum levels of n - terminal pro - b - type natriuretic peptide ( nt - probnp ) and the presence of atherosclerosis by the ankle brachial index ( abi ) were also determined as previously described.16,18 first , the differences between groups were analyzed univariately by using unpaired t - test , test , and analysis of variance ( anova ) with least significant difference post hoc analysis . then , a multivariate linear regression analysis was conducted with pai-1 as the dependent variable and copd as a predictor , and adjustments for possible confounders , such as age , sex , body mass index ( bmi ) , impaired fasting glucose ( ie , 100 mg / dl),19 dyslipidemia ( ie , triglycerides > 150 mg / dl or hdl < 40 mg / dl in male and < 50 mg / dl in female),19 cardiac dysfunction ( ie , nt - probnp > 125 pg / ml),21 atherosclerosis ( ie , abi 0.90),22 as well as history of diabetes , hypertension , and coronary artery disease , were performed . a second multivariate regression analysis with pai-1 as the dependent variable aimed at evaluating the best independent predictors for pai-1 within copd . therefore , a stepwise approach was chosen with a backward elimination . only the variables that showed at least a trend for statistical significance on the bivariate level , that is , p<0.10 , next , whether copd severity stages were still independently associated with pai-1 within the final model was proved . last , the interaction effects of gold stages with the remaining predictors of the final model were tested by using separate two - way anovas . impaired fasting glucose , dyslipidemia , cardiac dysfunction , hypertension , and history of diabetes ( p>0.05 , respectively ; table 1 ) did not differ between the patients with copd and controls . the frequency of atherosclerosis and history of coronary artery disease increased in patients with copd compared with controls ( p<0.05 ) . serum levels of pai-1 were significantly higher in copd patients than in controls ( figure 1a ) . stratified by severity stages , pai-1 levels were highest in gold stages ii and iii ( figure 1b ) . smoking status and statin use had no effect on the level of pai-1 in patients with copd ( p=0.51 and 0.50 , respectively ) . in a multivariate linear regression analysis , the presence of copd remained an independent predictor for higher pai-1 levels after adjustments for age , sex , bmi , impaired fasting glucose , dyslipidemia , cardiac dysfunction , hypertension , diabetes , atherosclerosis , and history of coronary artery disease ( regression coefficient b for copd patients versus controls = 2.9 , 95% confidence interval [ ci ] = 0.345.41 , significant correlations of pai-1 with hdl cholesterol , triglycerides , hs - crp , and adiponectin ( p<0.05 , respectively ; table 2 ) were found on the bivariate level . the association between age and pai-1 showed a trend for significance ( p=0.058 ) . in a multivariate regression analysis with a backward elimination , log - triglycerides ( 3.3 , 95% ci = 0.85.9 , p=0.012 ) and log - hs - crp ( 0.83 , 95% ci = 0.031.63 , p=0.043 ) were found to be the best independent predictors for pai-1 in copd . figure 2 visualizes the results from the multivariate regression analysis , that is , mean pai-1 levels in copd patients stratified according to triglycerides and hs - crp in comparison with controls . in a final model we studied whether copd severity stages add additional information as predictors for pai-1 independent of triglycerides and hs - crp within the total study cohort . it was found that gold stages ii and iii were independently associated with pai-1 ( table 3 ) . there were no significant interaction effects of gold stages either on triglycerides or on hs - crp . the main finding of the present study was that serum levels of pai-1 are higher in patients with copd independent of metabolic and cardiovascular disorders . furthermore , hyper - triglyceridemia and systemic inflammation were found to be the best independent predictors for higher pai-1 levels in copd patients , whereas the presence of moderate - to - severe airflow limitation remained to be independently associated with pai-1 . pai-1 is a multifunctional protein associated with thrombosis as well as with metabolic conditions , such as obesity , dyslipidemia , insulin resistance , and diabetes,5,7,9,10 which all are important concomitant conditions of copd.1,12 there are two very recent studies showing an association between elevated pai-1 levels and copd , but considered neither comorbidities of copd nor the whole spectrum of airflow limitation , from mild to very severe stage.14,15 the present study is the first to demonstrate that higher serum pai-1 levels in copd are independent of a broad spectrum of possible confounders , in particular established variables indicating metabolic and cardiovascular dysfunction . interestingly , this study found the highest pai-1 levels in patients with gold stages ii and iii . this observation is in good agreement with the finding of the previous study showing a significant association between higher levels of pai-1 and airflow limitation in patients with predominantly mild - to - moderate copd.15 moreover , it was also found that triglycerides and hs - crp are the best predictors for elevated pai-1 levels in copd patients . in general , an elevation in triglycerides and hs - crp plays an important role for cvd.23,24 moreover , an association of pai-1 levels with serum lipids and inflammatory markers has been found,9,25 although the mechanistic link behind this remains not fully understood.5,26 in patients with copd , hypertriglyceridemia and systemic inflammation are also found frequently and predict mortality.2729 the present study demonstrated an independent association of high pai-1 serum levels with moderate - to - severe airflow limitation , hypertriglyceridemia , and systemic inflammation . copd is characterized by airway and lung abnormalities and is frequently complicated by concomitant cardiovascular and metabolic disorders.1 local pathological changes of copd include parenchymal tissue destruction , resulting in emphysema , and disrupted repair and defense mechanisms , resulting in small airway fibrosis.1 pai-1 is synthesized by a variety of cells including vascular endothelial cells , macrophages , and fibroblasts and not only is involved in the pathogenesis of thrombosis but also activates tissue repair.3032 transiently increased levels of pai-1 may protect extracellular matrix proteins from proteolytic degradation , inducing matrix remodeling and enhancing wound healing.33,34 it is well documented that tissue homeostasis is maintained by the balance of extracellular matrix synthesis and degradation depending on the cellular proteolytic activities of urokinase - type / tissue - type plasminogen activator and plasmin , which mostly rely on the activity of pai-1.33,35 according to the data from the present study , the independent elevation of pai-1 in patients with copd may indicate a disease - specific mechanism . we speculate that higher pai-1 levels may to some extent reflect processes of airway remodeling in copd . these processes start early in the course of the disease and are less present in advanced copd,36 which might be the reason why the pai-1 level is highest in moderate copd . this concept is in good agreement with the findings of hogg et al showing that indicators of small airway remodeling are increased in moderate copd in comparison with controls and declined in very severe copd again.36 in line with this result , the plasminogen activator system has already been identified as a potential treatment target for chronic respiratory diseases including copd.37 on the other hand , the pivotal role of pai-1 in the pathogenesis of thrombosis , in general , seems to be clear,5,710 which allows to propose that higher levels of pai-1 in patients with moderate - to - severe copd ( gold stages ii and iii ) may be associated with an increased risk for cardiovascular events . this idea is supported by the observation that cvd and cardiovascular mortality are highest in patients with moderate - to - severe copd and drops in very severe copd.2,3 overall , pai-1 is recognized as one of the central molecules linking the metabolic syndrome / obesity to the increased cvd risk.26,38 beyond its function as a tissue - remodeling activator , pai-1 is also an adipocytokine , which is upregulated along with fat accumulation and , therefore , is associated with insulin resistance , dyslipidemia , and obesity.5,9,26,38 metabolic comorbidities are , in turn , especially prevalent in copd patients with gold stages ii and iii.2 altogether , pai-1 may serve as a cross - talk molecule between airway remodeling during the course of copd , accompanied metabolic comorbidities , and a subsequent increased risk for cardiovascular events , especially in moderate copd . first , this is a cross - sectional study and therefore does not allow any interpretation on causality . however , it was indicated that pai-1 may play a plausible role in copd , and this is the first study conducted in a well - characterized copd cohort to demonstrate a clear association of pai-1 with copd independent of a broad range of possible confounders . second , the sample size , especially in the control group , is small . despite these limitations , a significant difference in pai-1 between copd patients and controls was found . of note , the control subjects were free from lung diseases but not explicitly healthy and , therefore , were well matched in terms of metabolic comorbidities . nevertheless , further studies are needed to confirm these observations and to evaluate the role of pai-1 for cardiovascular outcomes in copd . the results of this study showed that pai-1 levels are higher in patients with copd , independent of metabolic disorders and cardiovascular function . hypertriglyceridemia and systemic inflammation may play a role for elevated levels of pai-1 in copd , even though they do not explain this elevation solely , as moderate - to - severe airflow limitation is independently associated with higher levels of pai-1 . these findings suggest pai-1 as a potential biomarker candidate that indicates a link between airway remodeling , metabolic comorbidities , and an increased cardiovascular risk . | introductionplasminogen activator inhibitor-1 ( pai-1 ) , a major inhibitor of fibrinolysis , is associated with thrombosis , obesity , insulin resistance , dyslipidemia , and premature aging , which all are coexisting conditions of chronic obstructive pulmonary disease ( copd ) .
the role of pai-1 in copd with respect to metabolic and cardiovascular functions is unclear.methodsin this study , which was nested within a prospective cohort study , the serum levels of pai-1 were cross - sectionally measured in 74 stable copd patients ( global initiative for chronic obstructive lung disease [ gold ] stages i iv ) and 18 controls without lung disease .
in addition , triglycerides , high - density lipoprotein cholesterol , fasting plasma glucose , waist circumference , blood pressure , smoking status , high - sensitive c - reactive protein ( hs - crp ) , adiponectin , ankle brachial index , n - terminal pro - b - type natriuretic peptide , and history of comorbidities were also determined.resultsthe serum levels of pai-1 were significantly higher in copd patients than in controls , independent of a broad spectrum of possible confounders including metabolic and cardiovascular dysfunction .
a multivariate regression analysis revealed triglyceride and hs - crp levels to be the best predictors of pai-1 within copd .
gold stages ii and iii remained independently associated with higher pai-1 levels in a final regression analysis.conclusionthe data from the present study showed that the serum levels of pai-1 are higher in patients with copd and that moderate - to - severe airflow limitation , hypertriglyceridemia , and systemic inflammation are independent predictors of an elevated pai-1 level .
pai-1 may be a potential biomarker candidate for copd - specific and extra - pulmonary manifestations . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
analysis of electrocardiogram ( ecg ) signals is so important in detecting heart diseases , because such diseases may cause sudden deaths . the electrical activity of the ventricles on ecg is in the range of qrs complex to t wave end . ventricular arrhythmias studied in this article are ventricular fibrillation ( vfi ) , ventricular tachycardia ( vt ) , and ventricular flutter ( vfl ) . premature ventricular contractions ( pvcs ) and ventricular escape beat ( veb ) are two ventricular abnormalities studied in this paper . first separated pvc beats from other healthy and cardiac abnormal beats by using wavelet transform and then classified them using time domain features and artificial neural networks . an overall accuracy of 95.6% was reported in this article . in martis et al . , the pca has been used as a tool for ecg beat classification ( normal bits , lbbb , pvc , rbbb ) . in this article , three features extraction methods [ ecg rate basic elements , the basic elements of the linear prediction error signals and the basic elements of discrete wavelet transform ( dwt ) coefficients ] were compared . in ebrahimzadeh and khazaee , the ecg signal has been investigated using wavelet coefficients approximation ( using three long features ) and rbf neural network as a classifier . in this paper , five types of ecg beats ( normal , lbbb , rbbb , pvc , and apc ) are classified . in korrek and doan , the authors used particle swann optimization and radial basis function neural network ( rbfnn ) for classifying six types of ecg beats . in de chazal et al . , the authors classified four types of ecg beats ( normal beat , ventricular ectopic beat ( veb ) , supraventricular ectopic beat ( sveb ) , and fusion of normal and veb ) . in this paper , the classification is performed using time domain features and linear kernel . in hosseini , the combination of independent features and compressed ecg data is used as an input to the multilayered perceptron network . the classification of five types of ecg beats ( nb , paced beat , lbbb , rbbb , and pvc ) is performed using wavelet coefficients in fourth and fifth scales and four features from qrs complex are considered in every cycle . the performance of the proposed method of this paper is compared with extracted statistical features using dwt . , we faced the dilemma of finding the location of r peaks , and no common method has proper functionality in finding r peaks . the data used in this study are as follows :
( 1)normal sinus database ( containing five 1-min signals or 350 beats ) with frequency of 128 hz.(2)sudden cardiac death database ( consisting of three 1-h signals ) with frequency of 256 hz . normal sinus database ( containing five 1-min signals or 350 beats ) with frequency of 128 hz . sudden cardiac death database ( consisting of three 1-h signals ) with frequency of 256 hz . in this study , this part of the article consists of two parts : how to detect r peaks , and feature selection , which will be explained in the following . to improve r peaks diagnosis , plenty of methods have been used . many methods such as derivative method , hamilton tompkins algorithm , wavelet transform method , and hilbert transform ( ht ) method are used to detect r peaks . some methods were applied for qrs detection by some translations , and more complex methods did not use from time dimension directly ; some of them were mentioned as following . in rodrguez , the authors were given new approach for the detection of qrs complex in different arrhythmias . et al . , the authors proposed peak synchronization that is novel method and it measures the simultaneity of occurrence of peaks in the signals . in ricardo ferro et al . , the authors employed processing of original signal , which included shannon energy envelope ( see ) estimator , ht , and moving average ( ma ) filter . data consisted of 10 records of 5 min length and different signal - to - noise ratios ( 15 , 12 , and 9 db ) . the accuracy was applied to 100% . in paper , the authors used a combination of some signal processing techniques , which were hilbert , wavelet transforms , and an adaptive thresholding method . this combination was named what . a common method for r peaks detection ( such as pan this study suggests a method based on a combination of pan tompkins and state logic machine which has improved r peaks detection through abnormalities . both algorithms detect the same location for r peaks in the ecg signal containing normal sinus rhythm and the signals not having acute abnormalities . the difference between r peak location detection via pan tompkins algorithm and state logic machine in every single beat for normal sinus signals shows no significant difference or a maximum of 10_samples difference . however , the locations of r peaks obtained from the pan tompkins algorithm and state logic machine are different in beats in the ecg signal having acute ventricular abnormalities . r peaks difference obtained from the two algorithms in two patient ( pvc and vfl ) and healthy cases are presented in figures 1 and 2 . rr intervals obtained from pan tompkins and state logic machine , respectively , for a patient rr intervals obtained from pan tompkins and state logic machine , respectively , for healthy case moreover , a template of normal sinus beat is used in the algorithm . for this beat , then , the cross - correlation is calculated at zero lag between each beat and normal sinus beat template . two conditions to classify data are studied in this paper . if both conditions are present in the meantime , that beat would be a normal sinus beat . the first condition is , when the cross - correlation between the normal sinus beat template and studied beats from signals in zero lag ( which is indicated by a2 ) is more than 0.3 of a1 and the difference of r peaks obtained from two algorithms ( pan tompkins and state logic machine ) is smaller than 10 , this beat is normal ; otherwise , this beat is abnormal . the coefficient ( 0.3 ) is obtained from the average of the division a2 to a1 . a significant point is that not all the beats are sampled with the same frequency . also , all beats are downsampled to the lowest sampling frequency ( 128 hz ) . sudden cardiac death database ( containing three 1-h signals ) with frequency of 256 hz and normal sinus database ( containing five 1-min signals ) with frequency of 128 hz are sampled . the second condition is that if a2 is less than 0.3 of a1 or if the difference of r peaks obtained from two algorithms in the studied beat is more than 10 , these beats have acute ventricular abnormalities . at intervals of some ventricular arrhythmias in the ecg signals ( such as pvc ) , no algorithm can find r peaks correctly ( as shown in figure 3 , r peaks in pvc beat was marked wrongly in seconds 1.3 and 3 ) . r peaks in pvc beats was detected wrongly then , to find r peaks locations in ventricular abnormal beats , 70% of the interval between two consequent r peaks obtained from state logic machine is considered and the maximum peaks and their locations are calculated . the differences between the amplitudes of previous and next samples of this sample ( obtained from previous step ) are calculated to insure that is a real peak . then , an interval of 0.06 s is considered after and before this sample and the minimum amplitudes are considered as q and s peaks , respectively . r peaks are obtained using this algorithm for pvc beats with the tp of 57% and fn of 43% , if none of the other algorithms ( state logic machine and pan tompkins ) are able to find r peaks . fp , fn , tp , and tn of detected r peaks for ventricular abnormalities in ecgs studied in this paper are according to table 2 . fp , fn , tp and tn of r peak detection in ventricular abnormalities studied morphological features are as the following : the amplitude of r peak , the rising and falling slopes of qrs complex , qs interval , positive and negative areas ( the sign of this value is negative ) of the complex qrs [ eqs . the positive and negative qrs complex area all of the features are obtained on lead ii . in state logic machine algorithm , then , a 1_second window has been determined on the ecg signal and the average of this window has been calculated ( m ) . the state logic machine considered two conditions for each of the r and s and t waves . estimating the location is done by considering the first condition , and definite presence of r peaks in this interval is determined by considering the second condition and its constraints . each condition has been different for peak detection in ecg at each step . at zero state , the product of m and an initial weight ( w=1.8 ) if this product is less than the mean value calculated in a 15_sample interval ( m1 ) from the one second interval , and also if the amplitude of the studied sample is larger than the rest of the samples , the amplitude and the location of the first sample from the one second window is stored as r peak ; then , we switch to state of one , which reports the definite presence of r peaks . in summary ( 3 ) ] : if ( m1 > m * weight s(i ) > s(j+1 ) ) ( j = 1 : 3 ; i in each step is fixed ) then s(i ) is stored as r peak . ( 3 ) in state of one , the stored amplitude from zero state is compared with the next four samples , to ensure that is the maximum one . each sample in this window must be less than its previous sample . if such a condition is imposed , the index from zero state is stored as the r peak amplitude and its location . an interval containing 0.04 s before this location until the location of this sample is considered , and minimum amplitude is assumed as q peak . also , weight is updated in this state , which means that if the number of detected r peaks is more than eight , 0.3 of mean of these eight r peaks is achieved and it is divided to m. then , the state will be equal to two . in the state of two , if m1 is less than m , we switch to state of three for finding s peak . the period of investigation for state of three is 0.2 s. if , among the eight samples , the studied sample is less than the rest of the samples and if each sample is less than its previous sample , we ensure that the studied sample is s. then , the state is equal to four . in the state of four , if m1 is less than m , we switch to state of six for finding t peaks . in state of six the threshold is considered based on the recent values of s and m. if m1 is more than this threshold [ eq . ( 4 ) ] , this condition is acceptable for three consecutive samples , and the peak of the studied sample is more than its subsequent eight samples ( each sample in this eight - sample window should be smaller than the previous sample ) , this sample is stored as t peak and state is updated to six . |samplitude(end)|)|*+samplitude(end ) ( 4 ) state of six is for prevention of finding several r peaks in a 0.4 s window . after this delay , the algorithm switches to zero state and this algorithm is repeated for each 1_second window . flowchart of state logic machine after extracting signal features , support vector machine ( svm ) by ( gaussian ) radial basis function kernel is used for classification of ventricular abnormalities . the method used in svm is one - against - all . to classify each arrhythmia , the accuracy of the data is obtained by six times shuffles . in this research , these signals include sudden cardiac death records for three patients and five normal sinus rhythms of five healthy persons . the feature extraction step extracted six features from more than 300 beats of mit - scd that included six acute abnormalities studied in this research , and 350 beats from normal sinus rhythm . these features , including the amplitude of r peak , the rising and falling slope of qrs complex , qs interval , and positive and negative areas of the complex qrs , were obtained from each beat . the classification accuracies of normal sinus rhythm , vt , vfl , vfi , pvc , and veb rhythm were evaluated . the accuracies achieved from this essay are compared with previous studies and the maximum accuracy is reported . by using the combined algorithm in this paper many methods such as derivative method , hamilton tompkins algorithm , wavelet transform method , and hilbert transform ( ht ) method are used to detect r peaks . some methods were applied for qrs detection by some translations , and more complex methods did not use from time dimension directly ; some of them were mentioned as following . in rodrguez , the authors were given new approach for the detection of qrs complex in different arrhythmias . et al . , the authors proposed peak synchronization that is novel method and it measures the simultaneity of occurrence of peaks in the signals . in ricardo ferro et al . , the authors employed processing of original signal , which included shannon energy envelope ( see ) estimator , ht , and moving average ( ma ) filter . data consisted of 10 records of 5 min length and different signal - to - noise ratios ( 15 , 12 , and 9 db ) . the accuracy was applied to 100% . in paper , the authors used a combination of some signal processing techniques , which were hilbert , wavelet transforms , and an adaptive thresholding method . this combination was named what . a common method for r peaks detection ( such as pan this study suggests a method based on a combination of pan tompkins and state logic machine which has improved r peaks detection through abnormalities . both algorithms detect the same location for r peaks in the ecg signal containing normal sinus rhythm and the signals not having acute abnormalities . the difference between r peak location detection via pan tompkins algorithm and state logic machine in every single beat for normal sinus signals shows no significant difference or a maximum of 10_samples difference . tompkins algorithm and state logic machine are different in beats in the ecg signal having acute ventricular abnormalities . r peaks difference obtained from the two algorithms in two patient ( pvc and vfl ) and healthy cases are presented in figures 1 and 2 . rr intervals obtained from pan tompkins and state logic machine , respectively , for a patient rr intervals obtained from pan tompkins and state logic machine , respectively , for healthy case moreover , a template of normal sinus beat is used in the algorithm . for this beat , then , the cross - correlation is calculated at zero lag between each beat and normal sinus beat template . two conditions to classify data if both conditions are present in the meantime , that beat would be a normal sinus beat . the first condition is , when the cross - correlation between the normal sinus beat template and studied beats from signals in zero lag ( which is indicated by a2 ) is more than 0.3 of a1 and the difference of r peaks obtained from two algorithms ( pan tompkins and state logic machine ) is smaller than 10 , this beat is normal ; otherwise , this beat is abnormal . the coefficient ( 0.3 ) is obtained from the average of the division a2 to a1 . a significant point is that not all the beats are sampled with the same frequency . also , all beats are downsampled to the lowest sampling frequency ( 128 hz ) . sudden cardiac death database ( containing three 1-h signals ) with frequency of 256 hz and normal sinus database ( containing five 1-min signals ) with frequency of 128 hz are sampled . the second condition is that if a2 is less than 0.3 of a1 or if the difference of r peaks obtained from two algorithms in the studied beat is more than 10 , these beats have acute ventricular abnormalities . at intervals of some ventricular arrhythmias in the ecg signals ( such as pvc ) , no algorithm can find r peaks correctly ( as shown in figure 3 , r peaks in pvc beat was marked wrongly in seconds 1.3 and 3 ) . r peaks in pvc beats was detected wrongly then , to find r peaks locations in ventricular abnormal beats , 70% of the interval between two consequent r peaks obtained from state logic machine is considered and the maximum peaks and their locations are calculated . the differences between the amplitudes of previous and next samples of this sample ( obtained from previous step ) are calculated to insure that is a real peak . then , an interval of 0.06 s is considered after and before this sample and the minimum amplitudes are considered as q and s peaks , respectively . r peaks are obtained using this algorithm for pvc beats with the tp of 57% and fn of 43% , if none of the other algorithms ( state logic machine and pan tompkins ) are able to find r peaks . fp , fn , tp , and tn of detected r peaks for ventricular abnormalities in ecgs studied in this paper are according to table 2 . fp , fn , tp and tn of r peak detection in ventricular abnormalities studied morphological features are as the following : the amplitude of r peak , the rising and falling slopes of qrs complex , qs interval , positive and negative areas ( the sign of this value is negative ) of the complex qrs [ eqs . the positive and negative qrs complex area all of the features are obtained on lead ii . in state logic machine algorithm , the ecg signal has passed through band pass filter with zero phase . then , a 1_second window has been determined on the ecg signal and the average of this window has been calculated ( m ) . the state logic machine considered two conditions for each of the r and s and t waves . estimating the location is done by considering the first condition , and definite presence of r peaks in this interval is determined by considering the second condition and its constraints . each condition has been different for peak detection in ecg at each step . at zero state , the product of m and an initial weight ( w=1.8 ) if this product is less than the mean value calculated in a 15_sample interval ( m1 ) from the one second interval , and also if the amplitude of the studied sample is larger than the rest of the samples , the amplitude and the location of the first sample from the one second window is stored as r peak ; then , we switch to state of one , which reports the definite presence of r peaks . in summary ( 3 ) ] : if ( m1 > m * weight s(i ) > s(j+1 ) ) ( j = 1 : 3 ; i in each step is fixed ) then s(i ) is stored as r peak . ( 3 ) in state of one , the stored amplitude from zero state is compared with the next four samples , to ensure that is the maximum one . each sample in this window must be less than its previous sample . if such a condition is imposed , the index from zero state is stored as the r peak amplitude and its location . an interval containing 0.04 s before this location until the location of this sample is considered , and minimum amplitude is assumed as q peak . also , weight is updated in this state , which means that if the number of detected r peaks is more than eight , 0.3 of mean of these eight r peaks is achieved and it is divided to m. then , the state will be equal to two . in the state of two , if m1 is less than m , we switch to state of three for finding s peak . the period of investigation for state of three is 0.2 s. if , among the eight samples , the studied sample is less than the rest of the samples and if each sample is less than its previous sample , we ensure that the studied sample is s. then , the state is equal to four . in the state of four , if m1 is less than m , we switch to state of six for finding t peaks . in state of six the threshold is considered based on the recent values of s and m. if m1 is more than this threshold [ eq . ( 4 ) ] , this condition is acceptable for three consecutive samples , and the peak of the studied sample is more than its subsequent eight samples ( each sample in this eight - sample window should be smaller than the previous sample ) , this sample is stored as t peak and state is updated to six . |samplitude(end)|)|*+samplitude(end ) ( 4 ) state of six is for prevention of finding several r peaks in a 0.4 s window . after this delay , the algorithm switches to zero state and this algorithm is repeated for each 1_second window . after extracting signal features , support vector machine ( svm ) by ( gaussian ) radial basis function kernel is used for classification of ventricular abnormalities . the method used in svm is one - against - all . to classify each arrhythmia , data segmentation includes 8% of training data and 20% of testing data . the accuracy of the data is obtained by six times shuffles . in this research , these signals include sudden cardiac death records for three patients and five normal sinus rhythms of five healthy persons . the feature extraction step extracted six features from more than 300 beats of mit - scd that included six acute abnormalities studied in this research , and 350 beats from normal sinus rhythm . these features , including the amplitude of r peak , the rising and falling slope of qrs complex , qs interval , and positive and negative areas of the complex qrs , were obtained from each beat . the classification accuracies of normal sinus rhythm , vt , vfl , vfi , pvc , and veb rhythm were evaluated . the accuracies achieved from this essay are compared with previous studies and the maximum accuracy is reported . by using the combined algorithm in this paper the algorithm used in this study was applied to increase the classification reliability of normal and abnormal beats in ecg real time signals and a combination of two algorithms ( pan tompkins and state logic machine ) was used for finding r peaks in beats containing acute ventricular abnormalities . in this research , morphological features were applied for classification of the aforementioned ventricular abnormalities . in this research , studying beat was done in beat - to - beat , real time for classification of ventricular arrhythmias . as a result , studying beat - to - beat may help the medicines for faster recognition of abnormalities rather than heart rate . if each abnormality is studied at each lead and the most effective features in each lead are obtained , the abnormality classification will have very high accuracy . independent features of leads will be very useful if they are diagnosed with high accuracy . | ventricular arrhythmias are one of the most important causes of annual deaths in the world , which may lead to sudden cardiac deaths .
accurate and early diagnosis of ventricular arrhythmias in heart diseases is essential for preventing mortality in cardiac patients .
ventricular activity on the electrocardiogram ( ecg ) signal is in the interval from the beginning of qrs complex to t wave end .
variations in the ecg signal and its features may indicate heart condition of patients .
the first step to extract features of ecg in time domain is finding r peaks . in this paper ,
a combination of two algorithms of pan tompkins and state logic machine has been used to find r peaks in heart signals for normal sinus signals and ventricular abnormalities .
then , a healthy or sick beat may be realized by comparing the difference between r peaks obtained from two algorithms in each beat .
the morphological features of the ecg signal in the range of qrs complex are evaluated .
ventricular tachycardia ( vt ) , ventricular flutter ( vfl ) , ventricular fibrillation ( vfi ) , ventricular escape beat ( veb ) , and premature ventricular contractions ( pvcs ) are abnormalities studied in this paper . in the classification step
, the support vector machine ( svm ) classifier with gaussian kernel ( one in front of everyone ) is used .
accuracy percentages of ventricular abnormalities mentioned above and normal sinus rhythm are respectively obtained as 95.8% , 92.8% , 94.5 , 98.9% , 91.5% , and 100% .
the database of this paper has been taken from normal sinus rhythm and mit - scd banks available on physionet.org . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
surgeries of all kinds and post - operative pain have been long shown to increase risk of hyperglycemia by triggering cytokines and stress hormones known to aggravate insulin action . thus , it is not surprising that hyperglycemia occurs after major operations required for solid organ transplant . but , as the numbers of solid organ transplants and bone marrow transplants performed each year continue to grow , and transplant recipients live longer , more transplant patients will require management of glucose not only after their initial transplant procedure but also after subsequent hospitalizations for episodes of care that may or may not be related to their transplant . current immunosuppression regimens contribute to hyperglycemia in organ transplant patients , as does preexisting risk . diabetes is common among transplant candidates , but some recipients are first recognized as having diabetes only after transplant . diabetes that is first diagnosed after transplant , previously called new onset diabetes after transplantation ( nodat ) , may be new onset but could also represent previously unrecognized diabetes , so a recent international consensus panel suggested changing the name to post - transplant diabetes mellitus ( ptdm ) [ 2 , 3 ] . hyperglycemia is also common immediately after solid organ and bone marrow transplant , with a frequency reported at 8090 % or more for kidney transplant recipients in the first days to week following transplant [ 4 , 5 ] but may resolve after immunosuppression doses are reduced . thus , the diagnosis of ptdm should be reserved for hyperglycemia that persists after the recipient has been reduced to maintenance immunosuppression doses . the incidence of ptdm using these new guidelines is as yet unknown , but the increasing frequency of obesity , the significant impact of current immunosuppression regimens on insulin secretion and action , and the greater scrutiny of glucose control in all hospitalizations have led to the recognition that significant hyperglycemia is very common in the immediate post - transplant hospitalization . with this background , whether treating preexisting diabetes , post - transplant diabetes , or new hyperglycemia that may be diagnosed as post - transplant diabetes at a later time , management of hyperglycemia in the transplant patient is common and important . this chapter will focus on the contributing factors to hyperglycemia in the hospitalized transplant patient , the current knowledge about outcomes and consequences of uncontrolled hyperglycemia after transplant , goals for glucose control , and practical strategies and considerations for glucose management in these populations , including the need to plan for care transitions to home after hospital discharge . diabetes is the most common cause of end - stage renal disease and need for kidney transplant in the usa [ 68 ] . diabetes is also present in 1422 % of heart transplant recipients [ 911 ] and up to a quarter of liver transplant recipients [ 12 , 13 ] . obesity is also very common in transplant populations . up to 60 % or more of kidney transplant recipients are overweight or obese at the time of transplant , and further weight gain after transplant is common , which can exacerbate hyperglycemia . obesity in the organ transplant recipient is directly linked to insulin resistance , as shown in kidney transplant recipients , where risk for hyperglycemia is associated with body mass index , though not necessarily in the immediate post - operative period after transplant [ 16 , 17 ] . ptdm , as defined by older 2003 guidelines , is estimated to occur in 17 to 74 % of kidney transplant recipients , up to 30 % of liver transplant recipients , and approximately a quarter of heart transplant recipients [ 1823 ] . newer consensus guidelines suggest that delaying diagnosis until persistent hyperglycemia is demonstrated after the recipient has been reduced to maintenance immunosuppression doses ( table 1 ) [ 3 ] . while hemoglobin a1c has been added as a diagnostic criteria for diabetes by the american diabetes association for diagnosis in the general population , it is associated with an unacceptable false - negative rate for diagnosis of diabetes after transplant due to the frequency of anemia and transfusion so should not be as the sole screening criteria in the first year after transplant . it should be noted that even with regular screening for diabetes , transplant patients can develop interval ptdm that may remain asymptomatic and unrecognized until they are hospitalized for another cause.table 1diagnosis of post - transplant diabetes mellitusdiagnosis of post - transplant diabetes mellitus is similar to the diagnosis of diabetes in non - transplant populations , where the testing should be performed outside of the hospital , with no recent history of restricted nutrition , while on maintenance immunosuppression doses . diagnosis can be made in the following ways [ 3 ] : fasting glucose 126 mg / dl ( 7 mmol / l ) on more than one occasion random glucose 200 mg / dl ( 11.1 mmol / l ) with symptoms oral glucose tolerance test where 2 h glucose 200 mg / dl ( 11.1 mmol / l)while hemoglobin a1c is allowed for diagnosis in non - transplant patients , it is not reliable for diagnosis of ptdm in the first year after transplant so should not be used for screening in the absence of other testing diagnosis of post - transplant diabetes mellitus one of the most significant contributing factors to hyperglycemia in the hospitalized transplant patient is the immunosuppression required by transplantation . corticosteroids have been long recognized to contribute to hyperglycemia by causing or exacerbating insulin resistance and increasing hepatic gluconeogenesis [ 17 , 25 , 26 ] . high - dose corticosteroids are commonly prescribed with induction , or to treat rejection or graft - versus - host disease , as after bone marrow transplant , in addition to being part of their long - term immunosuppression for many . other types of immunosuppressant agents also contribute to hyperglycemia including the calcineurin inhibitors ( tacrolimus more than cyclosporine ) and inhibitors of the mammalian target of rapamycin ( mtor ) , such as sirolimus . tacrolimus can reduce insulin secretion , increase islet apoptosis , and exacerbate insulin resistance [ 2730 ] . calcineurin - sparing strategies have also been shown to be associated with less risk of ptdm and better graft survival in a meta - analysis of 56 randomized control trials [ 31 ] . sirolimus has been shown to be independently associated with ptdm , in a large cohort of kidney transplant recipients in the us renal data system . a separate retrospective analysis of treatment regimens also demonstrated that sirolimus predisposes to ptdm after kidney transplantation [ 33 ] . studies in vitro show that sirolimus disrupts insulin action through multiple effects on the insulin signal transduction pathway and can reduce beta cell function and islet mass [ 28 , 32 , 3436 ] . nutritional support , often required immediately after both solid organ and bone marrow transplants , can also impact glycemic control , with or without a history of diabetes . total parenteral nutrition ( tpn ) is required after all small bowel transplants , often needed for management of graft - versus - host disease after bone marrow transplant which affects intestinal function [ 38 , 39 ] , and commonly used post - transplant for other solid organ recipients when enteral nutrition is contraindicated for an extended period of time . tpn can cause hyperglycemia in all populations , and risk is greater for those with significant insulin resistance , which describes many organ transplant recipients . immunosuppression increases risk for infections of all kinds after transplant , and severe infection , particularly sepsis , can precipitate hyperglycemia even in those patients with otherwise normal glucose tolerance . preexisting chronic infections like hepatitis c or cytomegalovirus , more common to some transplant populations , also increase risk of hyperglycemia . insulin requirements of diabetes patients decrease as renal disease progresses , due to reduced insulin degradation in the kidney . thus , after successful kidney transplant , insulin clearance by the kidney improves , unmasking preexisting glucose intolerance to contribute to the hyperglycemia observed in the immediate post - operative setting . glucose control improves outcomes for many groups of non - transplant hospitalized patients [ 4550 ] ptdm , whether originating before or immediately after transplant , also reduces overall graft and patient survival after kidney transplant [ 51 , 52 ] . there is also increasing recognition that perioperative glycemic control alone impacts outcomes after transplant even in the absence of ptdm , possibly because of its associated risk of ischemia reperfusion and delayed graft function after kidney transplant [ 53 , 54 ] . graft rejection is also more frequent in kidney transplant recipients with post - operative hyperglycemia , whether or not they have diabetes [ 5558 ] . in a retrospective review of liver and liver - kidney transplant recipients , patients with higher post - operative glucose levels ( more than 200 mg / dl ) had a higher rejection rate compared to those with glucose levels less than 200 mg / dl . however , to date , no studies suggest a benefit of perioperative glycemic control beyond that recommended for all hospitalized patients on mortality after kidney transplant [ 60 ] . in a retrospective analysis , van den berg et al . reported no correlation between post - operative blood glucose levels and rates of post - kidney transplant infections . however , a separate retrospective analysis revealed an increased risk of rehospitalizations for infectious complications in kidney transplant recipients who developed post - operative hyperglycemia . since the rate of post - operative hyperglycemia in this population ( defined as two blood glucose values > 126 mg / dl ) was quite low at 7.6 % , this study may not be representative of other kidney transplant populations or centers . reported a significantly higher rate of infections in kidney transplant recipients who developed hyperglycemia ( blood glucose > 200 mg / dl ) in the first 100 h post - operatively , again suggesting that post - operative hyperglycemia may be associated with increased risk for infections . in liver transplant recipients , ammori and colleagues reported a positive association between intraoperative glucose > 150 mg / dl and infection rate at 30 days when compared to an intensive glucose control group ( < 150 mg / dl ) . park and associates also reported that in patients undergoing liver transplantation , severe intraoperative hyperglycemia ( > 200 mg / dl ) was associated with increased risk of post - operative surgical site infection when compared to those with mean blood glucose 180 mg / dl . those with higher glucose values after transplant are also more likely to be given a diagnosis of ptdm , as suggested by a retrospective review of liver transplant recipients . elevated fasting glucose during the first 2 weeks was also associated with a greater incidence of diabetes after transplant . conversely , early tight glycemic control after kidney transplant with insulin may actually reduce risk of later ptdm [ 5 ] . glucose goals for the hospitalized transplant patient should be the same as other hospitalized patients ( icu 140180 mg / dl and non - icu premeal < 140 and random < 180 mg / dl ) . the one randomized control study of kidney transplant recipients designed to assess the impact of tighter control during the immediate hospitalization ( 70110 vs < 180 mg / dl ) did not improve any outcome , and the intensive group experienced not only more hypoglycemia but also more rejection episodes , although the latter was not statistically significant [ 60 ] . while it might be tempting to assume that tighter control would improve outcomes after heart transplant , in particular , there are no published studies . thus , to date , more stringent glucose control could result in worse rather than better outcomes after transplant . treatment of hyperglycemia immediately after transplant is generally initiated with and often requires an intravenous insulin infusion algorithm with frequent glucose monitoring . many types of intravenous insulin protocols can successfully achieve desired glucose goals , including algorithms that are nursing - driven . when the patient is ready for enteral or parenteral nutrition , the intravenous insulin infusion should be transitioned to subcutaneous insulin injections appropriate to their insulin requirements on the intravenous insulin infusion , planned size and frequency of meals , if eating , or planned parenteral or enteral tube feeding orders . the insulin treatment regimen required at the time of transition is highly variable so does not readily lend itself to a fixed insulin per kilogram dose , in part , because of the variation in kidney function , immunosuppression regimen , corticosteroid and other immunosuppressant doses required , severity of obesity , and other factors that drive insulin resistance in this group . if the patient is being given total parenteral nutrition ( tpn ) , 80 % of the intravenous insulin requirements over the last 24 h are usually administered as either neutral protamine hagedorn ( nph ) ( 1/3 delivered every 8 h ) or glargine ( either as a once daily dose or split with half given every 12 h ) . the first dose of long - acting insulin should be given at least two and preferably 4 h before the intravenous insulin is discontinued . if daily insulin requirements are decreasing rapidly , the daily requirements may be better estimated from that used in the last 8 h and multiplying by 3 to obtain the starting daily dose . after transitioning to subcutaneous insulin injections , glucose should be measured at the bedside at regular intervals , either every 4 or 6 h , with a scale provided to guide the administration of supplemental subcutaneous regular or fast - acting analog insulin . while there is no one standard , a starting point is often 12 units for every 4050 mg / dl above a defined glucose threshold value , that value being 150 mg / dl in our center . insulin requirements can change dramatically when transitioning off tpn , so insulin requirements should either be reassessed off tpn before transitioning to subcutaneous insulin or a weight - based dose of insulin should be calculated ( 0.20.4 units / kg ) , of which 50 % should be used as the starting basal insulin dose , whether the patient is being started on enteral tube feeding or meals . fast - acting insulin is then used for bolus tube feedings or for meals , usually ordered as a ratio of fast - acting insulin / grams of carbohydrates ( e.g. , 12 units/15 g as a starting dose ) . again , a supplemental scale of fast - acting insulin should be provided to address blood glucose values higher than the desired range . when reliability of food ingestion is a concern , as with frequent vomiting , the dose should be given only after it is clear what the patient actually ate and kept down , not based on what is ordered . if the patient is not on tpn and is being transitioned from clear liquids to full diet , 80 % of the intravenous insulin requirements over the last 24 h are usually given as basal insulin , most often as glargine or levemir , with fast - acting insulin ordered for ingested food based on an insulin/ carbohydrate ratio , as described above . an additional fixed dose of nph or glargine ( e.g. , 10 units ) is often added to cover intermittent corticosteroid doses given for induction or acute rejection to minimize glucose excursions from this dose alone . whenever possible , coordinating the timing of glucocorticosteroid dose , whether planned for oral or intravenous administration , with that of the long - acting insulin will improve the ability to titrate the insulin dose to cover the possible increased requirements over that interval . when changing to subcutaneous insulin dosing , glucose testing frequency and supplemental insulin orders should also be adjusted to the timing of the subcutaneous insulin injections . insulin doses should be adjusted daily based on the evidence of increasing or decreasing insulin resistance , planned changes in immunosuppression dosing , significant change in kidney function , or planned changes in nutrition strategy . regular conversations between the glucose monitoring team with the nursing staff should reinforce a need for rapid communication of any planned or unplanned changes in immunosuppression ; type , timing , frequency , or discontinuation of nutrition therapy ; or planned surgery . diabetes education should be considered early , to include glucose monitoring and even insulin administration training , as intermittent use of insulin is common enough that early training may prevent a delay in discharge . when discharge is imminent , some can be considered for oral hypoglycemic agents if insulin requirements are low or to simplify therapy if there is concern that the patient or the family is having difficulty in handling glucose monitoring and/or insulin therapy after discharge . there are few available studies to assess safety or risk of most oral hypoglycemic agents in transplant patients , and there are greater potential risks for many oral agents as outlined in table 2.table 2special considerations for use of oral and subcutaneous hypoglycemic agents in transplant patientsdiabetes medication classpotential restrictions or considerationssulfonylureas or repagliniderisk of hypoglycemia if gfr is reduced , less with repaglinide than sulfonylureas ; potential drug - drug interactions of sulfonylureas with cyclosporinemetforminshould not be used in the hospital or within 48 h of intravenous contrast administration , known heart failure , elevated liver function tests , or reduced gfrdpp - iv inhibitorshas not been studied with gfr < 40 ml / min ; linagliptin least likely to require dose adjustment for low gfrthiazolidinedionesmay be preferentially chosen for treatment of fatty liver after liver transplant but should generally be avoided in others with elevated liver function tests , heart failure , or significant peripheral edema . may also reduce hemoglobin and bone massacarboseavoid with low gfr ; less likely to be effective for most transplant patients because of mild benefitsglt-2 inhibitorsno studies to assure safety in transplant patients ; known to increase risk of genitourinary tract infections in women and balanitis in men so concern risk would be greater with immunosuppression . because these agents are known to reduce gfr and can reduce blood pressure , they may have a negative impact on kidney graftglp-1 agonists ( e.g. , exenatide , liraglutide)these agents have not been studied in transplant patients but are known to reduce intestinal motility , which may affect immunosuppressant pharmacokinetics . should not be used with gfr < 40 ml / minthere are few studies evaluating the safety or efficacy of many diabetes therapies other than insulin . while most transplant patients will be treated with insulin while in the hospital , some may be transitioned before discharge back to other agents that they were taking prior to the hospitalization . . gfr glomerular filtration rate , sglt-2 sodium - glucose co - transporter-2 special considerations for use of oral and subcutaneous hypoglycemic agents in transplant patients there are few studies evaluating the safety or efficacy of many diabetes therapies other than insulin . while most transplant patients will be treated with insulin while in the hospital , some may be transitioned before discharge back to other agents that they were taking prior to the hospitalization . frequent and sometimes severe shifts in renal function are common in this population and can cause rapid changes in insulin requirements those being transplanted for hepatitis c , particularly if on a tacrolimus - containing regimen , are much more likely to develop significant hyperglycemia after transplant . a retrospective review of patients who underwent liver transplantation concluded that post - operative infections were also lower in patients on insulin infusions managed by a glucose management service where average peri - operative glucose was 158 mg / dl , compared to those not on the glucose management service where average glucose was 189 mg / dl [ 67 ] . diabetes used to be a contraindication to heart transplant , but programs generally do not exclude diabetes outright , and there are no significant differences in outcomes between diabetic and non - diabetic recipients [ 68 , 69 ] . many lung transplant recipients are thin , so less likely to be considered at risk for diabetes , but hyperglycemia is still common . many patients receive lung transplant for cystic fibrosis , and many have developed cystic fibrosis - related diabetes prior to , or at risk for , hyperglycemia following transplant . it is common that patients receiving intestinal transplant require parenteral nutrition longer , but there is little data regarding the incidence of hyperglycemia or ptdm following intestinal transplant . the prevalence of post - transplant hyperglycemia has been reported as high as 71 % after bone marrow transplantation ( bmt ) and appears to be at least partially dependent on whether or not patients received parenteral nutrition [ 7072 ] . hyperglycemia after bmt is associated with higher rates of complications , specifically need for red blood cell and platelet transfusion and delay in granulocyte and platelet engraftment times . hyperglycemia during the neutropenic period following myeloablative allogeneic bmt is also associated with higher rates of organ dysfunction , acute graft - versus - host disease ( gvhd ) , and worse overall survival and non - relapse mortality . body mass index ( bmi ) and degree of hyperglycemia within the first 10 days after transplant appear to play a significant prognostic role for developing acute gvhd . a recent study showed that 14.2 % of normal to overweight subjects ( normal bmi 2124.9 ; overweight bmi 2529.9 ) developed severe hyperglycemia within 10 days of allogeneic bmt ( glucose > 9.99 more obese ( bmi > 30 kg / m ) and no lean ( bmi < 21 kg / m ) developed severe hyperglycemia , but the risk of gvhd was unrelated to severe hyperglycemia [ 74 ] . because skin swelling can be severe in some patients with gvhd , if subcutaneous insulin appears to be inadequately absorbed , intravenous insulin infusion may be required , as well as to achieve glucose goals in the setting of high - dose corticosteroids or with tpn . in some protocols , tpn is given as a 20-h infusion , which can be difficult to match with intravenous insulin . hyperglycemia is likely to be a risk with any large surgery and can not be completely avoided . however , there is considerable interest in reducing severity of hyperglycemia and strategies that might reduce the longer - term risk of developing ptdm . ptdm can reduce graft and patient survival , but rejection is the number one cause of reduced graft and patient survival . since graft survival remains the most important outcome overall , the immunosuppression regimen should be selected solely based on reducing graft failure , not diabetes risk . steroid - free regimens were first developed with the hope of reducing risk of ptdm but have not been associated with lower rates of ptdm because non - steroidal immunosuppressive agents also contribute to ptdm . developing immunosuppression agents or regimens with less risk of ptdm is still a goal for many manufacturers and transplant teams . multiple trials are in progress to determine if early introduction of hypoglycemic therapies can protect islets and prevent later ptdm , including dipeptidyl peptidase ( dpp)-iv inhibitors , metformin , and insulin . dpp - iv inhibitors and metformin are used for management of ptdm so are also attractive agents for prevention of ptdm [ 76 , 77 , 78 ] . thiazolidinediones ( tzd ) have been shown to be safe and efficacious in the management of diabetes after kidney transplantation [ 79 , 80 ] , but the potential side effects of tzds have not made it an attractive prevention agent . insulin has been studied in the prevention of ptdm , and basal insulin has been shown to decrease incidence of ptdm after kidney transplant [ 5 ] . metformin use can be difficult in the first year after kidney transplantation due to fluctuations in creatinine but is being studied for prevention . intensive lifestyle change can prevent type 2 diabetes in at risk non - transplant populations so has been suggested as a method to prevent ptdm . active lifestyle modification including dietitian referral , exercise program , and weight loss advice benefits kidney transplant recipients with impaired glucose tolerance and should be incorporated into any preventive measure . management of hyperglycemia in the immediate post - operative period is challenging because transplant recipients often have reduced or changing kidney function , experience unpredictable changes in nutrition due to nausea and vomiting and frequent need for parenteral and enteral nutrition , require treatment with types and doses of immunosuppression agents that can dramatically impact glucose intolerance , and have other events like surgery and infections or pain that can aggravate preexisting insulin resistance . glucose goals in the hospital are the same for transplant recipients as any other hospitalized patient , but transplant recipients are at greater risk for hypoglycemia because of the frequent and potentially rapid changes in factors that impact glucose ( renal function , nutrition , it is critical that all consulting teams work together as well as with the nursing team to anticipate and rapidly respond to any changes in status that might warrant a change in intravenous insulin protocol or other pharmacologic therapy . planning for discharge is even more important than other hospitalized patients , as ongoing changes in insulin resistance can continue to occur very rapidly . the patient needs to understand when and how to contact the team if significant changes in glucose , high or low , occur after discharge . | significant hyperglycemia is commonly observed immediately after solid organ and bone marrow transplant as well as with subsequent hospitalizations .
surgery and procedures are well known to cause pain and stress leading to secretion of cytokines and other hormones known to aggravate insulin action .
immunosuppression required for transplant and preexisting risk are also major factors .
glucose control improves outcomes for all hospitalized patients , including transplant patients , but is often more challenging to achieve because of frequent and sometimes unpredictable changes in immunosuppression doses , renal function , and nutrition . as a result , risk of hypoglycemia can be greater in this patient group when trying to achieve glucose control goals for hospitalized patients .
key to successful management of hyperglycemia is regular communication between the members of the care team as well as anticipating and rapidly implementing a new treatment paradigm in response to changes in immunosuppression , nutrition , renal function , or evidence of changing insulin resistance . |
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the origin of this condition remains unknown for the majority of cases , although several infectious , vascular , and immune causes have been proposed . a careful examination of the patient is required in order to exclude treatable causes and life - threatening situations such as vascular processes and tumour diseases . several different treatments have been used , including corticosteroids , antiviral drugs , rheology , and oxygen therapy . although no single treatment has demonstrated decisive results , a short schedule of oral corticosteroids is generally recommended . within the spectrum of possible causes of sudden deafness , vascular processes could be candidates for the pathogenesis of this condition because of their acute / abrupt nature and the audiovestibular symptoms ( deafness , vertigo , tinnitus , sensation of ear fullness , and hyperacusis ) that occur in ischaemia of the inner ear [ 25 ] . , an ischaemic process would explain the spontaneous recovery observed in over half of all cases , once vascular reperfusion is achieved . cerebral ischaemia , even the transient one , is characterised by an acute inflammatory response in the ischaemic area , which , in turn , produces an acute - phase response in the peripheral bloodstream , where polymorphonuclear cell concentrations are increased in an attempt to combat the damage caused by blood deficiency [ 710 ] . this study attempts to describe the peripheral acute - phase inflammatory response associated with the appearance of idiopathic sudden deafness . there is growing evidence of the association of an inflammatory process with sudden deafness . inflammatory cytokines such as interleukin-1 , interleukin-1 , interleukin 4 , interleukin 6 [ 14 , 15 ] , and matrix metalloproteinase-1 , as well as an increased neutrophil count in peripheral blood . there is also indirect evidence such as being improved after treatment with local hypothermia or worsening with vertebrobasilar flow decrease . fifty - four patients were cared for in the emergency department due to sudden hearing loss during 2011 . inclusion criteria were the occurrence of sudden deafness in one ear of 30 db or more in at least three consecutive frequencies the mean age of the patients was 45.4 years , with a range of 2478 years . the mean time of evolution between the appearance of the sudden deafness and the patient seeking medical care with the application of treatment was 6.4 days . the sudden deafness was unilateral in all cases : the right ear in 47% and the left ear in 53% . the emergency audiometric analysis revealed moderate sensorineural hearing loss ( 3545 db hl ) in 46% of patients and severe sensorineural hearing loss ( > 74 db hl ) in 54% . the audiometric curve produced was flat in 30% , ascending in 27% , and descending in 43% . the healthy ear had normal hearing in 44 patients , mild hearing loss in 10 cases ( 2534 db hl ) , and moderate sensorineural hearing loss in one . otorhinolaryngological and neurological examination , audiometry , tympanogram , haemogram , and cranial magnetic resonance ( t1 and t2 phases and flair fluid attenuated inversion recovery ) . the haemogram was performed in the emergency department before the patient started the corticosteroid treatment . a descriptive statistical analysis of the data , expressed as mean and standard deviation were carried out . mann - whitney u tests using spearman 's correlation coefficient ( rho ) using ibm spss software , version 19 , were performed . this study was performed in accordance with the principles of the helsinki declaration ( 1975 , 1983 ) . the research protocol , the patient information sheet , and the signed informed consent documents were all approved by the hospital ethics committee . the acute - phase response in the peripheral bloodstream due to the inflammatory process caused by the transient cerebral ischaemia is manifested in the change of white blood cells . figure 2 displays the mean values for this ratio during the first week following the appearance of the sudden deafness . no significant differences between the values for the neutrophil / lymphocyte ratio and sex , age , initial hearing loss , or type of audiometric curve were observed . it is defined as a sudden sensorineural hearing loss of at least 30 db and at least three consecutive frequencies , normally in one ear , with no previous background of ear disease . the causes of sudden deafness have been sought within the ear , with different proposed aetiologies such as a ruptured cochlear membrane , microangiopathic ear processes , viral ear infection , autoimmune diseases of the inner ear , mnire disease , or vestibular schwannoma , although none of these theories has been sufficiently proven or can be applied in all cases . the aetiology for this condition is defined in only 10% of cases , whereas the rest are labelled as idiopathic . in young patients ( younger than 50 ) , it is usually associated with viral problems , where the most relevant viruses include measles , mumps , herpes zoster , and adenovirus . after the age of 50 years , the viral infections are characterised by decreased neutrophil levels and higher concentrations of mononuclear cells , such as lymphocytes and monocytes , which would reduce the neutrophil / lymphocyte ratio far below normal values , ruling out a viral origin of sudden deafness . the incidence of sudden deafness has increased over time , and this condition has been estimated to reach 160 cases per year per 100,000 inhabitants . the diagnosis is made based on clinical symptoms , audiometry , and a magnetic resonance that includes the internal auditory canals where the auditory nerve passes . ischaemic processes of the inner ear have begun to be considered the pathogenic mechanism involved in sudden deafness . several publications have reported that patients diagnosed with idiopathic sudden deafness have a higher probability of suffering a stroke in the following 5 years [ 24 , 25 ] . genetic polymorphisms have been associated with inflammatory pathways in patients with sudden deafness [ 13 , 14 , 16 ] . under ischaemic conditions in the central nervous system , an inflammatory response is produced which attempts to improve the hypoxia . in the peripheral bloodstream , the inflammatory reaction presents an acute - phase response , increasing the concentrations of neutrophils and monocytes and decreasing lymphocytes [ 710 ] . the objective measurement of increased neutrophil / lymphocyte ratio values during the transient ischaemic process , and its subsequent normalisation , provides evidence that sudden deafness is associated with inflammatory processes secondary to transient cerebral ischaemia , that is to say a temporal link exists , which was corroborated by our statistical analysis ( figure 2 ) . the magnetic resonance images show the absence of organ damage , as in transient ischemic attacks . the acute - phase response reached its greatest intensity 4872 hours following the appearance of the sudden deafness . in all cases studied , any other process that could have caused an inflammatory reaction was ruled out using the patient 's clinical history . the acute - phase response caused by the inflammatory process of the transient cerebral ischaemia perhaps the most important result of acknowledging the presence of cerebral ischaemia in the development of sudden deafness is the severity of the process or , at least , the warning of the need to reexamine the patient for maintenance or advice by cardiology and then neurology specialists once again . the initial treatment of sudden deafness should be decided upon by the health professional that examines the patient , which would be an otorhinolaryngologist , provided the pathogenesis of cerebral ischaemia or any other organ disorder . these study findings relate to a nonspecific systemic inflammatory response in sudden deafness , which actually aligns with recent findings in this area [ 1118 ] . this study shows that there is an acute - phase response in the peripheral bloodstream with an increased neutrophil / lymphocyte ratio as an indirect evidence of an inflammatory process caused by transient cerebral ischaemia in sudden deafness . this same increase in the neutrophil / lymphocyte ratio also serves to rule out a viral origin for sudden deafness , since viral infections decrease neutrophil concentrations and increase lymphocyte counts , thus reducing the neutrophil / lymphocyte ratio . an objective measurement of these values aids in understanding the pathogenic mechanisms associated with sudden deafness , as well as in providing better care to the patients . | the acute - phase inflammatory response in the peripheral bloodstream can be an expression of transient cerebral ischaemia in idiopathic sudden deafness .
for this , a neurological and otorhinolaryngological examination of each patient , performing tests on audiometry , and tympanometry , haemogram , and cranial magnetic resonance were performed .
the acute - phase inflammatory response manifests as an increased neutrophil / lymphocyte ratio that is detected 4872 hours after the appearance of sudden deafness .
this study shows that there is an acute - phase response in the peripheral bloodstream with an increased neutrophil / lymphocyte ratio as an expression of an inflammatory process that can be caused by transient cerebral ischaemia in sudden deafness .
in addition , the increased neutrophil / lymphocyte ratio can rule out a viral origin of sudden deafness , since a viral infection lowers the neutrophil count and increases the lymphocyte count , thus reducing the neutrophil / lymphocyte ratio .
these findings aid in understanding the pathogenic mechanisms involved in sudden deafness and offer better treatment to the patient . |
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fungal infection of the paranasal sinuses with uncommon species is an increasingly recognized entity , both in normal and immunocompromised individuals . the pattern of fungal infection of the sinuses can be divided into invasive , noninvasive , and semi - invasive based on the presence of fungal hyphae in the tissue with associated granulomatous reaction or tissue necrosis [ table 1 ] . although fungal infections of the paranasal sinus are uncommon , 35% of cultured sinus samples are positive for fungi . aspergillus species are the most common causative agents of fungal sinusitis followed by candida and mucor . hyalohyphomycosis ( gk . hylos glass ) is an infection caused by a number of species in which the basic tissue form of fungi is hyaline without any pigment in their cell wall . . the disease may present as cutaneous , subcutaneous or systemic infection in the immunocompetent or immunocompromised patients . this article presents a case report of maxillary sinus hyalohyphomycosis caused by two opportunistic soil saprophytes of paecilomyces and scopulariopsis species . a 40-year - old adult male reported to the outpatient department with the chief complaint of a firm mass in the left cheek region . the duration of the swelling was approximately 6 months and the growth was continuous , slow , and painless . the patient was a farmer by profession and had no history of illness . on further probing , the patient gave the history of decay and infection in the left maxillary second molar a year ago . he underwent an unwarranted and irregular course of antibiotics and other drugs for almost 23 months prescribed by traditional practitioners in his village followed by extraction . the swelling appeared on the left cheek after approximately 2 months of extraction . on examination , the swelling was firm , nontender , sessile , mobile and oval in shape . it measured 4 3 cm and was not fixed to the surrounding soft tissue . there was no paraesthesia over the distribution of infraorbital nerve and the overlying skin was unaffected . intraorally , the left maxillary teeth were vital on pulp testing with no periapical radiolucency in dental radiographs . an occipito - mental view ( omv ) of skull showed generalized opacification of the left maxillary sinus . the computerized tomography ( ct ) scan showed evidence of well - defined , homogeneously enhancing soft tissue mass lesion seen in the left maxillary sinus [ figures 1 and 2 ] . the lesion was causing lytic destruction of anterior wall of the sinus and also the erosion of left floor of orbit . routine hematological evaluation showed mild leucocytosis with eosinophilia and an increased erythrocyte sedimentation rate ( esr ) [ table 2 ] . a differential diagnosis of a chronic sinusitis , benign cyst or tumor , and maxillary antrum mycosis was made , and an incisional biopsy was performed under local anesthesia . the pathological tissue was creamish in color , firm in consistency , and hyalinized in texture [ figure 3 ] . hematological values of the patient pathological specimen the histopathological picture showed diffuse areas of fibrosis and granulation tissue with patchy infiltrate of acute and chronic inflammatory cells . there were few septate organisms , giant cells , macrophages , epitheloid cells , and foci of hemorrhage . microbiological studies included koh ( potassium hydroxide ) wet mount , lcb ( lactophenol cotton blue ) preparation , and culture on sabouraud 's dextrose agar ( sda ) . one thin and white at first and then became gray tan in color and second golden colored , fast growing and powdery . microscopic finding in the first culture showed chains of single - celled conidia produced in basipetal succession from annellide ( basocatenate ) . annellide were solitary with globose , truncate , smooth , and brown colored conidia [ figure 4 ] . microscopic finding of second colony showed conidiophores bearing dense , verticillately arranged branches bearing phialides . chlamydospores were present in short chains , brown , sub spherical and thick walled [ figure 5 ] . based on all these findings , the isolates were identified as paecilomyces and scopulariopsis species . pictomicrograph showing paecilomyces species ( koh 10 40 magnification ) having elongated and tapering phialides with ovoid conidia in basipetal succession pictomicrograph showing scopulariopsis species ( koh 10 40 magnification ) having basipetal globose to pyriform conidia on annellides ( solitary and in groups ) the patient was put on oral itraconazole 200 mg twice daily for a week which resulted in a significant reduction of the extraoral swelling . a thorough sinus debridement and lavage the sinus was approached by the standard caldwell - luc method because of the evident bony erosion of the anterior wall which had to be removed simultaneously [ figure 6 ] . a plastic drain was fixed oroantrally for irrigation after sinus clearance . in the postoperative phase , the patient was again put on oral itraconazole 200 mg twice daily and the sinus was irrigated with 1% acetic acid once daily for 7 days . the patient is been followed regularly for the last 1 year and a check omv x - ray of skull confirmed repneumatization of the left maxillary sinus with complete resolution of clinical sign and symptoms . on examination , the swelling was firm , nontender , sessile , mobile and oval in shape . it measured 4 3 cm and was not fixed to the surrounding soft tissue . there was no paraesthesia over the distribution of infraorbital nerve and the overlying skin was unaffected . intraorally , the left maxillary teeth were vital on pulp testing with no periapical radiolucency in dental radiographs . an occipito - mental view ( omv ) of skull showed generalized opacification of the left maxillary sinus . the computerized tomography ( ct ) scan showed evidence of well - defined , homogeneously enhancing soft tissue mass lesion seen in the left maxillary sinus [ figures 1 and 2 ] . the lesion was causing lytic destruction of anterior wall of the sinus and also the erosion of left floor of orbit . routine hematological evaluation showed mild leucocytosis with eosinophilia and an increased erythrocyte sedimentation rate ( esr ) [ table 2 ] . a differential diagnosis of a chronic sinusitis , benign cyst or tumor , and maxillary antrum mycosis was made , and an incisional biopsy was performed under local anesthesia . the pathological tissue was creamish in color , firm in consistency , and hyalinized in texture [ figure 3 ] . the histopathological picture showed diffuse areas of fibrosis and granulation tissue with patchy infiltrate of acute and chronic inflammatory cells . there were few septate organisms , giant cells , macrophages , epitheloid cells , and foci of hemorrhage . microbiological studies included koh ( potassium hydroxide ) wet mount , lcb ( lactophenol cotton blue ) preparation , and culture on sabouraud 's dextrose agar ( sda ) . one thin and white at first and then became gray tan in color and second golden colored , fast growing and powdery . microscopic finding in the first culture showed chains of single - celled conidia produced in basipetal succession from annellide ( basocatenate ) . annellide were solitary with globose , truncate , smooth , and brown colored conidia [ figure 4 ] . microscopic finding of second colony showed conidiophores bearing dense , verticillately arranged branches bearing phialides . chlamydospores were present in short chains , brown , sub spherical and thick walled [ figure 5 ] . based on all these findings , the isolates were identified as paecilomyces and scopulariopsis species . pictomicrograph showing paecilomyces species ( koh 10 40 magnification ) having elongated and tapering phialides with ovoid conidia in basipetal succession pictomicrograph showing scopulariopsis species ( koh 10 40 magnification ) having basipetal globose to pyriform conidia on annellides ( solitary and in groups ) the patient was put on oral itraconazole 200 mg twice daily for a week which resulted in a significant reduction of the extraoral swelling . a thorough sinus debridement and lavage the sinus was approached by the standard caldwell - luc method because of the evident bony erosion of the anterior wall which had to be removed simultaneously [ figure 6 ] . a plastic drain was fixed oroantrally for irrigation after sinus clearance . in the postoperative phase , the patient was again put on oral itraconazole 200 mg twice daily and the sinus was irrigated with 1% acetic acid once daily for 7 days . the patient is been followed regularly for the last 1 year and a check omv x - ray of skull confirmed repneumatization of the left maxillary sinus with complete resolution of clinical sign and symptoms . chronic indolent fungal sinusitis can occur in both immunocompetent and immunocompromised individual and was first described by hora in 1965 . it is mostly invasive and is characterized histologically by a chronic granulomatous inflammation surrounding broken fungal hyphae . this type of fungal sinusitis may extend beyond the bony confines of the sinuses to the orbit or even to the anterior cranial fossa . therefore , clinically it may mimic a malignant neoplasm , wegener 's granulomatosis , osteomylitis , tuberculosis , and rhinoscleroma . paranasal sinus mycoses are common in north india , northern sudan , and south - western states of the north america which have warm and humid climate . saprophytic fungi are ubiquitous and omnipresent in nature , mostly surviving on dead organic plant matter . some species survive as commensals within the body cavities of humans , including the nose and paranasal sinuses . when a favorable situation ( immunocompromised status ) arises as in prolonged steroid or antibiotic therapy or postsurgical exposure of raw areas , these dimorphic fungi metamorphosize into invasive forms and present themselves in a panoramic spectrum of clinical manifestations [ table 3 ] . an unwarranted and uncontrolled misuse of drugs , especially antibiotics , steroids , and antihistamines , in semi - urban and rural population is an important factor to contribute to this disease in developing countries such as india . the maxillary and ethmoid sinuses are more commonly involved because drainage depends on mucocilliary propagation in these sites . criteria for sinonasal fungal infection during the recent decades , paranasal sinus mycosis has been recognized more frequently in different parts of the world mainly because of the following reasons : ( 1 ) increased awareness among clinicians ; ( 2 ) improved diagnostic tools ; and ( 3 ) increased host susceptibility . successful treatment of such indolent mycotic infections largely depends on the accurate identification of the pathogen and early , appropriate intervention by surgical debridement and sinus ventilation , supported with antifungal medications as per standardized regimen . | fungal infection of the paranasal sinuses is an increasingly recognized entity , both in normal and immunocompromised individuals .
the recent increase in mycotic nasal and paranasal infections is due to both improved diagnostic research and an increase in the conditions that favor fungal infection .
although fungal infections of the paranasal sinus are uncommon , 35% of incidence is reported .
aspergillus , candida , and mucor species are the most common causative agents of fungal sinusitis , but infection with lesser known species have been reported across the world infrequently .
this article reviews and presents a case report of chronic fungal sinusitis in an immunocompetent adult male infected with two species of hyalohyphomycosis group namely , paecilomyces and scopulariopsis which are opportunistic soil saprophytes , uncommon to humans . |
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understanding the evolutionary relationships between groups of organisms has become increasingly reliant on phylogenetic analysis . comparative sequence analysis is a useful method by which one can identify gene , infer the function of a gene 's product , and identify novel functional elements . by comparing several sequences along their entire length , researchers can find conserved residues that are likely preserved by natural selection . a reading frame is a set of consecutive , nonoverlapping triplets of three consecutive nucleotides . a codon is a triplet equating to an amino acid or stop signal during translation . an open reading frame ( orf ) is the section of reading frame containing no stop codons . a protein can not be made if rna transcription ceases prior to reaching the stop codon . therefore , to ensure that the stop codon is translated at the correct position , the transcription termination pause site is located after the orf . orfs also have been utilized to classify various virus families [ 13 ] , including members of norovirus [ 3 , 4 ] . the open reading frame finder ( orf finder ) is a graphical analysis tool that searches for open reading frames in dna sequences . the orf investigator program provides information on the coding and noncoding sequences and performs pairwise alignment of different dna regions . this tool efficiently identifies orfs and converts them to amino acid codes , declaring their respective positions in the sequence . first , the dna sequence is transcribed into rna , and all potential orfs are identified . these orfs are encoded within each of the six translation frames ( 3 in the forward direction and 3 in the reverse direction ) , so that users can identify the translation frame yielding the longest protein coding sequence . several biological organizations have implemented bioinformatics tools on websites . the national center for biotechnology information ( ncbi ) provides many tools for comparing database - stored nucleotide or protein sequences , including the well - known blast algorithms . ncbi also provides several databases , such as genbank and snp , in which biologists can seek homology or specific functions . the european molecular biology laboratory ( embl ) provides freely available data and online bioinformatics tools to all facets of the scientific community . cloud computing is a recently developed concept that delivers computing resources , either hardware or software , over the internet . many types of cloud computing have been proposed , such as infrastructure as a service ( iaas ) , platform as a service ( paas ) , software as a service ( saas ) , network as a service ( naas ) , and storage as a service ( staas ) . most of these services rely on virtualization technology the creation of virtual hardware platforms , operating systems , storage devices , and network resources . cloud computing is welcomed for its user friendliness , virtualization , internet - centric focus , resource variety , automatic adaptation , scalability , resource optimization , pay - per - use , service slas ( service - level agreements ) , and infrastructure slas . many cloud computing vendors distribute these resources on demand from large resource pools installed in data centers . amazon ec2 supplies an infrastructure service , while google app engine and microsoft 's azure services platform supply platform services . in academia , hadoop is an open - source software framework that supports data - intensive distributed computation . under hadoop the computational task is divided into many small tasks , each of which may be executed or reexecuted on a compute node in the hadoop cluster . mapreduce also provides a distributed file system , the hadoop distributed file system ( hdfs ) , that stores the data on compute nodes , enabling a very high aggregate bandwidth across the cluster . both map / reduce and the distributed file system are robust against failure several sequence analysis tools have been redeveloped as cloud tools based on the hadoop architecture , such as cloudblast and crossbow . such importing of preexisting tools constitutes the main goal of bioinformatics as a service ( baas ) . in this paper , we develop a high - availability , large - scale orf phylogenetic analysis cloud service based on virtualization technology and hadoop . this service provides phylogenetic analyses from orfs based on hadoop clusters to support multiple requests . the physical computing power is regarded as a user - pays utility that users can request as desired . users can upload their sequence data or files through the master node ( web portal ) and then submit a job . the job is assigned to the slave node containing the uploaded data , and the slave node completes the job . since orf comparisons have unambiguously established the homology of norovirus , we here adopt norovirus as a case study . the results show that the proposed cloud - based analysis tool , by virtue of virtualization technology and hadoop framework , can readily facilitate baas . in this paper , we propose a cloud - based orf phylogenetic analysis service combining hadoop framework , virtualization technology , phylogenetic tree tool , and diversity analysis . hadoop is performed on the vms created by virtualization technology such as kernel - based virtual machine ( kvm ) . the underlying architecture ensures elasticity , scalability , and availability of the proposed cloud - based service . the proposed cloud service integrates the orf finding process , phylogenetic tree contractions , and orf diversity analysis to generate a complete phylogenetic analysis . the procedure of the analysis is outlined below and shown in figure 1 .
step 1 : detecting open reading frames . the orf finder locates all open reading frames of a specified minimum size in a sequence . in this study , this tool identifies all open reading frames using the standard or alternative genetic codes . step 2 : constructing phylogenetic tree based on open reading frames . a phylogenetic tree ( or evolutionary tree ) is a branching ( tree ) diagram showing the inferred evolutionary relationships between biological species or other entities based on similarities and differences in their physical and/or genetic characteristics . virus orf alignments might reveal a common viral ancestor or an orf that is common to all viruses . this algorithm builds two phylogenetic trees ; one based on full sequences and the other for orfs only , thereby revealing the variance between the two trees . small diversity value at a position implies that protein sequences are very similar at that position . such high - variance frames can be used to observe protein structural differences and to aid vaccine development . in this paper , diversity is calculated from the entropy as follows :
( 1)h(i)=p(xi)log2p(xi ) , xi = { g , a , i , v , } ,
where h(i ) is the value of entropy and p(xi ) is the probability of finding a specified amino acid at position i. to find the significant position , entropy values under a certain threshold are filtered out . in this study , the cloud platform for proposed phylogenetic analysis tool is constructed on two important technologies : virtualization and the hadoop framework . the scalability and availability are guaranteed by hdfs , a self - healing distributed storage system and mapreduce , a specific fault - tolerant distributed processing algorithm . the master node consists of a job tracker , task tracker , name node , and data node . a slave node , or computing node , comprises a data node and a task tracker . the job tracker assigns map / reduce tasks to specific nodes within the cluster , ideally those already containing the data or at least within the same rack . a task - tracker node accepts map , reduce , and shuffle operations from a job - tracker . hadoop also creates multiple replicas of data blocks and distributes them to data nodes throughout a cluster to enable reliable , extremely rapid computations . the name node serves as both a directory namespace manager and a node metadata manager for the hdfs . the hdfs allows the data to spread across hundreds or thousands of nodes or machines , and the tasks are computed on data - holding nodes . hadoop replicates data , so that if one replica is lost , backup copies exist . when a node fails during computation , hadoop restarts the halted task on another node containing replicate data . in the hadoop framework , node failures are detected using the heartbeat mechanism , by which individual task nodes ( task trackers ) constantly communicate with the job tracker . if a task tracker fails to communicate with the job tracker for a period of time , the job tracker will assume that the task tracker has crashed . the job tracker knows which task trackers ( data nodes ) contain replicate data , and it issues a restart task . in this paper , the proposed cloud service was implemented by combining hadoop cluster distribution with a management model . in our cloud server , a submitted job is computed in a data node . rather than processing parallel data , therefore , submitted data are distributed to a data node by the hdfs , while the computing process is delivered to the task tracker and copied with the submitted data . the usual goal of virtualization is to improve scalability and overall hardware - resource utilization . virtualization permits the parallel running of several operating systems on a single physical computer . while a physical computer in the classical sense constitutes a complete and actual machine , a virtual machine ( vm ) is a completely isolated machine running a guest operating system within the physical computer . to ensure scalability and efficiency , all components job tracker , task tracker , name node , and data node in our cloud service operate as virtual machines . cloud - based orf phylogenetic analysis service was developed on a virtualization platform with the hadoop framework as described above . the master node ( name node ) and slave node ( data node ) are the master vm and slave vm , respectively . when a phylogenetic analysis request is submitted , it is saved in a job queue . the master node periodically extracts the jobs from the job queue and assigns them to slave nodes ( or mappers ) , which perform the task . at the completion of all jobs , the reducer collects the results and saves them in the network file system storage ( nfs ) . a single comparison result of a phylogenetic job is saved in a single file of nfs . as shown in figure 5 , a data node running in vm2 performs a phylogenetic analysis and a name node runs in vm1 . the reducer , running in vmn+2 , collates the results from the data nodes executing the phylogenetic analyses . in this service , the user uploads protein sequences and submits a phylogenetic analysis request on the website portal . all submitted analysis jobs are gathered in the job queue and sequence data are stored in different hosts by hdfs . the analysis results are sent to both data node and reducer to produce the final result stored in nfs . users either enter the comparative dna / rna sequences on the web portal or upload a file containing comparative rna sequences from a web portal .
step 2 : sequence translation . to detect the orf regions , all input rna sequences are translated to protein sequences based on the genetic code . the genetic code is the set of rules by which rna sequence information is translated into proteins . each codon in an rna sequence usually represents a single amino acid specified by the corresponding genetic code . this step identifies the functional orfs , recall that significant orfs are rare . in our service , the user can provide the length of orf that he / she regards as meaningful . , the first orf ( denoted as ab447445_1 ) extends from positions 3 to 5099 in the sequence ab447445 . in this step , two types of phylogenetic trees are built , one using the full sequence length and the other using orfs only . from the three orf regions identified in the analysis , three orf phylogenetic trees are built . these trees are recorded in ph format and are then transferred to and stored in the portal . meanwhile , step 4 : report result . in this step , the ph formatted trees are drawn as three diagrams and displayed on the portal . each server was equipped with two quad - core intel xeon 2.26 ghz cpus , 24 gb ram , and 296 gb hard disk , running under the ubuntu operating system version 10.4 , with 8 virtual machines on each server . one vm constituted the job tracker and name node ; the others are task trackers and data nodes . two of these vms are name node and data node running the reducer ; the remaining six are responsible for map operation . for the experiment , we randomly produced three datasets , each containing 20 sequences of different lengths ( 300 , 400 , and 600 nucleotides ) . clustalw and the proposed service were applied three times , for simulating three orf phylogenetic analyses . the computation time of the proposed service illustrated in figure 8 is proportional to the number of mappers . the execution time is considerably reduced when six mappers are used , relative to two mappers . figure 9 compares the performance between sequential phylogenetic analysis methods such as clustalw and the proposed service with six mappers , for different sequence lengths . clearly , the proposed service in the hadoop framework achieves better performance than standard sequential phylogenetic analysis . the nov genome is a single - stranded , positive sense , polyadenylated rna encoding three open reading frames , orf1 , orf2 , and orf3 . orf1 encodes a long polypeptide that is cleaved intracellularly into six proteins by the viral proteinase . orf2 encodes a viral capsid protein , vp1 , while orf3 encodes a vp2 protein that is regarded as a minor structural component of virus particles , apparently responsible for the expression and stabilization of vp1 . like the majority of rna viruses , the virus is tentatively divided into five genogroups and more than 25 genotypes , based on similarities between orf2 sequences [ 33 , 34 ] . therefore , the homology of this type of virus may be identified from orf similarities . identifying this homology will assist in viral drug and vaccine design . the phylogenetic trees constructed from full length sequences and three orfs are shown in figure 10 . obviously , these trees differ from each other . the tree constructed from the full length sequences ( figure 10(a ) ) demonstrates an evolutionary relationship between the viruses . however , different orfs yield distinctly different trees ( figures 10(b)10(d ) ) , suggesting that viruses can copy orfs from other viruses and alter their function by integrating them into their own sequences . therefore , by establishing evolutionary relationships for each orf , virologists can analyze the diseases caused by specific orfs . the residue position of high entropy is provided in figure 12 , which shows four phylogenetic trees and the diversity bar graph . the positions ( also the amino acids ) of high diversity are shown in the box . cloud computing is the online delivering of computing resources , such as hardware and software . users can access cloud - based applications through a web browser or via applications on mobile devices . although many bioinformatics tools have been developed as web applications , these are typically deployed in a server , which has limited computing power . currently , some tools have been redeveloped as distributed computing tools based on the hadoop framework . these tools are readily deployed on a cluster provided by a cloud computing vendor such as amazon ec2 . deployment of preexisting tools to the cloud environment is the current trend of bioinformatics as a service . in this paper , we propose a high - scale , available cloud - based open reading frame phylogenetic analysis service based on a hadoop cluster using virtualization technology . virtualization enables the proposed service to copy large quantities of jobs . because hadoop is strongly buffered against faults , the proposed cloud service guarantees that submitted jobs are recovered by task reassignment , ensuring a high - availability cloud service . our case study demonstrated that our service can construct different phylogenetic trees from comparisons of different orfs . | phylogenetic analysis has become essential in researching the evolutionary relationships between viruses .
these relationships are depicted on phylogenetic trees , in which viruses are grouped based on sequence similarity .
viral evolutionary relationships are identified from open reading frames rather than from complete sequences .
recently , cloud computing has become popular for developing internet - based bioinformatics tools .
biocloud is an efficient , scalable , and robust bioinformatics computing service . in this paper , we propose a cloud - based open reading frame phylogenetic analysis service .
the proposed service integrates the hadoop framework , virtualization technology , and phylogenetic analysis methods to provide a high - availability , large - scale bioservice . in a case study
, we analyze the phylogenetic relationships among norovirus .
evolutionary relationships are elucidated by aligning different open reading frame sequences .
the proposed platform correctly identifies the evolutionary relationships between members of norovirus . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
open nephron - sparing surgery is an accepted treatment modality for small renal masses less than 4 cm in size that provides oncological control comparable to that achieved with radical nephrectomy . the goal of a partial nephrectomy is complete local excision of the focal lesion with minimal complications and optimal functional preservation of the renal remnant . traditionally , partial nephrectomy is performed with renal pedicle clamping to facilitate tumor excision and decrease blood loss . however , renal vascular occlusion necessitates the dissection of the renal pedicle , which is technically challenging , time consuming , and potentially dangerous . furthermore , both renal vascular occlusion and the subsequent warm renal ischemia time increase the risk of postoperative renal failure . therefore , to facilitate the resection procedure and avoid hilar occlusion , a number of investigators have evaluated regional hypoperfusion using a variety of tourniquet - type methods or clamps [ 4 - 6 ] . others have reported using a hand - assisted approach to allow for manual compression of the parenchyma . these methods allow for the removal of renal tumors without clamping the renal pedicle , thus avoiding ischemia in the remaining renal tissue . however , despite favorable results with parenchymal compression methods , which have been reported as having shorter intra - operative times and less surgical blood loss , there have been no comparative studies with conventional vascular clamping methods directly addressing perioperative outcomes and maintenance of postoperative renal function . here , we evaluated the efficacy of the parenchymal compression method by directly comparing perioperative variables , including operative time , blood loss , and serial changes to serum creatinine levels , with those of the conventional vascular clamping method . open partial nephrectomy ( opn ) for small renal tumors less than 4 cm in size was performed by using one of two methods in our institution , according to the surgeon 's preference . the first method used was the modified parenchymal compression method using umbilical tape ( 1/8 " wide , ethicone , usa ) . the parenchymal compression technique was used from april 2006 ; the indications for using this opn technique were the presence of a small , exophytic , peripheral renal mass , including complicated cysts , located in a peripheral area of the kidney as evaluated by computed tomography ( ct ) or magnetic resonance imaging ( mri ) . we defined exophytic tumors as those where the lesion extended into more than 60% of the natural surface of the kidney . a peripheral tumor location was defined as a tumor peripherally located in the kidney and enveloped by the renal cortical or medullary parenchyma with no extension into the renal sinus . due to relative surgical inaccessibility , this method was not used for hilar tumors , which were defined as tumors within 10 mm of the main vascular structure of the renal pedicle . perioperative variables , including the american society of anesthesiology ( asa ) score , body mass index ( bmi ) , tumor size , tumor location , changes in serum creatinine , changes in hemoglobin , operative time , estimated blood loss ( ebl ) , transfusion rates , length of hospital stay , pathology findings , and complication rates , were evaluated prospectively . radiologic findings and serum creatinine , which was used as a surrogate for renal function , were also collected during the follow - up period . among the patients undergoing this technique , 20 patients with a normal preoperative serum creatinine level , defined as below 1.5 mg / dl ( group 1 ) , were exclusively enrolled in this study in an attempt to evaluate the effect of opn in patients with normal preoperative renal function . all patients who had opn at our institution were not only registered prospectively in a specific database that includes all pertinent information about the tumor and patient demographic characteristics , but were also followed - up under the same strict guidelines . this allowed us to match the parameters of group 1 patients against those of the other patient group , who had orn with a conventional vascular clamping method . according to preoperative characteristics of the tumors and patients from a database collected from april 2004 , 20 patients who had surgery with a vascular clamping method ( group 2 ) were carefully selected and enrolled in this series . regardless of the method used , the kidney was approached extraperitoneally from a standard flank incision between the 11th and 12th ribs in all cases . the kidney was mobilized while the perinephric fat overlying the tumor was left intact . in the modified parenchymal compression method the renal parenchyma was surrounded selectively with the umbilical tape and was then compressed with a kelly mosquito clamp ( fig . the umbilical tape was generally placed 2 to 3 cm from the tumor to allow for an excision with adequate surgical margins . to safeguard against inadvertent slippage of the umbilical tape , the line of renal parenchymal resection was 2 cm distal to the edge of the umbilical tape . the clamping pressure was carefully controlled manually by a second surgeon with a mosquito clamp ; the goal pressure was adequate to stop bleeding but not enough to damage the surrounding tissue . after identification of a negative surgical margin , the resected surface was then closed by using separated absorbable 2/0 sutures . in cases with positive surgical margins , more tissue was obtained from the previously resected site until identification of a negative surgical margin by cryosection . any breach of the pelvocaliceal system was suture - repaired in a dry operative field . after surgery , a closed drain was placed for all patients and left in place for 36 to 72 hours . in cases using the conventional vascular clamping method , the renal artery was occluded with a bulldog vascular clamp ; the renal vein was unclamped . patients were evaluated initially at one month postoperatively and then every 3 months during the first year , every 6 months during the second year , and then annually , with a medical history , physical examination , blood pressure , contrast - enhanced ct or mri , chest x - ray , serum electrolytes , liver function tests , and serum creatinine tests . the radiologic finding of local recurrence was defined as a mass with strong enhancement at the excision site or in the perinephric space , with an increase in size on subsequent follow - up imaging . a p - value of less than 0.05 was considered statistically significant . for comparing each group , a mann - whitney u test and a chi - square test were used . for comparing serial changes in serum creatinine in each group , a repeated - measures anova test , as an extension of the paired t - test to compare more than two repeated measures over time , was used . open partial nephrectomy ( opn ) for small renal tumors less than 4 cm in size was performed by using one of two methods in our institution , according to the surgeon 's preference . the first method used was the modified parenchymal compression method using umbilical tape ( 1/8 " wide , ethicone , usa ) . the parenchymal compression technique was used from april 2006 ; the indications for using this opn technique were the presence of a small , exophytic , peripheral renal mass , including complicated cysts , located in a peripheral area of the kidney as evaluated by computed tomography ( ct ) or magnetic resonance imaging ( mri ) . we defined exophytic tumors as those where the lesion extended into more than 60% of the natural surface of the kidney . a peripheral tumor location was defined as a tumor peripherally located in the kidney and enveloped by the renal cortical or medullary parenchyma with no extension into the renal sinus . due to relative surgical inaccessibility , this method was not used for hilar tumors , which were defined as tumors within 10 mm of the main vascular structure of the renal pedicle . perioperative variables , including the american society of anesthesiology ( asa ) score , body mass index ( bmi ) , tumor size , tumor location , changes in serum creatinine , changes in hemoglobin , operative time , estimated blood loss ( ebl ) , transfusion rates , length of hospital stay , pathology findings , and complication rates , were evaluated prospectively . radiologic findings and serum creatinine , which was used as a surrogate for renal function , were also collected during the follow - up period . among the patients undergoing this technique , 20 patients with a normal preoperative serum creatinine level , defined as below 1.5 mg / dl ( group 1 ) , were exclusively enrolled in this study in an attempt to evaluate the effect of opn in patients with normal preoperative renal function . all patients who had opn at our institution were not only registered prospectively in a specific database that includes all pertinent information about the tumor and patient demographic characteristics , but were also followed - up under the same strict guidelines . this allowed us to match the parameters of group 1 patients against those of the other patient group , who had orn with a conventional vascular clamping method . according to preoperative characteristics of the tumors and patients from a database collected from april 2004 , 20 patients who had surgery with a vascular clamping method ( group 2 ) were carefully selected and enrolled in this series . regardless of the method used , the kidney was approached extraperitoneally from a standard flank incision between the 11th and 12th ribs in all cases . the kidney was mobilized while the perinephric fat overlying the tumor was left intact . in the modified parenchymal compression method the renal parenchyma was surrounded selectively with the umbilical tape and was then compressed with a kelly mosquito clamp ( fig . the umbilical tape was generally placed 2 to 3 cm from the tumor to allow for an excision with adequate surgical margins . to safeguard against inadvertent slippage of the umbilical tape , the line of renal parenchymal resection was 2 cm distal to the edge of the umbilical tape . the clamping pressure was carefully controlled manually by a second surgeon with a mosquito clamp ; the goal pressure was adequate to stop bleeding but not enough to damage the surrounding tissue . after identification of a negative surgical margin , the resected surface was then closed by using separated absorbable 2/0 sutures . in cases with positive surgical margins , more tissue was obtained from the previously resected site until identification of a negative surgical margin by cryosection . any breach of the pelvocaliceal system was suture - repaired in a dry operative field . after surgery , a closed drain was placed for all patients and left in place for 36 to 72 hours . in cases using the conventional vascular clamping method , the renal artery was occluded with a bulldog vascular clamp ; the renal vein was unclamped . patients were evaluated initially at one month postoperatively and then every 3 months during the first year , every 6 months during the second year , and then annually , with a medical history , physical examination , blood pressure , contrast - enhanced ct or mri , chest x - ray , serum electrolytes , liver function tests , and serum creatinine tests . the radiologic finding of local recurrence was defined as a mass with strong enhancement at the excision site or in the perinephric space , with an increase in size on subsequent follow - up imaging . a p - value of less than 0.05 was considered statistically significant . for comparing each group , a mann - whitney u test and a chi - square test were used . for comparing serial changes in serum creatinine in each group , a repeated - measures anova test , as an extension of the paired t - test to compare the meansd age and tumor size was 55.610.5 years old and 2.20.9 cm in group 1 and 49.314.2 years and 2.311.0 cm in group 2 . the preoperative serum creatinine was 0.870.29 mg / dl in group 1 and 0.910.24 mg / dl in group 2 . the operative time was significantly less in group 1 than in group 2 ( 132.417.7 vs. 151.421.4 minutes , p=0.03 ) . ebl was slightly higher in group 2 , with borderline statistical significance ( 173.711.5 vs. 211.243.8 ml , p=0.06 ) ; consequently , the postoperative decrease in hemoglobin was lower in group 1 ( 1.291.18 vs. 2.111.08 g / dl , p=0.06 ) . histologic examination revealed that over 80% of the tumors in both groups were renal cell carcinomas . for all patients , the pathology results of the initial frozen specimens were negative ; the final pathologic report reconfirmed the initial results obtained by cryosectioning . the serum creatinine level during the surgical admission increased at postoperative day one and then gradually decreased . serum creatinine levels , checked at 1 , 3 , and 7 days after the operation , were significantly increased in both groups to a similar extent . however , 30 days after surgery , the pattern of serial serum creatinine levels in each group demonstrated statistical differences in repeated - measures anova testing ( p<0.001 ) . in mann - whitney u testing , the serum creatinine levels measured 30 days after surgery were statistically similar to preoperative levels in group 1 ( p=0.56 ) , although they were still elevated in group 2 ( p<0.001 ) . similar differences between preoperative and postoperative serum creatinine levels were found at 90 , 180 , and 360 days postoperatively ( fig . inadvertent slippage of the u - umbilical tape loop did not occur in any patients in group 1 . no major complications , including conversion to nephrectomy or major vascular injury , occurred in either group . three patients had minor complications : one patient in each group experienced urine leakage , but this was resolved after insertion of a temporary ureteral stent ; one patient in group 2 experienced a perirenal hematoma , which completely subsided , as determined by ct imaging , 3 months postoperatively . during a mean follow - up of 24.110.6 ( 13 - 47 ) months and 26.6614.9 ( 16 - 48 ) months for the renal cell carcinoma patients in groups 1 and 2 , respectively , there were no cases of metastasis or disease recurrence . the number of patients undergoing partial nephrectomy has increased due to the changing indications for this type of surgery , as well as the increased detection of incidental , small renal tumors . a partial nephrectomy is a technically demanding procedure that may lead to serious complications if not performed correctly . hemostasis of the resected surface is one of the most important aspects of partial nephrectomy surgery . intraoperative and delayed bleeding with the necessity of a secondary nephrectomy have been reported as complications of this procedure . therefore , a partial nephrectomy usually involves a wide dissection of the renal pedicle and artery , vein clamping , and renal cooling , all to provide better bleeding control . however , pedicle dissection is time - consuming and associated with a risk of vascular injury and profuse bleeding . renal pedicle clamping limits the available resection time to 30 - 60 min , during which time the lesion must be removed , prominent vessels sutured , the collecting system closed , and tissue removed for cryosection analysis . the most serious complications of this conventional surgical method are urinary fistulae ( 8 - 10% ) and acute renal failure , especially in 20 - 25% of patients having a solitary kidney . in these cases , ischemic renal failure following partial nephrectomy has been attributed to prolonged clamping of the renal artery leading to ischemia of the entire renal mass , intraoperative hypotension , and , rarely , postoperative thrombosis of the renal artery or vein . in light of these potential limitations , many alternative surgical methods have been developed in an attempt to decrease complication rates and improve renal function . after the initial introduction by gill et al , many other methods have been introduced , including the use of the reni - clamp , the satinsky clamp , vascular clamps , and the large curved debakey aortic clamp . gill et al , who used the tourniquet method , similar to the method we use here , reported that because the renal hilum was not occluded , blood flow to the remainder of the kidney was uninterrupted , thus minimizing the chances of ischemic injury to the renal remnant . they also reported that the use of the double loop apparatus eliminated the need for surgical mobilization of the renal hilar vessels . furthermore , because renal hypothermia was not required , the kidney did not need to be packed in ice slush intraoperatively . tsivian and sidi used a 1 cm wide vicryl mesh strip for parenchymal compression and reported that the resultant blood loss was minimal ; the maximal blood loss reported was 200 ml . among 61 patients undergoing a partial nephrectomy with this method , huyghe et al , who used the reni - clamp , reported the results of open partial nephrectomy in 33 patients . the mean operative and clamping times were 150 and 27 minutes , respectively ; the ebl was 150 ( range , 50 - 450 ) ml . similarly , mejean et al , who used a debakey aortic clamp in 10 patients with renal cell carcinoma , reported insignificant intraoperative blood loss with no complications , including urinary flank drainage or renal dysfunction . in the present series of patients using umbilical tape parenchymal compression , benefits consistent with those previously reported although not statistically significant , ebl was lower in the parenchymal compression group with a concurrent lower decrease in hemoglobin levels after surgery . because there was no need for dissection around the hilar vasculature using the parenchymal compression technique , the operative time for this approach the potential risk for bleeding was also decreased with the parenchymal compression technique . additionally , there was no risk of venous bleeding with the use of parenchymal compression because there was occlusion of the renal artery only . thus , we propose that the relative technical simplicity of the parenchymal compression technique positively affects surgical outcomes . one of the potential advantages of using the parenchymal clamping method compared with conventional vascular clamping is its positive effect on preservation of renal function . rodrguez - covarrubias et al , who used the debakey aortic clamp in seven patients with normal preoperative renal function , including one solitary kidney case , reported that the mean preoperative serum creatinine level was 0.74 mg / dl ( range , 0.58 - 1.26 mg / dl ) and the mean postoperative serum creatinine level was 0.81 mg / dl ( range , 0.69 - 1.21 mg / dl ) ; no patients required dialysis . in the current study , with a minimum follow - up of 12 months , although serum creatinine was temporarily increased during surgical admission in both groups , the pattern of serial changes in serum creatinine was significantly different between the groups . there was a trend toward an elevated serum creatinine in group 2 compared with group 1 ( fig . 2 ) . however , different from previous reports of an unchanged serum creatinine by use of the conventional vascular clamping method , the increased serum creatinine level observed in group 2 in this study merits discussion . although slippage of the umbilical tape did not occur in the current series , it is a critically important possibility to bear in mind when using the parenchymal compression technique . thus , although we attempted to position the umbilical tape in such a way as to be able to obtain a 2 - 3 cm margin from the tumor itself , the actual resection point using this technique tended to be minimized when compared with that of the vascular clamping method ( where we attempted to obtain a resectional margin approximately 1 cm from the tumor ) . although the size of the surgical margin in opn does not seem currently to affect the risk of local tumor recurrence , the patients enrolled in group 2 were a historical cohort . thus , this group contained patients undergoing opn with a different surgical margin policy , namely , the further the resection from the tumor margin , the greater advantage there was in tumor control . due to this policy , the amount of normally functional renal tissue resected in group 2 would be expected to be larger than in group 1 ; this may have affected the postoperative serum creatinine levels . despite positive findings in terms of perioperative and renal function outcomes , we recognize some limitations of this study series . this technique can not be applied to all renal tumors , as indicated by joung et al . to obtain sufficient compression of the kidney , it is imperative to circumscribe the mass with umbilical tape . however , for some tumors located in the hilar space , being endophytic and central , rather than exophytic and peripheral , makes it difficult to perform this technique . whereas our data included five cases of midpole lesions , all of these were small , peripherally located lesions ; this allowed for complete settlement of the umbilical tape in an oblique fashion . other limitations were intrinsic to the study design . although we attempted to collect data prospectively , the entire study design was not defined at the time of initiation . the margin goal was not initially standardized in our study , as previously mentioned , which may have negatively affected the serum creatinine levels in group 2 . also , serial serum creatinine measurements were used as a surrogate of renal function for simplicity , although this measure is not an accurate indicator for practical renal function . whereas differences in the pattern of serial serum creatinine measurements were noted , the increased serum creatinine levels were still within normal limits . this observation leaves unanswered the role of renal function preservation by use of the parenchymal compression technique in patients with abnormal renal function . whereas these data demonstrated practical advantages in terms of operative time , without compromising oncologic outcomes or increased complication rates , in order to evaluate the role of this technique on actual renal function , a study measuring serial glomerular filtration rates and having a strict operative procedural design is needed . opn using the parenchymal compression method had acceptable outcomes in terms of complete tumor control , avoidance of warm ischemic time , and minimizing blood loss , with good preservation of renal function and minimal complications . therefore , in selected tumors with peripheral nonhilar localization , this can be an alternative approach to traditional vascular clamping approaches . | purposewe evaluated the efficacy of parenchymal compression in open partial nephrectomies ( opns ) compared with that of the conventional vascular clamping method.materials and methodsopns were conducted by means of the parenchymal compression technique at our institution from april 2006 . among these
, the operative outcomes of 20 consecutive patients with normal preoperative renal function ( group 1 ) were matched with those of 20 control patients from the database of previous operations who underwent opn with a conventional vascular clamping method ( group 2).resultsall preoperative characteristics were similar in both groups .
the operative time was significantly higher for group 2 ( 132.417.7 vs. 151.421.4 minutes , p=0.031 ) . estimated blood loss was slightly higher for group 2 , with marginal statistical significance ( 173.711.5 vs. 211.243.8 ml , p=0.06 ) .
histologic examination revealed that over 80% of the tumors in both groups were renal cell carcinomas .
for all patients , the pathology results of specimens were negative .
serum creatinine , checked at 1 , 3 , and 7 days after the operation , was significantly increased in both groups to a similar degree .
however , 30 days after surgery , the patterns of serial serum creatinine levels demonstrated statistically significant differences by repeated - measures anova ( p<0.001 ) , with a trend of more elevated in group 2 than in group 1 , although values were within the normal range .
no major complications occurred in either group.conclusionsopn using the parenchymal compression method had acceptable outcomes in terms of complete tumor control , avoiding warm ischemic time , and minimizing blooding , with good preservation of renal function and minimal complications . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
as in most technical and medical fields , impressive developments have occurred in recent years in the technological aspects of digital photography and the possibilities of digital documentation . digital medical photography allows a professional view of novel clinical cases in cranio - maxillofacial surgery . visualization can be more effective than a verbal description and can aid in making appropriate decisions for treatment . one of the advantages of digital photography is the possibility of reviewing the picture immediately to judge technical aspects such as sharpness , illumination , color , and patient positioning . the immediate availability of digital images enables the treating physician to monitor a selected aspect in successive or serial shots in the presence of the patient . fewer appointments with patients may be necessary , as review of the accomplished or planned procedures is possible without waiting for photographs to be processed . due to the development of powerful data storage tools and software , clinical patient records can be supplemented with informative photographs , and these photographs can be integrated into digital patient files . these improvements along with technical innovations in photography have set the stage for high - quality results in maxillofacial surgery . in the literature , clinical photography is discussed from different viewpoints such as those of plastic and reconstructive surgery , dermatology , dentistry , and orthodontics.17 although human life unfolds in a 3-dimensional ( 3d ) setting , most observations and data are captured only in 2 dimensions , and information about the third dimension is left to our judgment . especially in the medical field , where surgery can change the appearance of a face , 3d assessment is becoming more and more essential . this new method will prove its value not only for planning of dental or surgical procedures , but also for predicting the outcome . several approaches have been investigated to open the third dimension to the medical world , starting with computerized tomography ( ct),810 ultrasonography,1113 stereolithography,14,15 and laser scanners.16,17 a detailed review of 3d craniofacial reconstruction imaging should describe modern imaging techniques most commonly used in medicine and dentistry . analysis of the whole craniofacial complex , virtual simulation , and real simulation of orthognatic surgery as well as laser scanning with use of stereolithographic biomodeling have been discussed.18 the aim of this article is to describe step - by - step the recent developments in medical photography , address solutions for data storage , and highlight the benefits as well as some of the technical and human pitfalls of this technology in the medical profession . in august 1981 , the digital camera revolution began when the sony corporation released the first commercial electronic handheld camera without film ( the sony mavica ) . this was designed as a point - and - shoot camera , which used a charge - coupled device - sensor ( ccd - sensor ) to record still images to mavipak diskettes with the equivalent of 0.3 megapixel ( mp ) resolution . because the pictures were viewed on a tv screen and could not be processed on a computer , the mavica was not considered a true digital camera . in 1988 , fuji unveiled the ds-1p as the first true digital camera , which recorded images to a removable static random - access memory ( sram ) card in a computerized file.19 the first commercially available digital camera was sold in 1990 as the dycam model 1 or logitech fotoman with a resolution of 376 240 pixels at 256 grayscale levels for a manufacturer s suggested retail price ( msrp ) of us$995.20 the next rung on the evolutionary ladder of digital photography was the kodak dsc-100 , shown publicly at the photokina in 1990 and marketed in 1991 for a msrp of us$25,000 . it was the first digital single - lens reflex camera ( dslr ) consisting of a modified nikon f3 slr body and a 1.3 mp digital back.21 although various companies such as canon , nikon , fujifilm , sigma , kodak , pentax , olympus , panasonic , samsung , and minolta released dslr cameras intended for professional photographers and early adopters , dslr cameras could not compete with film - based slr cameras due to their lack of speed and image resolution . dslr cameras began to compete with slr cameras in 1999 , when nikon introduced the nikon d1 , which employed autofocus lenses such as those in current use . in subsequent years , image resolution increased and prices decreased , until the canon eos digital rebel made dslr technology available to amateur photographers with a quality comparable to that of film cameras . with further development of ccd resolution , the question was often raised of when or if digital technology would exceed film technology in image quality . this issue has not yet been resolved and depends on numerous parameters . in summary , a resolution of 12 to 16 mp is equivalent to that of iso 100 color film , but this comparison can only be made when high - quality lenses are used . for image resolution exceeding 10 mp , the quality of the lenses and image compression seem to be the limiting factor for image quality.2224 for practical and clinical applications , more detailed image resolution does not yield further advantages , and thus the evolution of the dslr technique in clinical photography has apparently reached its end . considering digital imaging as a tool for routine work in dentistry and oral and maxillofacial surgery , acquired image data the amount and quality of image data determine the dimensions of the required image storage system . the best image quality is supplied by unprocessed raw - image data , which is not recommended in clinical photography due to the degree of post - processing needed and the large file sizes generated . the standardized jpg image format with variable compression , used with a resolution of 6 to 8 mp and low compression , fulfills the requirements of clinical photography and is manageable even for large numbers of images . in digital workflow , the sharpness , white balance , brightness , and orientation of images should be verified before they are stored in the database . images should not be post - processed for these parameters , but primarily should be exposed correctly , due to the time - consuming nature of post - processing and the possibility of falsifying the document . thus , the ability to immediately control the quality of the picture is a valuable advantage of the digital era . the requirements for storage of patient images are complex . a patient image database should have a hierarchical structure for user administration , support key - wording , indexing , and savable queries , have a programmable interface for linking image data to a clinical information system ( cis ) , and be fast , scalable , and intuitive to use . some of the ciss that are currently commercially available support structured data systems with the ability to link an image to a patient file . for more advanced storage and administrative functions , professional digital asset management systems ( eg , the canto cumulus ) must be integrated into the cis via a programmable interface . a good compromise for a low - priced image database is to use software such as adobe photoshop lightroom or acdsee pro , which can be used separately from the cis with few limitations of convenience and function . as the importance of photography in routine work increases , long - term storage , reliability , and availability become an issue . although image data can be stored to digital media such as dvds and blu - ray discs , the durability of the image data is threatened by the possibility of hardware failure ( due to wear , electrical surge , flood , or fire ) , accidental deletion , theft , and malicious software . to guarantee permanent availability and safe long - term storage of image data , a multistage strategy must be followed including daily automated backup on a physically separate device , firewalls , a virus scanner , an uninterruptible power source ( ups ) , surge protection , access control , and a documented emergency and disaster recovery plan . a meaningfully defined standard picture set is necessary and can be adapted to the concerns of the respective users . a full - face front view , oblique , submental oblique , and lateral views have been described as a useful basic picture set . intraoral documentation includes upper and lower occlusal , buccal left and right , and frontal views.2,25 additional picture sets can be obtained for orthognathic surgery , skull deformities , synostotic or positional plagiocephaly , facial palsy , aesthetic surgery , and dental implantology . in dental implantology , the frontal region of the upper jaw is particularly and aesthetically important , and additional close - ups showing neighboring structures are essential . the attention of the surgeon should not focus on the tooth or implant alone , since an implant usually also has effects on the lip and cheek contours of the patient at various ages . a preoperative assessment with the aid of photographs one of the fundamental parameters should be the patient s position with the head at the same level as the camera . for each picture , the patient s position and distance from the camera should remain the same , and rotation of the head and tilting must be avoided . the image should be aligned horizontally and vertically to the middle axis of the occlusion plane . for facial pictures , the frankfort horizontal plane should be parallel to the floor and aligned vertical to the occlusion plane . the deformity can be exaggerated or masked if the patient is wrongly position , and this is especially likely to happen with orthognatic patients , as shown in figure 1 . the photograph should be adjusted so that the mid - sagittal plane of the patient is orientated perpendicular to the optical axis . interfering cosmetics and jewelry should be removed as well as blood or saliva in intraoral views . the brain can achieve 3d perception by interpreting the difference in depth of 2 pictures with the right and left eye . the establishment of the next dimension in photography lies in the use of more than one camera at a time . the easiest way to achieve a 3d image is to take 2 pictures of the same object by moving the camera to one side without changing the level . these 2 pictures can now be viewed with 2 eyes using the cross - eye method , looking at the left picture with the left eye and at the right picture with the right eye . more professional ways of producing real 3d pictures require additional camera viewpoints , and several camera systems have been introduced with this capacity . in 2008 , a 3d digital imaging system , the fuji finepix real 3d , was announced , with dual lenses that capture images simultaneously . for medical concerns , other systems with more than two cameras have been investigated , for example the 3d capture systems by genex or 3dmd ( figure 2 ) . the 3dmd cranial system , for example , works with five camera viewpoints to obtain a full 360 picture of the head ( figures 3 and 4 ) . these systems have been analyzed with regard to their anthropometric precision and accuracy of digital 3d photogrammetry of the face , and can be combined or compared with direct anthropometry using statistical methods.26 furthermore , these 3d applications are useful in the description of cranial and facial soft tissues . a meaningful example of their use in medical treatment is the identification of common features in children with craniofacial deformities . the capacity for 3d visualization supports the ability to distinguish synostotic and non - synostotic plagiocephaly . the addition of this feature adds significant information in the diagnosis and treatment of these children . the use of 3d photography is of interest in all fields dealing with the treatment of obvious changes in the appearance of facial morphology , both for evaluating changes and predicting surgical results . applications of 3d imaging for assessment of facial changes have been described in orthodontics as well as in the related discipline of orthognathic surgery.2731 other authors have described applications in patients with cleft lip and palate3235 or with craniofacial malformations to aid in recognizing the key components of particular syndromes.36 new technologies are being implemented in 3d photogrammetry for collecting phenotypic measurements of the face.37 photogrammetry is more than simply making measurements using stereoscopic photographs , but can capture 3d images with the ability to estimate coordinates of points , linear or surface distances , and volumetric measurements . the more sophisticated computerized stereophotogrammetry , c3d , has been introduced as a useful technique for 3d recording of monochrome and color stereo images32,3840 in the field of maxillofacial surgical planning . as previously mentioned , standardization is an essential requirement in clinical and scientific photography , and this has been demonstrated in the field of 3d photography as well . more information is gained with the added dimension , but the number of possible mistakes increases accordingly . the changeover from analog to digital photography in medicine has occurred gradually and without major difficulties , and the advantages of technologies for digital photography in the dental and maxillofacial field have been clearly outlined ; however , the availability of these digital technologies represents both an opportunity and a challenge . the physician is expected to provide sufficient image processing and to ensure the high quality of images . meaningful archiving and secure storage can be achieved using a professional keyword - indexed asset management system . such a system provides easy access for presentations and lectures , as well as for forensic purposes . the capability for digital post - processing , however , has the disadvantage of enabling falsification of images . many published papers define a basic picture set in 2 dimensions for different uses including dentistry , orthodontics , and maxillofacial and plastic surgery.2,3,6,25,41 furthermore , supplemental picture sets for special circumstances have been described , which are useful in the field of maxillofacial surgery.25 beyond the function of documentation , attempts have been made to use photography as a means of identifying landmarks and measure distances on two - dimensional photographs . measurements of photographs have been carried out by various specialists , for example , for computerized eyelid measurement analysis in ophthalmology.42 other attempts to characterize facial morphology in orthodontics using standardized photographs have been examined and compared to cephalometric measurements.43,44 photographic methods have also been used to identify landmarks or digitally optimize appliances such as head bands.4547 nevertheless , reducing the picture set to a minimum will increase acceptance and feasibility . knowledge of common mistakes can prevent pitfalls and help in achieving professional skills in digital photography.48,49 manipulation of the patient s head position49 or changes in illumination50 can make a difference in the surgical outcome . the advantages of digital photography such as saving time , lower costs , speed of storage , and reduced storage space with easier access to the photographs , have been described in the literature.2,51 the use of 3d photography supports clinical diagnosis and treatment in various fields . in medical genetics , it has demonstrated high levels of sensitivity and specificity in discriminating between controls and individuals diagnosed with noonan syndrome , and has the potential for use in training physicians.36 precision and error of 3d phenotypic measures from 3dmd photogrammetric images have also been described in the field of clinical dysmorphology in medical genetics . here the precision is specified as highly repeatable with an error for placement of landmarks in the sub - millimeter range.37 the development of ct has revolutionized diagnostic and treatment purposes in medicine . especially the field of orthognatic surgery has major benefits in the three - dimensional analysis.52 the combination of ct - based 3d data sets with 3d photographs could add significant information for tissue landmarks requiring information of hairline or eyelids . it could be shown that the registration of 3d photographs with ct images could provide an accurate match between the 2 surfaces.53 recently this group was able to confirm the accuracy of matching 3d photographs with skin surfaces from cone - beam cts with an error within 1.5 mm.54 using 3d stereophotogrammetry for the soft tissue analysis 2 observers showed a high reliability coefficient with 0.97 for intraobserver and 0.94 for intraobserver reliability in 20 patients.55 however , it been reported that the accuracy of 3d facial imaging in orthodontics using the genex camera system showed substantial image distortion when images of sharp angles 90 were captured . this system , the genex rainbow 3d camera model , is a technology with 2 cameras . the accuracy was greater the less that the z - coordinate was incorporated in the image . this limitation was to be expected , given the camera configuration . because the lenses were located somewhat close to each other , resulting in a limited field of view , it was difficult to get an accurate z - coordinate measurement.31 in the medical literature several 3d imaging systems in photography have been introduced . besides commercially offered systems like 3dmd and genex , other 3d custom - made systems and software developments have presented.3840 the validation of the systems has been published independently.28,32,37,56 the only comparison of measurement data of different 3d photogrammetric systems was performed by weinberg et al26 and showed that both systems are sufficiently concordant ( relative to one another ) , accurate ( relative to direct anthropometry ) , and precise to meet the needs of most clinical and basic research designs . the evolution of photography has resulted in easy - to - use and affordable digital photography for the practitioner . in the specialty of dentistry , medical photography has become a high - quality tool for health care professionals using a defined standard picture set for documentation in a standard reproducible set - up . the newest innovation in photography , incorporating the third dimension , offers detailed studies of the facial surface and soft tissue morphology . the advantages of digital photography include improved capabilities for diagnostics , planning of surgery and treatment , follow - up , and interdisciplinary communication between physicians and other specialists . | in maxillofacial surgery , digital photographic documentation plays a crucial role in clinical routine .
this paper gives an overview of the evolution from analog to digital in photography and highlights the integration of digital photography into daily medical routine .
the digital workflow is described and we show that image quality is improved by systematic use of photographic equipment and post - processing of digital photographs .
one of the advantages of digital photography is the possibility of immediate reappraisal of the photographs for alignment , brightness , positioning , and other photographic settings , which aids in avoiding errors and allows the instant repetition of photographs if necessary .
options for avoiding common mistakes in clinical photography are also described and recommendations made for post - processing of pictures , data storage , and data management systems .
the new field of 3d digital photography is described in the context of cranial measurements . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
idiopathic inflammatory myopathies ( iim ) encompass three subsets : dermatomyositis ( dm ) , polymyositis ( pm ) , and sporadic inclusion body myositis ( ibm ) with distinct immunopathological patterns . in dm , complement deposition on endomysial capillaries , loss of microvessels , and mainly perivascular inflammation are observed , whereas pm and ibm show a t - cell invasion of muscle cells associated with degenerative features in ibm . recently , necrotizing autoimmune myopathy ( nam ) , with scarce or no inflammation , has been recognized as a fourth iim entity . the expression of inflammatory effectors in iim is extensively investigated , since pharmacological targeting of immunoregulatory factors is increasingly recognized as an effective treatment tool [ 24 ] . vascular endothelial growth factor ( vegf ) family comprises a group of potent endothelial cell mitogens . vegf - a , an angiogenic growth factor , proinflammatory mediator , and promoter of vascular permeability , is produced by various cell types , among which are myocytes and inflammatory cells . its expression is transcriptionally regulated by multiple molecules : growth factors , hormones , and oncogenes . alternative splicing from eight exons within the vegf - a gene generates a family of proteins , named according to their amino acid number , vegfxxx . vegf family encompasses proangiogenicxxx and antiangiogenicxxxb isoforms , originating by alternate splice site selection in the terminal exon . molecules have identical length but differing c - terminal amino acid sequences and opposing biological effect on angiogenesis : proangiogenic vegfxxx isoforms originate by proximal splice site selection and antiangiogenic vegfxxxb isoforms by distal splice site selection . inhibition of angiogenesis by vegfxxxb isoforms is due to competitive receptor binding , inhibition of receptor phosphorylation , and downstream intracellular signalling . in the present work , we investigated tissue localization of vegf - a165b and vegf - a in iim as well as transforming growth factor- ( tgf- ) , involved in the angiogenic and proliferative processes and a prominent vegf - a165b inductor , along with pathological parameters of myositides . muscle samples were archival diagnostic biopsy specimens from twenty - one patients affected with iim , diagnosed by current clinicopathological criteria , as dm ( n = 8) , pm ( n = 6 ) , or ibm ( n = 7 ) . demographic data are presented in table 1 . as control samples , we processed biopsy muscle specimens ( n = 6 ) from subjects complaining of muscle pain , or cramps , with normal or mildly increased levels of serum creatine kinase and normal or mild myopathic electromyographic findings . we selected as controls only subjects in which muscle histology , histochemistry , immunohistology , and ultrastructural morphology resulted as normal , ruling out inflammation , degeneration - regeneration , denervation , or changes suggestive of metabolic storage . all subjects signed an informed consent with allowance to scientific utilization of muscle samples for research purposes . specimens were frozen in liquid nitrogen - cooled isopentane and stored at 80c until use . cryostat sections , 10 m thick , were submitted to diagnostic routine histological and histochemical stains . wb analysis in muscle samples from control subjects and iim patients was performed following standard procedures . muscle samples were lysed in radioimmunoprecipitation assay ( ripa ) buffer ; proteins ( 40 g load ) were separated by sds - page 420% tris - glycine pre - cast gel ( invitrogen s.r.l . , after blocking , samples were incubated with the primary antibodies for vegf - a ( cod . mab3734 ; millipore corporation , billerica , ma , usa ) , vegf - a165b ( abcam plc , cambridge , uk ) , tgf- ( santa cruz biotechnology , inc . , santa cruz , ca , usa ) , and -actin ( cell signaling technology , inc . , danvers , ma , usa ) and then with the appropriate secondary antibodies . bound antibodies were visualized by enhanced chemiluminescence ( ecl detection kit , ge healthcare , milan , italy ) , and the band densities were quantified using nih image software by using -actin band for normalization . immunohistology for diagnostic analysis of inflammation ( hla - abc , c5-b9 , cd4 , cd8 , cd11b , cd20 , cd45ro dako , glostrup , denmark ; carpinteria , ca , usa ) was carried out on 7 m thick cryostat sections on silane - coated slides ( starfrost ; knittel glser , braunschweig , germany ) . immunohistochemistry for tgf- and endothelial marker cd31 ( dako ) , for vessel detection , were also carried out . regenerating fibres were identified by antifoetal myosin heavy chain antibody ( novocastra laboratories ltd , newcastle upon tyne , uk ) . analysis was performed by immunoperoxidase technique , by hrp - labeled polymer ( dako ) , and 3,3-diaminobenzidine ( sigma - aldrich , milan , italy ) for visualization . morphometry was carried out by zeiss axioplan2 microscope equipped with axiovision 4.6 software ( carl zeiss vision gmbh , hallbergmoos , germany ) . the endomysial vessel density , the degree of inflammation , and the entity of muscle regeneration were evaluated , as histopathological parameters of myositis . density of endomysial vessels was expressed as number / mm of muscle area by counting the whole sections immunostained for cd31 . inflammatory score was assessed on sections stained for marker of activated leukocytes cd45ro . for each sample , three randomly selected fields at 100x magnification were analyzed by automatized colorimetric pixel evaluation , detecting the peroxidase reaction product . cd45ro peroxidase labeled area and the area of the whole field were measured : inflammatory score was expressed as the cd45ro+ percentage of the total area . regeneration index was expressed as the percentage of foetal myosin heavy chain reactive fibres by examination of at least 700 fibres . intergroup variabilities were analyzed by mann - whitney test for paired analysis , and analysis of variance was performed by kruskal - wallis test for multiple groups . spearman rank test was used to analyze correlations between wb band densities and the other investigated pathological parameters . significance was set at p < 0.05 . the protein levels of both vegf isoforms were higher in iim subjects ( figure 1 ) . an increase of circa 30% for vegf - a and up to 2.5-fold for vegf - a165b versus controls ( figure 1(b ) ) was observed . vegf - a165b was significantly upregulated in the iim subgroups dm , ibm , and pm versus controls ; analysis of intergroup variance evidentiated significantly higher levels of vegf - a165b in ibm versus pm and dm ( figure 1(c ) ) . the ratio vefg - a165b / vegf - a was significantly increased in iim ( figure 1(d ) ) , indicating a preferential upregulation of the antiangiogenic subunit : iim 0.70 0.40 ( min . 0.15 , max 2.09 ) ; ctrls : 0.31 0.25 ( min 0.03 , max 0.68 ) . pm and ibm subgroups were significantly different from controls ( figure 1(e ) ) . iim samples displayed an almost 2-fold increase in tgf- levels versus controls ( figure 2(b ) ) , with statistical significance in all the iim subsets ( figure 2(c ) ) . tgf- protein levels in iim were positively related with both vegf isoforms content ( figures 2(d ) and 2(e ) ) . muscle fibres showed a constitutive diffuse cytoplasmic expression of vegf - a , either in controls ( figure 3(a ) ) and in iim ( figures 4(1b ) ; 4(2b ) ; 4(3b ) ; 4(4a ) ) . cytoplasmic localization of vegf - a165b was very faint in control muscle , where endomysial vessels appear the main source of the protein ( figure 3(b ) ) and in morphologically normal fibres of iim ( figures 4(1c ) ; 4(2c ) ; 4(3c ) ; 4(4b ) ) . in myositis , atrophic and regenerating fibres ( figure 4(1a ) ) , identified by foetal myosin ( figure 4(1d ) ) , were strongly reactive for either isoforms . a milder vegf - a165b upregulation was observed in most nonregenerating , morphologically normal fibres ( figure 4(1c ) ) , with neolocalization of mhc i molecule hla - abc ( figure 4(1e ) ) . necrotic fibres , identified by deposits of the terminal complex of complement ( figure 4(2a ) ) , displayed no vegf or vegf - a165b expression ( figures 4(2b)-4(2c ) ) . vacuolated fibres of ibm ( figures 4(3a)4(3c ) ) strongly expressed vegf - a165b in adjacency of rimmed vacuoles , a pathological hallmark of ibm . perifascicular atrophic fibres of dm also showed an increased expression of both vegf isoforms ( figures 4(4a)-4(4b ) ) . areas of vegf upregulation displayed a substantial vegf - a / vegf - a165b co - localization but a stronger diffuse immunostain and a high occurrence of focal reactive deposits resulted distinctive vegf - a165b features ( figures 4(1b)-4(1c ) , 4(3b)-4(3c ) ) . both vegfs were also expressed by endothelium of most capillaries ( figures 4(1b)-4(1c ) ) or larger endomysial vessels ( figures 4(3e)-4(3f ) ) . occurrence of mononuclear infiltrates with scarce vegf - a stain was observed in subjects submitted to steroid treatment ( figure 4(3b ) ) , whereas vegf - a165b expression was maintained ( figure 4(3c ) ) . in biopsies performed prior to therapy tgf- was detected in inflammatory cells and muscle fibres in adjacency of infiltrates , as well as in vessel walls ( figure 4(3d ) ) , in substantial colocalization with vegfs ( figures 4(3e)-4(3f ) ) . ibm cases showed a significantly higher vessel density than controls ( figure 5(a ) ) . analysis of variance among iim subsets evidentiated a significantly higher endomysial vascularization in ibm versus pm and dm . endomysial vessel density in iim was in positive correlation with vegf - a ( r = 0.585 , p = 0.017 , ) and vegf - a165b levels ( r = 0.503 , p = 0.047 ) , as well as with vegf - a165b / vegf - a ratio ( r = 0.812 , p < 0.001 ) . ibm cases showed the highest degree of inflammation compared to the other groups ( figure 5(b ) ) . inflammatory score in iim was in positive correlation with vegf - a165b levels ( r = 0.630 , p = 0.009 ) , as well as with vessel density ( r = 0.499 , p = 0.049 ) . the occurrence of regenerating fibres was highly variable among and within the subgroups ( figure 5(c ) ) : ibm and pm showed higher regeneration percentage as compared to dm . the regeneration index was positively related to tgf- protein level in the whole group of iim ( r = 0.473 , p = 0.03 ) and to vegf - a165b / vegf - a ratio ( r = 0.523 , p = 0.012 ) . the present report demonstrates that vegf - a165b is expressed in human skeletal muscle and its level robustly increases in iim . constitutive expression of vegf by myocytes is found to be essential for regulation of capillarity and , in aged humans , the lower density of endomysial capillaries is associated with vegf local decrease . therefore , the vegf increase that we report in iim is not biased , but rather stressed , by the younger median age of our noninflammatory controls . increase of vegf in toto in skeletal muscle following injury , in association with fibres regeneration , is documented . a recent fundamental study highlighted increased muscle expression of vegf - a in iim , in correlation with clinicopathological stages of disease , and it also specifically addressed the issue of therapy influence . glucocorticoid treatment ( 36 months ) in pm and dm lowered vegf - a expression , which stayed anyway significantly higher than in controls , by decreasing the number of vegf - a reactive capillaries and mononuclear inflammatory cells , as we also observed in our treated patients . therefore a role of steroids is likely also in the modulation of muscle vegf - a165b ; however , the highest levels of both vegf isoforms were detected in our group of ibm patients , typically unresponsive to steroids , all submitted to 14 months glucocorticoid treatment , prior to biopsy and histological diagnosis . they are downregulated in highly angiogenic tissues , such as placenta , but they may reach 90% of the total vegf protein in colon . a previous study investigating antiangiogenic isoforms of vegf - a in human muscle did not detect vegf - a165b , basally or after submaximal exercise , in healthy subjects , by pcr , whereas previous pcr , and elisa identifications of vegf165b in skeletal muscle are reported . as skeletal muscle is richly vascularized , either western blot or elisa , at the protein level , and pcr , at the mrna level , do not rule out detection of endothelial molecules . as we agree that , within normal muscle , the antiangiogenic vegf subunit is mainly expressed by endothelial cells , the events of inflammation and coexisting atrophy / regeneration in iim seem to trigger a substantial vegf165b upregulation in muscle . a basal constitutive vegf - a165b synthesis by myocytes , against a diffusion from adjacent inflammatory cells , is also suggested by the absent stain , for either vegf isoforms , in necrotic fibres . the specificity of the utilized vegf - a165b antibody , directed against the antiangiogenic cooh - terminal sequence , was assessed by complete lack of recognition of angiogenic vegf - a165 protein isoform by wb , as documented by manufacturer ( http://www.abcam.com/vegf-165b-antibody-mrvl56-1-ab14994.html ) . studies addressing the alternate distal / proximal site splicing in human disease describe a splicing switch to pro - angiogenic isoform in angiogenesis - associated disorders , such as solid tumors and diabetic retinopathy [ 17 , 18 ] . opposingly , a selective upregulation of vegf - a165b is reported in retinal detachment associated with proliferative vitreoretinopathy , glaucoma , and fibrosing autoimmune disorder systemic sclerosis [ 20 , 21 ] , where decrease of angiogenesis occurs . in our iim samples , a local vegf - a165b preferential upregulation is suggested by the increased vegf - a165b / vegf - a ratio . research data on vegf - a165b offer clues to elucidate its increase in iim : growth factors and related signal pathways act on vegfxxx / vegfxxxb alternative splicing [ 5 , 17 ] , and pleiotropic fibrogenic tgf- is a key factor in switch to distal site splice selection for synthesis of vegf - a165b [ 5 , 20 ] . in our iim samples , as previously described [ 2 , 22 ] , tgf- was upregulated , and we observed a positive correlation tgf-/vegf - a165b by western blot . therefore , the peculiar vegf - a165b staining of degenerating / regenerating fibres may be linked to molecular events of muscle regeneration , where upregulation of tgf- occurs , as supported by correlation of extent of regeneration and vegf - a165b / vegf - a ratio . nevertheless , the lack of a direct correlation vegf - a / regeneration index and a milder vegf - a165b localization in nonregenerating fibres neoexpressing major histocompatibility complex - i , a change consistent with myofiber activation ( reviewed in ) , suggest that other factors contribute significantly to vegf - a165b upregulation in iim . immunohistology shows that tgf- reactive endothelium and inflammatory cells are also a prominent source of vegf - a165b . the finding is associated to inflammatory score / vegf - a165b and both vegf isoforms / vessel density positive correlations . though our patients ' subgroups are numerically too small to allow general conclusions , ibm higher inflammatory score and density of endomysial vessels , an acknowledged pathological feature of ibm , which resulted in our study strictly related to vegf - a165b / vegf - a ratio , appear to concur to higher vegf - a165b expression . this apparently controversial issue might depend on a negative feedback triggered by vegf - a165b . in fact , as it has been proposed [ 6 , 25 ] , vegf - a165b can inhibit vegf - a - mediated proliferation and migration of endothelial cells , via a counterregulatory mechanism , limiting the rate of de novo muscle capillarization . moreover , hypoxia , a strong vegf modulating agent [ 14 , 21 , 26 , 27 ] , due to microvascular involvement , has a role in all the major iim subsets by activating circuitries of cytokines , adhesion molecules , and leukocyte recruitment . skeletal muscle reacts with increased vegf - a gene expression to hypoxia caused by acute exercise . as an inductor of tgf- , hypoxia might result in an additional causative factor of local vegf - a165b increase in iim . interestingly , besides antiangiogenic effect , a cytoprotective activity of vegf - a165b against ischaemic damage has been recently documented on colon epithelium and on retinal epithelium and endothelium , so that its compound biological effects still need to be elucidated . local vegf antiangiogenic switch in inflammation and necrosis / regeneration of skeletal muscle is likely to depend on a cohort of humoral and cellular effectors , possibly involved in tissue protection against inflammatory process . as therapeutic modulation of vegf isoforms is currently investigated in cancer [ 12 , 14 ] and in angiogenic eye disorders , and considering a potential development in autoimmune disorder systemic sclerosis [ 20 , 21 ] , further studies are needed for a complete understanding of the balance between antiangiogenic and proangiogenic vegf isoforms in inflammatory myopathies . | objective . to investigate expression of vascular endothelial growth factor ( vegf ) antiangiogenic isoform a-165b on human muscle in idiopathic inflammatory myopathies ( iim ) and to compare distribution of angiogenic / antiangiogenic vegfs , as isoforms shifts are described in other autoimmune disorders .
subjects and methods .
we analyzed vegf - a165b and vegf - a by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with iim ( polymyositis , dermatomyositis , and inclusion body myositis ) and 6 control muscle samples .
tgf- , a prominent vegf inductor , was analogously evaluated .
intergroup differences of western blot bands density were statistically examined .
endomysial vascularization , inflammatory score , and muscle regeneration , as pathological parameters of iim , were quantitatively determined and their levels were confronted with vegf expression .
results .
vegf - a165b was significantly upregulated in iim , as well as tgf-. vegf - a was diffusely expressed on unaffected myofibers , whereas regenerating / atrophic myofibres strongly reacted for both vegf - a isoforms .
most inflammatory cells and endomysial vessels expressed both isoforms .
vegf - a165b levels were in positive correlation to inflammatory score , endomysial vascularization , and tgf-. conclusions .
our findings indicate skeletal muscle expression of antiangiogenic vegf - a165b and preferential upregulation in iim , suggesting that modulation of vegf - a isoforms may occur in myositides . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the outer membrane of gram - negative bacteria constitutes a semipermeable barrier that slows the penetration of antibiotics . the permeability of this barrier is known to vary greatly among species , with the outer membrane of pseudomonas aeruginosa only 8% as permeable as that of escherichia coli ( 1 ) . this decreased permeability severely restricts the uptake of nutrients and other important compounds into the cell , which consequently must be imported into cells using a collection of water - filled protein channels called porins . the p. aeruginosa family of porin proteins , defined based on their apparent sequence homology within the p. aeruginosa genome , plays an important physiological role in the transport of substances required for metabolism . however , these proteins also exhibit an affinity for certain hydrophilic antibiotics , such as -lactams , aminoglycosides , tetracyclins , and some fluoroquinolones , allowing these compounds to transverse the otherwise insoluble outer bacterial membrane ( 2 , 3 ) . deletion of one or more porin proteins has been shown to reduce the susceptibility of p. aeruginosa to certain antibacterial agents ( 4 ) . the p. aeruginosa porin oprd is a substrate - specific porin that facilitates the diffusion of basic amino acids , small peptides , and carbapenems into the cell ( 5 ) . oprd - mediated resistance occurs as a result of decreased transcriptional expression of oprd and/or loss of function mutations that disrupt protein activity . specific mechanisms resulting in decreased transcriptional expression of oprd include ( i ) disruption of the oprd promoter , ( ii ) premature termination of oprd transcription , ( iii ) co - regulation with trace metal resistance mechanisms , ( iv ) salicylate - mediated reduction , and ( v ) decreased transcriptional expression via co - regulation with the multidrug efflux pump encoded by mexef - oprn ( 6 ) . in this study , we examined the level of oprd expression in p. aeruginosa clinical isolates to determine the contribution of oprd porins in carbapenem resistance . clinical isolates were further examined using additional molecular methods to determine the degree of variability among isolates . antibiotic susceptibility testing was carried out using the kirby - bauer disc diffusion method ( 7 ) . isolates were divided into two groups according to their resistance status : multiple - drug resistant ( mdr ) and isolated carbapenem resistant ( icr ) . minimum inhibitory concentrations ( mic ) of ceftazidime ( caz ) , gentamicin ( cn ) , piperacillin - tazobactam ( tzp ) , ciprofloxacin ( cip ) , imipenem ( imp ) , and meropenem ( mem ) were determined using the vitek 2 system ( biomerieux , france ) . the densi - check 2 system ( biomrieux , france ) susceptibility tests were performed on the vitek 2 system using ast - n174 cards , according to the manufacturer s instructions . mic values of 32 g / ml for ceftazidime , 16 g / ml for gentamicin , 128 g / ml for piperacillin - tazobactam , 4 g / ml for ciprofloxacin , 16 g / ml for imipenem , and 16 g / ml for meropenem were defined as resistance ( 7 ) . transcript levels of oprd were analyzed by real - time polymerase chain reaction ( qpcr ) using a lightcycler instrument ( roche diagnostics , germany ) . total rna was extracted using the high pure rna isolation kit ( roche diagnostics , germany ) and converted into cdna for qpcr using the transcriptor high - fidelity cdna synthesis kit ( roche diagnostics , germany ) . the quality and purity of the rna obtained was evaluated spectrophotometrically ( maestrogen nanodrop , usa ) . as a result of evaluation , quantitative pcr was performed in capillary glass using the lightcycler faststart dna master sybr green i kit ( roche diagnostics , germany ) with primers specific for oprd , and rpsl ( table 1 ) . amplification of triplicate cdna samples from each isolate was performed under the following conditions : initial denaturation for 10 minutes at 95c , followed by 45 cycles of denaturation at 95c for 20 seconds , annealing at 68c for 10 seconds , and elongation at 72c for 15 seconds . a final melting curve analysis was performed using a single read at 90c . to evaluate the similarities among strains , arbitrarily primed pcr ( ap - pcr ) was performed using an m13 primer of sequence 5-gagggtggcggttct-3. pcr was carried out under the following conditions : 2 cycles of 94c for 5 minutes , 40c for 5 minutes , and 72c for 5 minutes , followed by 40 cycles of 94c for 1 minute , 40c for 1 minute , and 72c for 2 minutes . amplification products were identified by agarose gel electrophoresis , and similarities among isolates were evaluated by comparison of the band profiles . relative expression values ( r ) were determined using the ct method ; the gene encoding ribosomal protein rpsl was used as a control ( 10 ) . p. aeruginosa strain pao1 was used as a standard for normalization of relative mrna levels . reduced oprd expression was defined as transcription levels 70% of those of the pao1 isolate ( 11 ) . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification had occurred , the melting curves of each amplicon were assessed and compared with tm values obtained using pao1 dna as the template . antibiotic susceptibility testing was carried out using the kirby - bauer disc diffusion method ( 7 ) . isolates were divided into two groups according to their resistance status : multiple - drug resistant ( mdr ) and isolated carbapenem resistant ( icr ) . minimum inhibitory concentrations ( mic ) of ceftazidime ( caz ) , gentamicin ( cn ) , piperacillin - tazobactam ( tzp ) , ciprofloxacin ( cip ) , imipenem ( imp ) , and meropenem ( mem ) were determined using the vitek 2 system ( biomerieux , france ) . the densi - check 2 system ( biomrieux , france ) susceptibility tests were performed on the vitek 2 system using ast - n174 cards , according to the manufacturer s instructions . mic values of 32 g / ml for ceftazidime , 16 g / ml for gentamicin , 128 g / ml for piperacillin - tazobactam , 4 g / ml for ciprofloxacin , 16 g / ml for imipenem , and 16 g / ml for meropenem were defined as resistance ( 7 ) . transcript levels of oprd were analyzed by real - time polymerase chain reaction ( qpcr ) using a lightcycler instrument ( roche diagnostics , germany ) . total rna was extracted using the high pure rna isolation kit ( roche diagnostics , germany ) and converted into cdna for qpcr using the transcriptor high - fidelity cdna synthesis kit ( roche diagnostics , germany ) . the quality and purity of the rna obtained was evaluated spectrophotometrically ( maestrogen nanodrop , usa ) . as a result of evaluation , quantitative pcr was performed in capillary glass using the lightcycler faststart dna master sybr green i kit ( roche diagnostics , germany ) with primers specific for oprd , and rpsl ( table 1 ) . amplification of triplicate cdna samples from each isolate was performed under the following conditions : initial denaturation for 10 minutes at 95c , followed by 45 cycles of denaturation at 95c for 20 seconds , annealing at 68c for 10 seconds , and elongation at 72c for 15 seconds . to evaluate the similarities among strains , arbitrarily primed pcr ( ap - pcr ) was performed using an m13 primer of sequence 5-gagggtggcggttct-3. pcr was carried out under the following conditions : 2 cycles of 94c for 5 minutes , 40c for 5 minutes , and 72c for 5 minutes , followed by 40 cycles of 94c for 1 minute , 40c for 1 minute , and 72c for 2 minutes . amplification products were identified by agarose gel electrophoresis , and similarities among isolates were evaluated by comparison of the band profiles . transcription data were analyzed using the lightcycler relative quantification software . relative expression values ( r ) were determined using the ct method ; the gene encoding ribosomal protein rpsl was used as a control ( 10 ) . p. aeruginosa strain pao1 was used as a standard for normalization of relative mrna levels . reduced oprd expression was defined as transcription levels 70% of those of the pao1 isolate ( 11 ) . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification had occurred , the melting curves of each amplicon were assessed and compared with tm values obtained using pao1 dna as the template . mdr isolates were defined as those exhibiting resistance to ceftazidime ( mic 32 ) , piperacillin ( mic 128 ) , imipenem ( mic 16 ) , and gentamicin ( mic 16 ) ( 12 ) . all icr isolates were resistant to imipenem , and an additional three isolates ( 33% ) also exhibited resistance to meropenem . group 1 : multiple drug resistant ( mdr ) ; group 2 : isolated carbapenem resistant ( icr ) p. aeruginosa isolates . ap - pcr category : classification of p.aeruginosa isolates following ap - pcr analysis . relative gene expression : expression levels decreased oprd expression was observed in 16 of 18 p. aeruginosa clinical isolates , with significant decreases detected in 13 isolates ( 72% ) . within the mdr group , oprd expression oprd levels were decreased in 7 icr isolates , with significant decreases found in 4 of these isolates . detailed expression data for each group are shown in table 2 . due to the consistently low expression seen in the mdr group , differences in mrna expression between the groups were statistically significant ( p = 0.001 ) . the genetic similarity among isolates was determined using ap - pcr . among the 18 clinical isolates tested , classifications of p. aeruginosa isolates based upon ap - pcr analysis are shown in table 2 . mdr isolates were defined as those exhibiting resistance to ceftazidime ( mic 32 ) , piperacillin ( mic 128 ) , imipenem ( mic 16 ) , and gentamicin ( mic 16 ) ( 12 ) . all icr isolates were resistant to imipenem , and an additional three isolates ( 33% ) also exhibited resistance to meropenem . group 1 : multiple drug resistant ( mdr ) ; group 2 : isolated carbapenem resistant ( icr ) p. aeruginosa isolates . ap - pcr category : classification of p.aeruginosa isolates following ap - pcr analysis . relative gene expression : expression levels decreased oprd expression was observed in 16 of 18 p. aeruginosa clinical isolates , with significant decreases detected in 13 isolates ( 72% ) . within the mdr group , oprd expression oprd levels were decreased in 7 icr isolates , with significant decreases found in 4 of these isolates . detailed expression data for each group are shown in table 2 . due to the consistently low expression seen in the mdr group , differences in mrna expression between the groups the genetic similarity among isolates was determined using ap - pcr . among the 18 clinical isolates tested , classifications of p. aeruginosa isolates based upon ap - pcr analysis are shown in table 2 . pseudomonas aeruginosa represents a phenomenon of bacterial resistance , and most of the known antimicrobial resistance mechanisms are displayed in this species , and multiple resistance mechanisms may be expressed simultaneously within the same isolate ( 4 ) . due to the increase in multi - drug resistant p. aeruginosa infections , a greater emphasis has been placed on identifying genetic characteristics underlying bacterial resistance and the clinical implications of these mutations . outer membrane protein oprd is considered the preferred portal of entry for carbapenems and similar drugs such as imipenem and meropenem also enter the cell via oprd ( 3 , 13 , 14 ) . any loss of oprd expression from the outer membrane significantly decreases the susceptibility of p. aeruginosa to carbapenems and has been shown to play a major role in the acquired resistance to imipenem and , to a lesser extent , meropenem ( 15 ) . one study showed that oprd expression was decreased in the vast majority of 29 multi - drug resistant p. aeruginosa isolates ( 97% ) and played a significant role in their carbapenem resistance ( 16 ) . expression of oprd was decreased significantly in 13 of the 18 imipenem - resistant p. aeruginosa clinical isolates examined in this study , including all 9 mdr isolates . the frequency of decreased oprd expression in mdr isolates was significantly higher than that of icr isolates ( p = 0.001 ) , suggesting that oprd also plays an important role in the emergence of both carbapenem and non - carbapenem resistance . the impact of oprd - mediated resistance on carbapenems can be quantified relative to its effect on the antibacterial potency of carbapenems ( 6 ) . in a study evaluating isogenic wild - type and oprd - deficient mutant pairs , the loss of oprd decreased the susceptibility of p. aeruginosa to meropenem 4- to 32-fold , compared with 4- to 16-fold for imipenem and 8- to 32-fold for doripenem ( 14 ) . zeng et al . ( 17 ) investigated relative gene expression in 29 carbapenem - resistant and ceftazidime- and cefepime - sensitive p. aeruginosa clinical isolates and found that the loss of oprd was directly related to carbapenem resistance . in another study , fournier et al . ( 18 ) detected loss of oprd , as a result of mutations or gene disruptions , in 94 of 109 ( 86.2% ) imipenem - resistant p. aeruginosa isolates . in this study , oprd mrna levels were decreased in 7 of 9 icr isolates evaluated , even though significance was observed in only 4 of these isolates , in contrast with prior studies . although our data are consistent with the basic mechanism of imipenem resistance mediated by diminished oprd protein levels in the outer membrane , the poor correlation between oprd mrna expression and carbapenem resistance suggests involvement of additional resistance mechanisms in these isolates . impermeability was long thought to be the driver of intrinsic resistance in p. aeruginosa ; however , resistance has since been found to involve a more complex interplay between impermeability and multi - drug efflux pumps ( 19 ) . while it is believed that both meropenem and imipenem are able to enter the cell via the oprd pathway , only meropenem is a substrate of the mexab - oprm efflux pump ( 19 ) . furthermore this mechanism plays a role in the emergence of resistance to fluoroquinolones and other -lactams , increasing the likelihood of cross - resistance ( 20 ) . however , despite this additional mechanism , meropenem resistance is less likely to be acquired than imipenem resistance ( 77 vs. 68% sensitivities for meropenem and imipenem , respectively ) , as it requires both the loss of oprd expression and upregulation of mexab - oprm ( 6 , 21 ) . in our study , all 18 p. aeruginosa clinical isolates were resistant to imipenem , compared with only 12 isolates exhibiting meropenem resistance , consistent with previously published reports . the relationship between oprd deficiency and imipenem resistance has been well established ; however , cases of discordant oprd expression and carbapenem susceptibility , due to genetic versatility and multiple resistance mechanisms displayed in this pathogen , have been reported ( 6 ) . el amin et al . ( 15 ) identified four imipenem - susceptible isolates with significant reductions in oprd mrna levels caused by severe oprd mutations that resulted in frame shifts or premature termination . furthermore , they reported that the oprd mrna levels did not always correlate with imipenem resistance , and differences in imipenem susceptibility could not be explained by oprd mutations or efflux pump genes ( 15 ) . in our study , increased oprd levels were detected in 2 of the 18 imipenem - resistant p. aeruginosa clinical isolates analyzed . also we could not find any relation between genotype and resistance pattern in ap - pcr study . further studies will be necessary to understand the mechanisms underlying this apparent discordance between oprd levels and imipenem resistance . while oprd porin proteins play an important role in carbapenem resistance in p. aeruginosa , this resistance can not be explained by oprd levels alone , and other important interactions may influence carbapenem susceptibility . characterization of carbapenem resistance mechanisms could provide additional therapeutic targets or allow for alternative strategies to enhance the efficacy of carbapenems . | background : the pseudomonas aeruginosa porin oprd is a substrate - specific porin that facilitates the diffusion of basic amino acids , small peptides , and carbapenems into the cell . oprd - mediated resistance occurs as a result of decreased transcriptional expression of oprd and/or loss of function mutations that disrupt protein activity.objectives:in this study , we examined the level of oprd expression in p. aeruginosa clinical isolates to determine the contribution of oprd porins in carbapenem resistance.materials and methods : included strains were divided into two groups , comprised of multidrug - resistant ( mdr ) and isolated carbapenem - resistant ( icr ) strains .
the transcription product level of oprd was identified using real - time polymerase chain reaction ( qpcr).results : of the 18 clinical isolates , a decrease in the oprd level was found to be significant in 13 isolates .
nine of eighteen isolates with a significant decrease were determined in the first group and comprised mdr isolates that showed a statistically significant difference compared with the icr group ( p = 0.001 ) . in the icr group ,
oprd levels were found to be significantly low in 4 isolates .
six different patterns were determined by comparing band profiles in ap-pcr.conclusions:although the data support the idea that the basic mechanism of imipenem resistance could be via the loss of oprd , they do not fully explain the role of oprd and indicate that other mechanisms may play an important role .
additionally , the significant decrease in the oprd levels in mdr strains suggests that oprd also plays a role in the emergence of both carbapenem and non - carbapenem resistance . |
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infection by rubella virus in early pregnancy leads to a constellation of devastating effects on multiple organs characteristically called as gregg syndrome . cutaneous manifestations are varied and infrequent but the presence of dermal erythropoiesis indicates a severe disease . we report a case of a preterm newborn with microcephaly , hepatosplenomegaly along with violaceous lesions all over the body . dermatological opinion was sought by neonatal care unit for a preterm male newborn with a generalized violaceous skin rash . the baby was delivered at 35 weeks of gestation after an emergency caesarian section performed for premature rupture of membranes . the neonate was tachypnoeic but acyanotic and had microcephaly ( head circumference 30 cm , less than third centile ) , and low birth weight ( 2 kg ) . on cutaneous examination , there were multiple , discrete , 0.5 0.5 to 2 1 cm sized , violaceous macules and barely palpable nodules found predominantly over the face and upper trunk [ figure 1 ] . palms , soles and mucosae were spared . based on cutaneous manifestations , a diagnosis of blueberry muffin baby syndrome was made . purple macules and barely palpable nodules over the body blood investigations showed severe thrombocytopenia ( platelet count : 17,000/mm ) , raised prothrombin ( test : 21.3 s , control : 11.7 s ) and activated partial thromboplastin time ( test : 180 s , control : 30 ) with inr of 1.82 . skeletal survey revealed metaphyseal lucencies of long bones and enlarged right atrium [ figure 2 ] . a small patent ductus arteriosus ( pda ) of 2 mm was detected on echocardiography , whereas electrocardiogram was normal . ultrasonography of the head showed linear echogenic foci , suggestive of calcification in the chordothalamic region . skeletal survey showing metaphysial lucencies of long bones histopathology from a cutaneous nodule showed several ill - defined nodules of extramedullary hematopoiesis in the dermis , reaching up to subcutis suggestive of blue berry muffin lesions [ figure 3 ] . serology of the neonate showed the presence of igm antibody ( 42.20 iu / ml ) to rubella virus . the mother was also found to be positive for rubella igm , 79.70 iu / ml , and cytomegalovirus igg , 209 iu / ml . rest of the serological tests for torch ( toxoplasma , cytomegalovirus , herpes virus ) and venereal disease research laboratory ( vdrl ) were non - reactive in both mother and the neonate . on the basis of the above findings , a final diagnosis of congenital rubella syndrome ( crs ) was made . high power view ( 100 ) of a skin biopsy ( h and e stain ) showing nucleated red blood cells , megakaryocytes and metamyelocytes suggestive of dermal erythropoiesis in this era of widespread vaccination , rubella still accounts for a high number of intrauterine infections in the developing countries . in an indian study , 1 - 15% of all infants , suspected to have intra - uterine infection , had laboratory evidence of crs . spectrum of crs includes ocular abnormalities ( cataracts , retinopathy , microphthalmos , glaucoma ) , sensorineural deafness , neurological defects ( microcephaly , motor defects , mental retardation ) , cardiovascular abnormalities ( pda , ventricular septal defect , aortic stenosis ) , genitourinary and bone defects . the neonate had bone involvement and hepatosplenomegaly that have been reported only in 10 - 20% of these cases . the osseous lesions present as transverse radiolucent zones and longitudinal bands of demineralization , which appear because of abnormal mineralization . the cutaneous findings of crs include a thrombocytopenic purpura , adult like maculopapular rash and less frequently blueberry muffin lesions . only 5% of the crs cases manifest with extramedullary dermal erythropoiesis as seen in this case . usually present at birth , clearing of the lesions mostly occurs by 3 to 6 weeks . extracutaneous signs and symptoms depend on the cause and can be severe . thus , these neonates should be thoroughly worked up to rule out the underlying systemic disease and its extent . prognosis of blueberry muffin syndrome is thus variable , although dermal erythropoiesis is usually associated with severe disease . no specific therapy for congenital rubella has been established , so treatment is primarily supportive . we emphasize the importance of universal rubella immunization especially in females of reproductive age group to prevent the mortality and disability arising out of the congenital rubella infection . blueberry muffin lesions are a rare presentation of congenital rubella syndrome and portends a poor prognosisrare but characteristic osseous and hepatic involvement is reported in this case . blueberry muffin lesions are a rare presentation of congenital rubella syndrome and portends a poor prognosis rare but characteristic osseous and hepatic involvement is reported in this case . | congenital rubella syndrome involves a configuration of systemic and cutaneous manifestations in a neonate due to in utero infection caused by the rubella virus . the case of a preterm neonate with blueberry muffin lesions and classical as well as rare systemic features of congenital rubella syndrome
is reported . |
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